Association between the gut microbiome and carotenoid plumage phenotype in an avian hybrid zone.

Vertebrates host complex microbiomes that impact their physiology. In many taxa, including colourful wood-warblers, gut microbiome similarity decreases with evolutionary distance. This may suggest that as host populations diverge, so do their microbiomes, because of either tight coevolutionary dynamics, or differential environmental influences, or both. Hybridization is common in wood-warblers, but the effects of evolutionary divergence on the microbiome during secondary contact are unclear. Here, we analyse gut microbiomes in two geographically disjunct hybrid zones between blue-winged warblers (Vermivora cyanoptera) and golden-winged warblers (Vermivora chrysoptera). We performed 16S faecal metabarcoding to identify species-specific bacteria and test the hypothesis that host admixture is associated with gut microbiome disruption. Species identity explained a small amount of variation between microbiomes in only one hybrid zone. Co-occurrence of species-specific bacteria was rare for admixed individuals, yet microbiome richness was similar among admixed and parental individuals. Unexpectedly, we found several bacteria that were more abundant among admixed individuals with a broader deposition of carotenoid-based plumage pigments. These bacteria are predicted to encode carotenoid biosynthesis genes, suggesting birds may take advantage of pigments produced by their gut microbiomes. Thus, host admixture may facilitate beneficial symbiotic interactions which contribute to plumage ornaments that function in sexual selection.

Genomics-informed conservation units reveal spatial variation in climate vulnerability in a migratory bird.

Identifying genetic conservation units (CUs) in threatened species is critical for the preservation of adaptive capacity and evolutionary potential in the face of climate change. However, delineating CUs in highly mobile species remains a challenge due to high rates of gene flow and genetic signatures of isolation by distance. Even when CUs are delineated in highly mobile species, the CUs often lack key biological information about what populations have the most conservation need to guide management decisions. Here we implement a framework for CU identification in the Canada Warbler (Cardellina canadensis), a migratory bird species of conservation concern, and then integrate demographic modelling and genomic offset to guide conservation decisions. We find that patterns of whole genome genetic variation in this highly mobile species are primarily driven by putative adaptive variation. Identification of CUs across the breeding range revealed that Canada Warblers fall into two evolutionarily significant units (ESU), and three putative adaptive units (AUs) in the South, East, and Northwest. Quantification of genomic offset, a metric of genetic changes necessary to maintain current gene-environment relationships, revealed significant spatial variation in climate vulnerability, with the Northwestern AU being identified as the most vulnerable to future climate change. Alternatively, quantification of past population trends within each AU revealed the steepest population declines have occurred within the Eastern AU. Overall, we illustrate that genomics-informed CUs provide a strong foundation for identifying current and future regional threats that can be used to inform management strategies for a highly mobile species in a rapidly changing world.

Dispersal-Limited Symbionts Exhibit Unexpectedly Wide Variation in Host Specificity.

A fundamental aspect of symbiotic relationships is host specificity, ranging from extreme specialists associated with only a single host species to generalists associated with many different species. Although symbionts with limited dispersal capabilities are expected to be host specialists, some are able to associate with multiple hosts. Understanding the micro- and macro-evolutionary causes of variations in host specificity is often hindered by sampling biases and the limited power of traditional evolutionary markers. Here, we studied feather mites to address the barriers associated with estimates of host specificity for dispersal-limited symbionts. We sampled feather mites (Proctophyllodidae) from a nearly comprehensive set of North American breeding warblers (Parulidae) to study mite phylogenetic relationships and host-symbiont codiversification. We used pooled-sequencing (Pool-Seq) and short-read Illumina technology to interpret results derived from a traditional barcoding gene (cytochrome c oxidase subunit 1) versus 11 protein-coding mitochondrial genes using concatenated and multispecies coalescent approaches. Despite the statistically significant congruence between mite and host phylogenies, mite-host specificity varies widely, and host switching is common regardless of the genetic marker resolution (i.e., barcode vs. multilocus). However, the multilocus approach was more effective than the single barcode in detecting the presence of a heterogeneous Pool-Seq sample. These results suggest that presumed symbiont dispersal capabilities are not always strong indicators of host specificity or of historical host-symbiont coevolutionary events. A comprehensive sampling at fine phylogenetic scales may help to better elucidate the microevolutionary filters that impact macroevolutionary processes regulating symbioses, particularly for dispersal-limited symbionts. [Codiversification; cophylogenetics; feather mites; host switching; pooled sequencing; species delineation; symbiosis, warblers.].

Gut microbiome composition better reflects host phylogeny than diet diversity in breeding wood-warblers.

Understanding the factors that shape microbiomes can provide insight into the importance of host-symbiont interactions and on co-evolutionary dynamics. Unlike for mammals, previous studies have found little or no support for an influence of host evolutionary history on avian gut microbiome diversity and instead have suggested a greater influence of the environment or diet due to fast gut turnover. Because effects of different factors may be conflated by captivity and sampling design, examining natural variation using large sample sizes is important. Our goal was to overcome these limitations by sampling wild birds to compare environmental, dietary and evolutionary influences on gut microbiome structure. We performed faecal metabarcoding to characterize both the gut microbiome and diet of 15 wood-warbler species across a 4-year period and from two geographical localities. We find host taxonomy generally explained ~10% of the variation between individuals, which is ~6-fold more variation of any other factor considered, including diet diversity. Further, gut microbiome similarity was more congruent with the host phylogeny than with host diet similarity and we found little association between diet diversity and microbiome diversity. Together, our results suggest evolutionary history is the strongest predictor of gut microbiome differentiation among wood-warblers. Although the phylogenetic signal of the warbler gut microbiome is not very strong, our data suggest that a stronger influence of diet (as measured by diet diversity) does not account for this pattern. The mechanism underlying this phylogenetic signal is not clear, but we argue host traits may filter colonization and maintenance of microbes.

Whole genome assessment of a declining game bird reveals cryptic genetic structure and insights for population management.

Population genomics applied to game species conservation can help delineate management units, ensure appropriate harvest levels and identify populations needing genetic rescue to safeguard their adaptive potential. The ruffed grouse (Bonasa umbellus) is rapidly declining in much of the eastern USA due to a combination of forest maturation and habitat fragmentation. More recently, mortality from West Nile Virus may have affected connectivity of local populations; however, genetic approaches have never explicitly investigated this issue. In this study, we sequenced 54 individual low-coverage (~5X) grouse genomes to characterize population structure, assess migration rates across the landscape to detect potential barriers to gene flow and identify genomic regions with high differentiation. We identified two genomic clusters with no clear geographic correlation, with large blocks of genomic differentiation associated with chromosomes 4 and 20, likely due to chromosomal inversions. After excluding these putative inversions from the data set, we found weak but nonsignificant signals of population subdivision. Estimated gene flow revealed reduced rates of migration in areas with extensive habitat fragmentation and increased genetic connectivity in areas with less habitat fragmentation. Our findings provide a benchmark for wildlife managers to compare and scale the genetic diversity and structure of ruffed grouse populations in Pennsylvania and across the eastern USA, and we also reveal structural variation in the grouse genome that requires further study to understand its possible effects on individual fitness and population distribution.

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.

Comparing divergence landscapes from reduced-representation and whole genome resequencing in the yellow-rumped warbler (Setophaga coronata) species complex.

Researchers seeking to generate genomic data for non-model organisms are faced with a number of trade-offs when deciding which method to use. The selection of reduced representation approaches versus whole genome resequencing will ultimately affect the marker density, sequencing depth, and the number of individuals that can multiplexed. These factors can affect researchers' ability to accurately characterize certain genomic features, such as landscapes of divergence-how FST varies across the genomes. To provide insight into the effect of sequencing method on the estimation of divergence landscapes, we applied an identical bioinformatic pipeline to three generations of sequencing data (GBS, ddRAD, and WGS) produced for the same system, the yellow-rumped warbler species complex. We compare divergence landscapes generated using each method for the myrtle warbler (Setophaga coronata coronata) and the Audubon's warbler (S. c. auduboni), and for Audubon's warblers with deeply divergent mtDNA resulting from mitochondrial introgression. We found that most high-FST peaks were not detected in the ddRAD data set, and that while both GBS and WGS were able to identify the presence of large peaks, WGS was superior at a finer scale. Comparing Audubon's warblers with divergent mitochondrial haplotypes, only WGS allowed us to identify small (10-20 kb) regions of elevated differentiation, one of which contained the nuclear-encoded mitochondrial gene NDUFAF3. We calculated the cost per base pair for each method and found it was comparable between GBS and WGS, but significantly higher for ddRAD. These comparisons highlight the advantages of WGS over reduced representation methods when characterizing landscapes of divergence.

The Evolution and Genetics of Carotenoid Processing in Animals.

Coloration is one of the most conspicuous traits that varies among organisms. Carotenoid pigments are responsible for many of the red, orange, and yellow colors in the natural world and, at least for most animals, these molecules must be acquired from their environment. Identifying genes important for carotenoid transport, deposition, and processing has been difficult, in contrast to the well-characterized genes involved in the melanogenesis pathways. We review recent progress in the genetics of carotenoid processing, advances owing in part to the application of high-throughput sequencing data. We focus on examples from several classes of genes coding for scavenger receptors, ß-carotene oxygenases, and ketolases. We also review comparative studies that have revealed several important findings in the evolution of these genes. Namely, that they are conserved across deep phylogenetic timescales, are associated with gene/genome duplications, and introgression has contributed to their movement between several taxa.

A comparison of genomic islands of differentiation across three young avian species pairs.

Detailed evaluations of genomic variation between sister species often reveal distinct chromosomal regions of high relative differentiation (i.e., "islands of differentiation" in FST ), but there is much debate regarding the causes of this pattern. We briefly review the prominent models of genomic islands of differentiation and compare patterns of genomic differentiation in three closely related pairs of New World warblers with the goal of evaluating support for the four models. Each pair (MacGillivray's/mourning warblers; Townsend's/black-throated green warblers; and Audubon's/myrtle warblers) consists of forms that were likely separated in western and eastern North American refugia during cycles of Pleistocene glaciations and have now come into contact in western Canada, where each forms a narrow hybrid zone. We show strong differences between pairs in their patterns of genomic heterogeneity in FST , suggesting differing selective forces and/or differing genomic responses to similar selective forces among the three pairs. Across most of the genome, levels of within-group nucleotide diversity (πWithin ) are almost as large as levels of between-group nucleotide distance (πBetween ) within each pair, suggesting recent common ancestry and/or gene flow. In two pairs, a pattern of the FST peaks having low πBetween suggests that selective sweeps spread between geographically differentiated groups, followed by local differentiation. This "sweep-before-differentiation" model is consistent with signatures of gene flow within the yellow-rumped warbler species complex. These findings add to our growing understanding of speciation as a complex process that can involve phases of adaptive introgression among partially differentiated populations.

A wood-warbler produced through both interspecific and intergeneric hybridization.

Hybridization between divergent taxa can provide insight into the breakdown of characters used in mate choice, as well as reproductive compatibility across deep evolutionary timescales. Hybridization can also occur more frequently in declining populations, as there is a smaller pool of conspecific mates from which to choose. Here, we report an unusual combination of factors that has resulted in a rare, three-species hybridization event among two genera of warblers, one of which is experiencing significant population declines. We use bioacoustic, morphometric and genetic data, to demonstrate that an early generation female hybrid between a golden-winged warbler (Vermivora chrysoptera) and a blue-winged warbler (V. cyanoptera) went on to mate and successfully reproduce with a chestnut-sided warbler (Setophaga pensylvanica). We studied the product of this event-a putative chrysoptera × cyanoptera × pensylvanica hybrid-and show that this male offspring sang songs like S. pensylvanica, but had morphometric traits similar to Vermivora warblers. The hybrid's maternal parent had V. chrysoptera mitochondrial DNA and, with six plumage-associated loci, we predicted the maternal parent's phenotype to show that it was likely an early generation Vermivora hybrid. That this hybridization event occurred within a population of Vermivora warblers in significant decline suggests that females may be making the best of a bad situation, and that wood-warblers in general have remained genetically compatible long after they evolved major phenotypic differences.

Admixture mapping in a hybrid zone reveals loci associated with avian feather coloration.

Identifying the genetic bases for colour patterns has provided important insights into the control and expression of pigmentation and how these characteristics influence fitness. However, much more is known about the genetic bases for traits based on melanin pigments than for traits based on another major class of pigments, carotenoids. Here, we use natural admixture in a hybrid zone between Audubon's and myrtle warblers (Setophaga coronata auduboni/S. c. coronata) to identify genomic regions associated with both types of pigmentation. Warblers are known for rapid speciation and dramatic differences in plumage. For each of five plumage coloration traits, we found highly significant associations with multiple single-nucleotide polymorphisms (SNPs) across the genome and these were clustered in discrete regions. Regions near significantly associated SNPs were enriched for genes associated with keratin filaments, fibrils that make up feathers. A carotenoid-based trait that differs between the taxa-throat colour-had more than a dozen genomic regions of association. One cluster of SNPs for this trait overlaps the Scavenger Receptor Class F Member 2 (SCARF2) gene. Other scavenger receptors are presumed to be expressed at target tissues and involved in the selective movement of carotenoids into the target cells, making SCARF2 a plausible new candidate for carotenoid processing. In addition, two melanin-based plumage traits-colours of the eye line and eye spot-show very strong associations with a single genomic region mapping to chromosome 20 in the zebra finch. These findings indicate that only a subset of the genomic regions differentiated between these two warblers are associated with the plumage differences between them and demonstrate the utility of reduced-representation genomic scans in hybrid zones.

From song dialects to speciation in white-crowned sparrows.

The behavioural signals used in mate selection are a key component in the evolution of premating isolating barriers and, subsequently, the formation of new species. The importance of mating signals has a long tradition of study in songbirds, where many species differ in their song characteristics. In oscine songbirds, individual birds usually learn their songs from a tutor. Mistakes during learning can help generate geographic dialects, akin to those within human language groups. In songbirds, dialect differences can often be substantial and there is an intuitive connection between the evolution of song amongst populations at a small scale, and the more substantive song differences between bird species and presumably used in species recognition. However, studies investigating the concordance between putative genetic and behavioural boundaries have generated mixed results. In many cases, this is possibly a function of the poor resolving power of the genetic markers employed. In this issue of Molecular Ecology, Lipshutz et al. () combine genomic markers with a robust behavioural assay to address the importance of song variation amongst white-crowned sparrow (Zonotrichia leucophrys) subspecies.

Genomes of the extinct Bachman's warbler show high divergence and no evidence of admixture with other extant Vermivora warblers.

Bachman's warbler1 (Vermivora bachmanii)-last sighted in 1988-is one of the only North American passerines to recently go extinct.2,3,4 Given extensive ongoing hybridization of its two extant congeners-the blue-winged warbler (V. cyanoptera) and golden-winged warbler (V. chrysoptera)5,6,7,8-and shared patterns of plumage variation between Bachman's warbler and hybrids between those extant species, it has been suggested that Bachman's warbler might have also had a component of hybrid ancestry. Here, we use historic DNA (hDNA) and whole genomes of Bachman's warblers collected at the turn of the 20th century to address this. We combine these data with the two extant Vermivora species to examine patterns of population differentiation, inbreeding, and gene flow. In contrast to the admixture hypothesis, the genomic evidence is consistent with V. bachmanii having been a highly divergent, reproductively isolated species, with no evidence of introgression. We show that these three species have similar levels of runs of homozygosity (ROH), consistent with effects of a small long-term effective population size or population bottlenecks, with one V. bachmanii outlier showing numerous long ROH and a FROH greater than 5%. We also found-using population branch statistic estimates-previously undocumented evidence of lineage-specific evolution in V. chrysoptera near a pigmentation gene candidate, CORIN, which is a known modifier of ASIP, which is in turn involved in melanic throat and mask coloration in this family of birds. Together, these genomic results also highlight how natural history collections are such invaluable repositories of information about extant and extinct species.

A complex genomic architecture underlies reproductive isolation in a North American oriole hybrid zone.

Natural hybrid zones provide powerful opportunities for identifying the mechanisms that facilitate and inhibit speciation. Documenting the extent of genomic admixture allows us to discern the architecture of reproductive isolation through the identification of isolating barriers. This approach is particularly powerful for characterizing the accumulation of isolating barriers in systems exhibiting varying levels of genomic divergence. Here, we use a hybrid zone between two species-the Baltimore (Icterus galbula) and Bullock's (I. bullockii) orioles-to investigate this architecture of reproductive isolation. We combine whole genome re-sequencing with data from an additional 313 individuals amplityped at ancestry-informative markers to characterize fine-scale patterns of admixture, and to quantify links between genes and the plumage traits. On a genome-wide scale, we document several putative barriers to reproduction, including elevated peaks of divergence above a generally high genomic baseline, a large putative inversion on the Z chromosome, and complex interactions between melanogenesis-pathway candidate genes. Concordant and coincident clines for these different genomic regions further suggest the coupling of pre- and post-mating barriers. Our findings of complex and coupled interactions between pre- and post-mating barriers suggest a relatively rapid accumulation of barriers between these species, and they demonstrate the complexities of the speciation process.

The biogeography of mitochondrial and nuclear discordance in animals.

Combining nuclear (nuDNA) and mitochondrial DNA (mtDNA) markers has improved the power of molecular data to test phylogenetic and phylogeographic hypotheses and has highlighted the limitations of studies using only mtDNA markers. In fact, in the past decade, many conflicting geographic patterns between mitochondrial and nuclear genetic markers have been identified (i.e. mito-nuclear discordance). Our goals in this synthesis are to: (i) review known cases of mito-nuclear discordance in animal systems, (ii) to summarize the biogeographic patterns in each instance and (iii) to identify common drivers of discordance in various groups. In total, we identified 126 cases in animal systems with strong evidence of discordance between the biogeographic patterns obtained from mitochondrial DNA and those observed in the nuclear genome. In most cases, these patterns are attributed to adaptive introgression of mtDNA, demographic disparities and sex-biased asymmetries, with some studies also implicating hybrid zone movement, human introductions and Wolbachia infection in insects. We also discuss situations where divergent mtDNA clades seem to have arisen in the absence of geographic isolation. For those cases where foreign mtDNA haplotypes are found deep within the range of a second taxon, data suggest that those mtDNA haplotypes are more likely to be at a high frequency and are commonly driven by sex-biased asymmetries and/or adaptive introgression. In addition, we discuss the problems with inferring the processes causing discordance from biogeographic patterns that are common in many studies. In many cases, authors presented more than one explanation for discordant patterns in a given system, which indicates that likely more data are required. Ideally, to resolve this issue, we see important future work shifting focus from documenting the prevalence of mito-nuclear discordance towards testing hypotheses regarding the drivers of discordance. Indeed, there is great potential for certain cases of mitochondrial introgression to become important natural systems within which to test the effect of different mitochondrial genotypes on whole-animal phenotypes.

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.

Molecular assays of pollen use consistently reflect pollinator visitation patterns in a system of flowering plants.

Determining how pollinators visit plants vs. how they carry and transfer pollen is an ongoing project in pollination ecology. The current tools for identifying the pollens that bees carry have different strengths and weaknesses when used for ecological inference. In this study we use three methods to better understand a system of congeneric, coflowering plants in the genus Clarkia and their bee pollinators: observations of plant-pollinator contact in the field, and two different molecular methods to estimate the relative abundance of each Clarkia pollen in samples collected from pollinators. We use these methods to investigate if observations of plant-pollinator contact in the field correspond to the pollen bees carry; if individual bees carry Clarkia pollens in predictable ways, based on previous knowledge of their foraging behaviors; and how the three approaches differ for understanding plant-pollinator interactions. We find that observations of plant-pollinator contact are generally predictive of the pollens that bees carry while foraging, and network topologies using the three different methods are statistically indistinguishable from each other. Results from molecular pollen analysis also show that while bees can carry multiple species of Clarkia at the same time, they often carry one species of pollen. Our work contributes to the growing body of literature aimed at resolving how pollinators use floral resources. We suggest our novel relative amplicon quantification method as another tool in the developing molecular ecology and pollination biology toolbox.

Genetic confirmation of a hybrid between two highly divergent cardinalid species: A rose-breasted grosbeak (Pheucticus ludovicianus) and a scarlet tanager (Piranga olivacea).

Using low-coverage whole-genome sequencing, analysis of vocalizations, and inferences from natural history, we document a first-generation hybrid between a rose-breasted grosbeak (Pheucticus ludovicianus) and a scarlet tanager (Piranga olivacea). These two species occur sympatrically throughout much of eastern North America, although were not previously known to interbreed. Following the field identification of a putative hybrid, we use genetic and bioacoustic data to show that a rose-breasted grosbeak was the maternal parent and a scarlet tanager was the paternal parent of the hybrid, whose song was similar to the latter species. These two species diverged >10 million years ago, and thus it is surprising to find a hybrid formed under natural conditions in the wild. Notably, the hybrid has an exceptionally heterozygous genome, with a conservative estimate of a heterozygous base every 100 bp. The observation that this hybrid of such highly divergent parental taxa has survived until adulthood serves as another example of the capacity for hybrid birds to survive with an exceptionally divergent genomic composition.

Pigmentation Genes Show Evidence of Repeated Divergence and Multiple Bouts of Introgression in Setophaga Warblers.

Species radiations have long served as model systems in evolutionary biology.1,2 However, it has only recently become possible to study the genetic bases of the traits responsible for diversification and only in a small number of model systems.3 Here, we use genomes of 36 species of North, Central, and South American warblers to highlight the role of pigmentation genes-involved in melanin and carotenoid processing-in the diversification of this group. We show that agouti signaling protein (ASIP) and beta-carotene oxygenase 2 (BCO2) are predictably divergent between species that differ in the distribution of melanin and carotenoid in their plumages, respectively. Among species, sequence variation at ASIP broadly mirrors the species' phylogenetic history, consistent with repeated, independent mutations generating melanin-based variation. In contrast, BCO2 variation is highly discordant from the species tree, with evidence of cross-lineage introgression among species like the yellow warbler (Setophaga petechia) and magnolia warbler (S. magnolia) with extensive carotenoid-based coloration. We also detect introgression of a small part of the BCO2 coding region (<3 kb) in S. discolor and S. vitellina, including an amino acid substitution that is unique to warblers but otherwise highly conserved across birds. Lateral transfer of carotenoid-processing genes has been documented in arthropods, but introgression of BCO2 as demonstrated here-presumably adaptive-represents the first example of carotenoid gene transfer among vertebrates. These contrasting genomic patterns show that both independent evolution in a common set of genes and past hybridization have fueled plumage diversification in this colorful avian radiation.

Extensive historical and contemporary hybridization suggests premating isolation in Vermivora warblers is not strong: A reply to Confer et al.

We present comments on an article published by Confer et al. (Ecology and Evolution, 10, 2020). Confer et al. (2020) aggregate data from multiple studies of social pairing between Vermivora chrysoptera and V. cyanoptera, two wood warblers in the family Parulidae that hybridize extensively where they co-occur. From analysis of these data, they conclude there is near-complete reproductive isolation between these two species. In our reply, we show that this finding is not supported by other lines of evidence, and significant drawbacks of their study design preclude such strong conclusions. In our critique, we show that (a) coarse-scale plumage classifications cannot be used to accurately estimate hybrid ancestry in Vermivora; (b) extra-pair paternity is very high in Vermivora and is likely facilitating hybridization, yet was not considered by Confer et al. (2020), and we suggest this will have a substantial influence on the interpretation of reproductive isolation in the system; and (c) the central finding of strong total reproductive isolation is not compatible with the results of other long-term studies, which demonstrate low isolation and high gene flow. We conclude with a more comprehensive interpretation of hybridization and reproductive isolation in Vermivora warblers.