Corticosterone exposure is associated with long-term changes in DNA methylation, physiology and breeding decisions in a wild bird.

When facing challenges, vertebrates activate a hormonal stress response that can dramatically alter behaviour and physiology. Although this response can be costly, conceptual models suggest that it can also recalibrate the stress response system, priming more effective responses to future challenges. Little is known about whether this process occurs in wild animals, particularly in adulthood, and if so, how information about prior experience with stressors is encoded. One potential mechanism is hormonally mediated changes in DNA methylation. We simulated the spikes in corticosterone that accompany a stress response using non-invasive dosing in tree swallows (Tachycineta bicolor) and monitored the phenotypic effects 1 year later. In a subset of individuals, we characterized DNA methylation using reduced representation bisulfite sequencing shortly after treatment and a year later. The year after treatment, experimental females had stronger negative feedback and initiated breeding earlier-traits that are associated with stress resilience and reproductive performance in our population-and higher baseline corticosterone. We also found that natural variation in corticosterone predicted patterns of DNA methylation. Finally, corticosterone treatment influenced methylation on short (1-2 weeks) and long (1 year) time scales; however, these changes did not have clear links to functional regulation of the stress response. Taken together, our results are consistent with corticosterone-induced priming of future stress resilience and support DNA methylation as a potential mechanism, but more work is needed to demonstrate functional consequences. Uncovering the mechanisms linking experience with the response to future challenges has implications for understanding the drivers of stress resilience.

Population structure and connectivity among coastal and freshwater Kelp Gull (Larus dominicanus) populations from Patagonia.

The genetic identification of evolutionary significant units and information on their connectivity can be used to design effective management and conservation plans for species of concern. Despite having high dispersal capacity, several seabird species show population structure due to both abiotic and biotic barriers to gene flow. The Kelp Gull is the most abundant species of gull in the southern hemisphere. In Argentina it reproduces in both marine and freshwater environments, with more than 100,000 breeding pairs following a metapopulation dynamic across 140 colonies in the Atlantic coast of Patagonia. However, little is known about the demography and connectivity of inland populations. We aim to provide information on the connectivity of the largest freshwater colonies (those from Nahuel Huapi Lake) with the closest Pacific and Atlantic populations to evaluate if these freshwater colonies are receiving immigrants from the larger coastal populations. We sampled three geographic regions (Nahuel Huapi Lake and the Atlantic and Pacific coasts) and employed a reduced-representation genomic approach to genotype individuals for single-nucleotide polymorphisms (SNPs). Using clustering and phylogenetic analyses we found three genetic groups, each corresponding to one of our sampled regions. Individuals from marine environments are more closely related to each other than to those from Nahuel Huapi Lake, indicating that the latter population constitutes the first freshwater Kelp Gull colony to be identified as an evolutionary significant unit in Patagonia.

How structural variants shape avian phenotypes: Lessons from model systems.

Despite receiving significant recent attention, the relevance of structural variation (SV) in driving phenotypic diversity remains understudied, although recent advances in long-read sequencing, bioinformatics and pangenomic approaches have enhanced SV detection. We review the role of SVs in shaping phenotypes in avian model systems, and identify some general patterns in SV type, length and their associated traits. We found that most of the avian SVs so far identified are short indels in chickens, which are frequently associated with changes in body weight and plumage colouration. Overall, we found that relatively short SVs are more frequently detected, likely due to a combination of their prevalence compared to large SVs, and a detection bias, stemming primarily from the widespread use of short-read sequencing and associated analytical methods. SVs most commonly involve non-coding regions, especially introns, and when patterns of inheritance were reported, SVs associated primarily with dominant discrete traits. We summarise several examples of phenotypic convergence across different species, mediated by different SVs in the same or different genes and different types of changes in the same gene that can lead to various phenotypes. Complex rearrangements and supergenes, which can simultaneously affect and link several genes, tend to have pleiotropic phenotypic effects. Additionally, SVs commonly co-occur with single-nucleotide polymorphisms, highlighting the need to consider all types of genetic changes to understand the basis of phenotypic traits. We end by summarising expectations for when long-read technologies become commonly implemented in non-model birds, likely leading to an increase in SV discovery and characterisation. The growing interest in this subject suggests an increase in our understanding of the phenotypic effects of SVs in upcoming years.

Genomic islands of speciation harbor genes underlying coloration differences in a pair of Neotropical seedeaters.

Incomplete speciation can be leveraged to associate phenotypes with genotypes, thus providing insights into the traits relevant to the reproductive isolation of diverging taxa. We investigate the genetic underpinnings of the phenotypic differences between Sporophila plumbea and Sporophila beltoni. Sporophila beltoni has only recently been described based, most notably, based on differences in bill coloration (yellow vs. black in S. plumbea). Both species are indistinguishable through mtDNA or reduced-representation genomic data, and even whole-genome sequencing revealed low genetic differentiation. Demographic reconstructions attribute this genetic homogeneity to gene flow, despite divergence in the order of millions of generations. We found a narrow hybrid zone in southern Brazil where genetically, yet not phenotypically, admixed individuals appear to be prevalent. Despite the overall low genetic differentiation, we identified 3 narrow peaks along the genome with highly differentiated SNPs. These regions harbor 6 genes, one of which is involved in pigmentation (EDN3) and is a candidate for controlling bill color. Within the outlier peaks, we found signatures of resistance to gene flow, as expected for islands of speciation. Our study shows how genes related to coloration traits are likely involved in generating prezygotic isolation and establishing species boundaries early in speciation.

Comparative genomics of two Empidonax flycatchers reveal candidate genes for bird song production.

Whole-genome-level comparisons of sister taxa that vary in phenotype against a background of high genomic similarity can be used to identify the genomic regions that might underlie their phenotypic differences. In wild birds, this exploratory approach has detected markers associated with plumage coloration, beak and wing morphology, and complex behavioral traits like migration. Here, we use genomic comparisons of two closely related suboscine flycatchers (Empidonax difficilis and E. occidentalis) and their hybrids to search for candidate genes underlying their variation in innate vocal signals. We sequenced the genomes of 20 flycatchers that sang one of two species-specific pure song types and 14 putative hybrid individuals with intermediate song types. In the resulting genomic comparisons, we found six areas of high differentiation that may be associated with variation in nonlearned songs. These narrow regions of genomic differentiation contain a total of 67 described genes, of which three have been previously associated with forms of language impairment and dyslexia in humans and 18 are known to be differentially expressed in the song nuclei regions of the avian brain compared with adjacent parts of the avian brain. This "natural experiment" therefore may help identify loci associated with song differences that merit further study across bird lineages with both learned and innate vocalizations.

Selective sweeps on different pigmentation genes mediate convergent evolution of island melanism in two incipient bird species.

Insular organisms often evolve predictable phenotypes, like flightlessness, extreme body sizes, or increased melanin deposition. The evolutionary forces and molecular targets mediating these patterns remain mostly unknown. Here we study the Chestnut-bellied Monarch (Monarcha castaneiventris) from the Solomon Islands, a complex of closely related subspecies in the early stages of speciation. On the large island of Makira M. c. megarhynchus has a chestnut belly, whereas on the small satellite islands of Ugi, and Santa Ana and Santa Catalina (SA/SC) M. c. ugiensis is entirely iridescent blue-black (i.e., melanic). Melanism has likely evolved twice, as the Ugi and SA/SC populations were established independently. To investigate the genetic basis of melanism on each island we generated whole genome sequence data from all three populations. Non-synonymous mutations at the MC1R pigmentation gene are associated with melanism on SA/SC, while ASIP, an antagonistic ligand of MC1R, is associated with melanism on Ugi. Both genes show evidence of selective sweeps in traditional summary statistics and statistics derived from the ancestral recombination graph (ARG). Using the ARG in combination with machine learning, we inferred selection strength, timing of onset and allele frequency trajectories. MC1R shows evidence of a recent, strong, soft selective sweep. The region including ASIP shows more complex signatures; however, we find evidence for sweeps in mutations near ASIP, which are comparatively older than those on MC1R and have been under relatively strong selection. Overall, our study shows convergent melanism results from selective sweeps at independent molecular targets, evolving in taxa where coloration likely mediates reproductive isolation with the neighboring chestnut-bellied subspecies.

The genomics of adaptation in birds.

Organismal adaptations are the hallmark of natural selection. Studies of adaptations in avian systems have been central to key conceptual and empirical advances in the field of evolutionary biology and, over the past decade, leveraged the proliferation of a diversity of genomic tools. In this synthesis, we first discuss how the different genomic architectures of avian traits are relevant to adaptive phenotypes. A mutation's chromosomal location (e.g., microchromosomes or sex chromosomes) or its specific nature (e.g., nucleotide substitution or structural variant) will determine how it may evolve and shape adaptive phenotypes, and we review different examples from the avian literature. We next describe how the source of adaptive variation, whether from de novo mutations, existing genetic variation, or introgression from another species, can affect the evolutionary dynamics of a trait. Our third section reviews case studies where the genetic basis of key avian adaptive phenotypes (e.g., bill morphology or plumage coloration) have been revealed. We end by providing an outlook and identifying important challenges to this field, both by focusing on technical aspects, such as the completeness of genomic assemblies and the ability to validate genetic associations with new sources of data, as well as by discussing the existential threat posed to birds from habitat alteration and climate change.

Interspecific forced copulations generate most hybrids in broadly sympatric ducks.

Although rare, hybrids are more common in broadly sympatric waterfowl than in any other avian family; yet, the behavioral ecology explaining their generation has remained controversial. Leading hypotheses are forced interspecific copulations, mis-imprinting caused by mixed broods, and scarcity of conspecific mates. Using a large sample of hybrid ducks solicited from North American hunters we evaluated these hypotheses by genetically determining the mother and father species of F1 hybrids. Based on abundances in areas where their breeding ranges overlap, the frequency of hybrids varied greatly from expectations, with hybrids between species within recently derived clades being much more frequent than those between more divergent clades. Forced copulations, as measured by large phallus-length asymmetries between parentals, strongly predicted the father species of most F1 hybrids. Thus, most Anas acuta x A. platyrhynchos (Northern Pintail x Mallard) F1s were sired by A. acuta, and most A. platyrhynchos x Mareca strepera (Mallard x Gadwall) F1s were sired by A. platyrhynchos. Siring asymmetries were consistent with phallus length asymmetries in five additional parental combinations, but none had samples large enough to be individually statistically significant. The exception to this trend was our sample of nine A. platyrhynchos x Mareca americana (Mallard x Gadwall) F1s, for which a large phallus asymmetry failed to predict the father species. Hybrids were rare in brood parasitic species, suggesting mis-imprinting to be an unlikely cause of most hybrids; however, our samples of hybrids from regular brood parasites were inadequate to strongly address this hypothesis. We could test the scarcity of mates hypothesis for only a single hybrid combination and it contradicted our prediction: most F1 M. Penelope x M. americana (Eurasian x American Wigeon) were sired by M. penelope, strongly contradicting our prediction that female M. penelope wintering in enormous flocks of M. americana (American Wigeon) on the west coast of North America would have difficulty finding conspecific mates. In general, our results support interspecific forced copulations as the predominant behavioral mechanism generating hybrids in North temperate waterfowl.

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.

A Deep-Learning Approach for Inference of Selective Sweeps from the Ancestral Recombination Graph.

Detecting signals of selection from genomic data is a central problem in population genetics. Coupling the rich information in the ancestral recombination graph (ARG) with a powerful and scalable deep-learning framework, we developed a novel method to detect and quantify positive selection: Selection Inference using the Ancestral recombination graph (SIA). Built on a Long Short-Term Memory (LSTM) architecture, a particular type of a Recurrent Neural Network (RNN), SIA can be trained to explicitly infer a full range of selection coefficients, as well as the allele frequency trajectory and time of selection onset. We benchmarked SIA extensively on simulations under a European human demographic model, and found that it performs as well or better as some of the best available methods, including state-of-the-art machine-learning and ARG-based methods. In addition, we used SIA to estimate selection coefficients at several loci associated with human phenotypes of interest. SIA detected novel signals of selection particular to the European (CEU) population at the MC1R and ABCC11 loci. In addition, it recapitulated signals of selection at the LCT locus and several pigmentation-related genes. Finally, we reanalyzed polymorphism data of a collection of recently radiated southern capuchino seedeater taxa in the genus Sporophila to quantify the strength of selection and improved the power of our previous methods to detect partial soft sweeps. Overall, SIA uses deep learning to leverage the ARG and thereby provides new insight into how selective sweeps shape genomic diversity.

Variable Signatures of Selection Despite Conserved Recombination Landscapes Early in Speciation.

Recently diverged taxa often exhibit heterogeneous landscapes of genomic differentiation, characterized by regions of elevated differentiation on an otherwise homogeneous background. While divergence peaks are generally interpreted as regions responsible for reproductive isolation, they can also arise due to background selection, selective sweeps unrelated to speciation, and variation in recombination and mutation rates. To investigate the association between patterns of recombination and landscapes of genomic differentiation during the early stages of speciation, we generated fine-scale recombination maps for six southern capuchino seedeaters (Sporophila) and two subspecies of White Wagtail (Motacilla alba), two recent avian radiations in which divergent selection on pigmentation genes has likely generated peaks of differentiation. We compared these recombination maps to those of Collared (Ficedula albicollis) and Pied Flycatchers (Ficedula hypoleuca), non-sister taxa characterized by moderate genomic divergence and a heterogenous landscape of genomic differentiation shaped in part by background selection. Although recombination landscapes were conserved within all three systems, we documented a weaker negative correlation between recombination rate and genomic differentiation in the recent radiations. All divergence peaks between capuchinos, wagtails, and flycatchers were located in regions with lower-than-average recombination rates, and most divergence peaks in capuchinos and flycatchers fell in regions of exceptionally reduced recombination. Thus, co-adapted allelic combinations in these regions may have been protected early in divergence, facilitating rapid diversification. Despite largely conserved recombination landscapes, divergence peaks are specific to each focal comparison in capuchinos, suggesting that regions of elevated differentiation have not been generated by variation in recombination rate alone.

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.

Rapid speciation via the evolution of pre-mating isolation in the Iberá Seedeater.

Behavioral isolation can catalyze speciation and permit the slow accumulation of additional reproductive barriers between co-occurring organisms. We illustrate how this process occurs by examining the genomic and behavioral bases of pre-mating isolation between two bird species (Sporophila hypoxantha and the recently discovered S. iberaensis) that belong to the southern capuchino seedeaters, a recent, rapid radiation characterized by variation in male plumage coloration and song. Although these two species co-occur without obvious ecological barriers to reproduction, we document behaviors indicating species recognition by song and plumage traits and strong assortative mating associated with genomic regions underlying male plumage patterning. Plumage differentiation likely originated through the reassembly of standing genetic variation, indicating how novel sexual signals may quickly arise and maintain species boundaries.

Patterns of genetic divergence and demographic history shed light on island-mainland population dynamics and melanic plumage evolution in the white-winged Fairywren.

The existence of distinct traits in island versus mainland populations offers opportunities to gain insights into how eco-evolutionary processes operate under natural conditions. We used two island colonization events in the white-winged fairywren (Malurus leucopterus) to investigate the genomic and demographic origin of melanic plumage. This avian species is distributed across most of Australia, and males of the mainland subspecies (M. l. leuconotus) exhibit a blue nuptial plumage in contrast to males of two island subspecies - M. l. leucopterus on Dirk Hartog Island and M. l. edouardi on Barrow Island - that exhibit a black nuptial plumage. We used reduced-representation sequencing to explore differentiation and demographic history in this species and found clear patterns of divergence between mainland and island populations, with additional substructuring on the mainland. Divergence between the mainland and Dirk Hartog was approximately 10 times more recent than the split between the mainland and Barrow Island, supporting two independent colonizations. In both cases, estimated gene flow between the mainland and the islands was low, contributing to signals of divergence among subspecies. Our results present demographic reconstructions of mainland-island dynamics and associated plumage variation in white-winged fairywrens, with broader implications regarding our understanding of convergent evolution in insular populations.

An avian dominance hierarchy at a supplemental water source in the Patagonian steppe.

Birds often compete and engage in interspecific agonistic interactions for access to resources such as food and breeding territories. Based on the observed outcomes from such interactions (i.e., patterns of displacements) dominance hierarchies can be established. Knowing which species can outcompete others for essential resources allows researchers to make predictions about the broader ecological impacts of interspecific interactions. We constructed an interspecific dominance hierarchy of twelve avian species which visited an artificial water source in an arid region of coastal Patagonia, Argentina. Displacements were categorized into four types, based on the behaviors involved in the interaction, and we tested if they could predict the difference in dominance between the interacting species (the difference between calculated dominance coefficients for the two focal species). Indirect displacements, involving only the arrival of the dominant species to the water source without direct aggression toward the subordinate bird, occurred more frequently between species with a large difference in dominance. The most dominant bird observed was the kelp gull (Larus dominicanus), which, due to an increasing population and expanding range, in part due to food supplementation from fisheries waste, is likely to outcompete terrestrial and marine avian species for other scarce resources.

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.

Metapopulation dynamics and foraging plasticity in a highly vagile seabird, the southern rockhopper penguin.

Population connectivity is driven by individual dispersal potential and modulated by natal philopatry. In seabirds, high vagility facilitates dispersal yet philopatry is also common, with foraging area overlap often correlated with population connectivity. We assess the interplay between these processes by studying past and current connectivity and foraging niche overlap among southern rockhopper penguin colonies of the coast of southern South America using genomic and stable isotope analyses. We found two distinct genetic clusters and detected low admixture between northern and southern colonies. Stable isotope analysis indicated niche variability between colonies, with Malvinas/Falklands colonies encompassing the species entire isotopic foraging niche, while the remaining colonies had smaller, nonoverlapping niches. A recently founded colony in continental Patagonia differed in isotopic niche width and position with Malvinas/Falklands colonies, its genetically identified founder population, suggesting the exploitation of novel foraging areas and/or prey items. Additionally, dispersing individuals found dead across the Patagonian shore in an unusual mortality event were also assigned to the northern cluster, suggesting northern individuals reach southern localities, but do not breed in these colonies. Facilitated by variability in foraging strategies, and especially during unfavorable conditions, the number of dispersing individuals may increase and enhance the probability of founding new colonies. Metapopulation demographic dynamics in seabirds should account for interannual variability in dispersal behavior and pay special attention to extreme climatic events, classically related to negative effects on population trends.

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.

Genome-wide variation in DNA methylation is associated with stress resilience and plumage brightness in a wild bird.

Individuals often differ in their ability to cope with challenging environmental and social conditions. Evidence from model systems suggests that patterns of DNA methylation are associated with variation in coping ability. These associations could arise directly if methylation plays a role in controlling the physiological response to stressors by, among other things, regulating the release of glucocorticoids in response to challenges. Alternatively, the association could arise indirectly if methylation and resilience have a common cause, such as early-life conditions. In either case, methylation might act as a biomarker for coping ability. At present, however, relatively little is known about whether variation in methylation is associated with organismal performance and resilience under natural conditions. We studied genome-wide patterns of DNA methylation in free-living female tree swallows (Tachycineta bicolor) using methylated DNA immunoprecipitation (MeDIP) and a tree swallow genome that was assembled for this study. We identified areas of the genome that were differentially methylated with respect to social signal expression (breast brightness) and physiological traits (ability to terminate the glucocorticoid stress response through negative feedback). We also asked whether methylation predicted resilience to a subsequent experimentally imposed challenge. Individuals with brighter breast plumage and higher stress resilience had lower methylation at differentially methylated regions across the genome. Thus, widespread differences in methylation predicted both social signal expression and the response to future challenges under natural conditions. These results have implications for predicting individual differences in resilience, and for understanding the mechanistic basis of resilience and its environmental and social mediators.

Gradual evolution towards flightlessness in steamer ducks.

Flightlessness in birds is the product of changes in suites of characters-including increased body size and reduced anterior limbs-that have evolved repeatedly and independently under similar ecological conditions (generally insularity). It remains unknown whether this phenotypic convergence extends to the genomic level, partially because many losses of flight occurred long ago (such as in penguins or ratites), thus complicating the study of the genetic pathways to flightlessness. Here, we use genome sequencing to study the evolution of flightlessness in a group of ducks that are current and dynamic exemplars of this major functional transition. These recently diverged Tachyeres steamer ducks differ in their ability to fly: one species is predominantly flighted and three are mainly flightless. Through a genome-wide association analysis, we identify two narrow candidate genomic regions implicated in the morphological changes that led to flightlessness, and reconstruct the number of times flightlessness has evolved in Tachyeres. The strongest association is with DYRK1A, a gene that when knocked out in mice leads to alterations in growth and bone morphogenesis. These findings, together with phylogenetic and demographic analyses, imply that the genomic changes leading to flightlessness in Tachyeres may have evolved once, and that this trait remains functionally polymorphic in two species.