The benefit of “broken” hybrids

Using mismatched hybrids to find plumage genes.

The first sentence of a paper can be very important. It should capture the attention of the reader and spark curiosity. A recent piece in the journal Ecology nailed it: “We study hybrids, in part, because they are broken.” What a great way to start a paper (although you had me at hybrids).

To be clear, the authors were not referring to the reduced fitness of hybrids. Instead, they were writing about recombination, the molecular process that breaks up the genome and reassembles it into novel combinations. This genetic mixing partly explains the mosaic nature of hybrids. They receive genomic regions from both parental species and express them in previously unseen combinations. The disassociation of certain traits in hybrids can help scientists to uncover the genetic basis of these traits. This approach has been used to find “plumage genes” in several species, such as Colaptes woodpeckers and Setophaga warblers.

Black Masks

The Ecology-paper focused on the Golden-winged Warbler (Vermivora chrysoptera) and the Blue-winged Warbler (V. cyanoptera). Hybrids between these species have already provided insights into the genetic basis of a black throat patch. The presence or absence of this trait could be linked to regulatory sequences of the ASIP-gene (see this blog post). However, the black throat patch almost always co-occurs with a black face mask, even in hybrids. It was thus impossible to disentangle the genetic variants that underlie these plumage patterns. Logically, previous researchers assumed that the throat patch and face mask are controlled by the same genetic locus.

Occasionally, however, a special hybrid is born. A bird with a mismatched throat/mask phenotype. In 1934, Kenneth Parkes collected such a rare specimen in Michigan. He speculated that both traits might be controlled by different genetic loci that are tightly linked. Very rarely, recombination would break the linkage between these loci. Unfortunately, he did not have the genetic tools to test this idea. But in June 2020, another mismatched hybrid showed up. And now, we do have the tools to explore its genetic make-up.

Plumage traits of Vermivora warblers and their hybrids. The arrows indicate the co-occurrence of the black throat patch and the black face mask. The hybrid in the picture shows a rare mismatch of these traits. From: Baiz et al. (2021).


Marcella Baiz and her colleagues sequenced the genome of this mismatched hybrid. Comparing its genetic make-up with the parental species and other hybrids allowed the researchers to find the genetic basis of these traits. The analyses pointed to a genomic region in front of (or upstream in the genomic jargon) of the ASIP-gene. This locations indicates that it concerns a promotor, the regulatory on-and-off-switch of a gene. Within this promotor region, the researchers found several genetic variants that were associated with the two traits. Close inspection of these variants suggested “the mask promotor is adjacent to the throat promotor upstream of ASIP.”

It thus seems that multiple regulatory sequences determine the deposition of pigments in different plumage patches. Because the mask promotor and the throat promotor are so close together, they are mostly inherited as a single unit. Very rarely, however, they are broken up by recombination, giving rise to a mismatched hybrid.

Next, you only need an observant ornithologist with access to genome sequencing technology.

The genomic region upstream of the ASIP-gene contains the regulatory sequences for the throat and the mask phenotype. From: Baiz et al. (2021).


Baiz, M. D., Wood, A. W., & Toews, D. P. (2021). Rare hybrid solves “genetic problem” of linked plumage traits. Ecology102(10), e03424.

Featured image: Golden-winged warbler (Vermivora chrysoptera) © Bettina Arrigoni | Wikimedia Commons

Does mitochondrial introgression also impact the nuclear genome?

Looking for nuclear-encoded mitochondrial genes in Audubon’s Warbler.

Some individuals of the Audubon’s Warbler (Setophaga [coronata] auduboni) have acquired mitochondrial DNA from the closely related Myrtle Warbler (S. [c.] coronata). The transfer of this circular DNA might even have conferred an advantage on the receiving species. In 2014, David Toews and his colleagues found that Myrtle-type mitochondria were metabolically more efficient than ancestral ones. Moreover, these variants might be linked to changes in migratory behavior. Having a cellular powerhouse that produces more energy is certainly an asset during migration.

This interesting discovery was, however, limited to mitochondrial DNA. In the 2014 paper, the authors did speculate about potential patterns in the nuclear genome: “Are there small portions of the nuclear genome that covary with mtDNA, consistent with a pattern of cryptic genomic regions of isolation between individuals with the two mitochondrial types?” Indeed, some nuclear genes still interact with mitochondrial ones during energy production, and might thus show genetic signs of coevolution. A recent study in the journal Molecular Ecology finally put this idea to the test.

Nuclear Genes

Stephanie Szarmach and her colleagues compared whole genome sequences for Audubon’s Warblers that carried different mitochondrial variants. For clarity, the Myrtle-type mitochondria were referred to as “northern haplotypes” whereas the ancestral mitochondria were called “southern haplotypes.” If certain nuclear genes coevolved with these mitochondrial variants, they might show genetic differentiation between the northern and southern haplotypes. And indeed, the researchers reported “three previously unidentified regions that were moderately differentiated (FST > 0.2) between Audubon’s warblers that had mitochondrial haplotypes from the introgressed northern clade versus those from the ancestral southern clade.” Interestingly, one of these genomic regions houses the gene NDUFAF3, which is a nuclear-encoded mitochondrial gene. Exactly what you would expect.

A differentiated region on chromosome 12 contained the nuclear-encoded mitochondrial gene NDUFAF3. From: Szarmach et al. (2021).

Comparing Techniques

Apart from this biological investigation, the researchers also asked a more technical question: Would you be able to find these differentiated regions with less powerful sequencing techniques? As the saying goes, to pose the question is to answer it (apparently this figure of speech is known as hypophora). So, the researchers compared three genomic sequencing techniques: Whole Genome Sequencing (WGS), Genotyping-by-Sequencing (GBS) and double-digest restriction-site associated DNA (ddRAD). These three approaches differ dramatically in the amount of data that they produce. In the warblers of this study, the average number of reads varied accordingly: 0.6 million per individual for ddRAD, 4.8 million per individual for GBS, and 31.0 million per individual for WGS.

When the researchers scanned the data for regions of genetic differentiation, they noticed that “the reduced representation approaches [GBS and ddRAD] were much less effective at identifying regions with elevated FST at the fine scale and do not provide the same detailed picture of the landscape of divergence that is found using WGS.” This result might not be that surprising – it has been found in other study systems, such as Sporophila seedeaters and Colaptes woodpeckers – but it does lead to an intruiging insight. If you are mainly interested in characterizing the genomic landscape of differentiation, it will be worthwhile to directly opt for WGS instead of first exploring the genome with reduced representation approaches.

Exploring the genomic landscape of differentiation with different sequencing techniques revealed that WGS is far superior compared to reduced representation approaches (GBS and ddRAD). From: Szarmach et al. (2021).


Szarmach, S. J., Brelsford, A., Witt, C. C., & Toews, D. P. (2021). Comparing divergence landscapes from reduced‐representation and whole genome resequencing in the yellow‐rumped warbler (Setophaga coronata) species complex. Molecular Ecology30(23), 5994-6005.

Toews, D. P., Mandic, M., Richards, J. G., & Irwin, D. E. (2014). Migration, mitochondria, and the yellow‐rumped warbler. Evolution68(1), 241-255.

Featured image: Audubon’s Warbler (Setophaga [coronata] auduboni) © Pterzian | Wikimedia Commons

Divergence in a crowded space: How sympatry influences the evolution of wood-warblers

The number of sympatric species can constrain the evolutionary process.

When closely related species occur in the same area, they might occasionally interbreed. If the resulting hybrids are unfit – for example, sterile or unviable – there will be a strong selection against hybridization. This selective pressure would then promote increased divergence between the species. In other words, the species will look or sound more different over time. This reasoning makes intuitive sense, but that doesn’t mean we should not test it. We might have missed a crucial aspect in our reasoning. Armchair theorizing can be valuable, but needs to be validated with direct observations or experiments. That is exactly what a recent study in the Proceedings of the Royal Society B did, using wood-warblers (family Parulidae) as a study system.

First, Richard Simpson and his colleagues tested the prediction that sympatric species are more divergent in sexual signals, such as plumage and song, compared to allopatric species. In addition, they went one step further and assessed the influence of the number of co-occurring species on the evolution of these sexual signals. When multiple species co-exist, the options for divergence become more constrained. The evolution of different plumage patterns or songs might push a species into the signal space of another related species. You can compare this situation with a busy party where you are trying to avoid some obnoxious guests. As you move away from one annoying person, you might accidently bump into another one. Again, this idea makes sense, but is it supported by the data?

A graphical representation showing how an increase in the number of species can limit the possibilities for divergence. From: Simpson et al. (2021).

Signal Space

The researchers amassed an impressive dataset on the morphological and acoustic features of numerous wood-warblers. For 818 museum specimens (representing 93 species), the plumage reflectance of 15 body regions was measured. The resulting patterns did not support the first prediction: allopatric wood-warbler species showed more divergent plumage patterns compared to sympatric ones. The researchers attributed this finding to the effect of habitat: “Species that occur in allopatry likely do not share similar habitats, while those in sympatry likely do (i.e. light environment, visual background, predatory species.” This explanation sounds reasonable, but requires further investigation.

When limiting the analyses to sympatric species, the prediction was borne out though. The more species overlapped in distribution, the more divergent their plumage patterns. Moreover, the number of sympatric species had a clear effect on the evolution of sexual signals. As more wood-warbler species were found in the same area, the occupied space of colors tended to reach a plateau. This pattern suggests that further divergence becomes constrained by the available signal space. To return to our analogy: the more annoying people at the party, the less room you will have to more around safely.

The number of sympatric species influences the evolution of sexual signals. As more species occur in the same area, the occupied space of color volume tends to level off. This pattern holds for males (left) and females (right). From: Simpson et al. (2021).

Acoustic Adaptation

The predictions formulated at the beginning of this blog post seem to hold for plumage patterns. But what about song features: do sympatric birds sing more divergent songs? Based on the analyses of 494 song recordings – representing 102 species – the answer is a resounding no. In fact, the exact opposite pattern was observed. Species with more overlapping distributions produced similar songs. The researchers called upon the “acoustic adaptation hypothesis” to explain this finding: “Species that exhibit higher degrees of sympatric overlap likely occur in more similar habitats, and these habitats are driving song evolution so that songs are optimally transmitted within the local environment.” A reasonable explanation that needs to be tested in future studies.

These findings nicely show that we should take into account alternative explanations and test our ideas with observations and experiments. Reasoning from a comfortable armchair only gets you so far. Don’t forget to test your ideas. I cannot help but think of these words by physicist Richard Feynman:

If it disagrees with experiment, it’s wrong. In that simple statement is the key to science. It doesn’t make any difference how beautiful your guess is, it doesn’t matter how smart you are who made the guess, or what his name is … If it disagrees with experiment, it’s wrong. That’s all there is to it.


Simpson, R. K., Wilson, D. R., Mistakidis, A. F., Mennill, D. J., & Doucet, S. M. (2021). Sympatry drives colour and song evolution in wood-warblers (Parulidae). Proceedings of the Royal Society B288(1942), 20202804.

Featured image: Bay-breasted Warbler (Setophaga castanea) © Mdf | Wikimedia Commons

Morphological and genetic evidence for hybrids between Magnolia Warbler and American Redstart

But why would these two clearly distinct species interbreed?

The bird family Parulidae – the wood warblers – is a hybrid hotspot, with more than 50% of species known to hybridize. Indeed, numerous species combinations have been reported (check out this page for an overview), providing ornithologists with valuable insights into the origin and evolution of species (see for example here and here). You would think that by now, we would have uncovered all hybrid combinations among these warblers. However, a recent study in The Wilson Journal of Ornithology adds another hybrid to the list: Magnolia Warbler (Setophaga magnolia) x American Redstart (S. ruticilla). Using morphological and genetic analyses, ornithologists managed to confirm the hybrid nature of two peculiar individuals.

Plumage and Genetics

It is interesting that although this hybrid combination had not been reported before, two independent observations are described in the paper. The first hybrid was found by two birdwatchers – Daniel Néron and Joël Coutu – in Quebec, Canada. They detected a Magnolia Warbler-like individual that produced the song of an American Redstart. This aberrant bird was later captured and banded. The second hybrid was caught during a routine ringing session of the Black Swamp Bird Observatory in Ohio, USA. One of the staff members took detailed measurements and collected feathers from this bird. In both cases, the hybrid showed plumage characteristics of both species, namely:

Magnolia Warbler plumage features include white eye arcs, yellow wash on the belly, narrow white wing bars, and black lores. American Redstart plumage features include a dark yellow-orange coloration on the sides of the breast and under the wing, yellow on the base of most of the secondaries, and pale yellow patches on the outer tail feathers.

The morphological suggestions of hybridization were confirmed with genetic data. The researchers obtained DNA sequences for three genes: the mitochondrial ND2, the Z-linked MUSK and the nuclear MYO2. For both hybrids, analyses of these markers pointed to a Magnolia Warblers as the mother and American Redstart as the father.

Two hybrid Magnolia Warbler x American Redstart. Images to the left (a–d) show the male hybrid discovered in Laval, Québec, Canada (photos courtesy of Simon Duval). Images to the right (e–h) show the female hybrid discovered at the Navarre Banding Station, Ottawa County, Ohio, USA (photos courtesy of Ryan Jacob). From: Brennan et al. (2020).

A Rare Occasion

The strikingly different plumage patterns of these two species raise the question why they would engage in hybridization. Surely, they look different enough to avoid any confusion. The researchers speculate that the immature plumage of young male American Redstarts is similar to that of Magnolia Warblers. Moreover, both species sing similar songs which could interfere with species recognition. In most cases, hybridization will be unlikely. But under particular circumstances – such as a lonely male unable to locate a conspecific partner – interbreeding might occur. And hopefully, an observant ornithologist will be around to spot such a rare sight.


Brennan, C. L., Boulanger, E., Duval, S., Frei, B., Gorbet, A., Head, J., Shieldcastle, M. & Jones, A. W. (2020). Two cases of a previously undocumented New World warbler hybrid (Setophaga magnolia x S. ruticilla) in eastern North America. The Wilson Journal of Ornithology132(3), 537-547.

Featured image: Magnolia Warbler (Setophaga magnolia) © William H. Majoros | Wikimedia Commons

Similar migration strategies explain the evolution of flight calls in wood warblers

Although phylogenetic relatedness plays a major role as well.

Several species of wood warbler (family Parulidae) migrate at night. During their nightly travels, they produce short, simple calls known as “flight calls”. In some species, these flight calls are so similar that they have been grouped into bio-acoustic categories, such as the “Zeep complex” and the “Upsweep complex.” Interestingly, members of these complexes tend to exhibit similar migration strategies. Could there be an evolutionary link between these traits? Given that wood warblers often migrate in mixed species flocks, shared flight calls might facilitate interspecific communication during migration. In a recent Evolution study, Zach Gayk and his colleagues tested this hypothesis by comparing the flight calls and migration strategies of 36 wood warbler species.

Migratory Variables

The researchers quantified the similarity of flight calls in the different species and correlated this with several features of migration, such as overlap in wintering range and total migration distance. The analyses indicated that phylogenetic relatedness explains a large part of the variation in flight calls (similar to this study on the evolution of bird song). In other words, closely related species produce similar flight calls. After correcting for phylogenetic relationships, some migratory variables were associated with similarity in flight calls. When considering all 36 species, flight calls were similar between species that breed at similar latitudes and that show a temporal overlap in migration timing. A more detailed analysis on the “Zeep complex” found significant effects of migration length and overlap in wintering range on flight call similarity. Taken together, these findings support the hypothesis that migratory similarity is a driving factor in the evolution of flight calls.

Range maps and flight call spectrograms for six species of migratory wood warblers. The top three species have similar long-distance migrations and acoustically similar flight calls, whereas the bottom three species have more varied migrations and dissimilar flight calls. From: Gayk et al. (2021) Evolution.


Intuitively, these findings make sense. Efficient communication during migration can be a life-saver. Literally. Similar flight calls can help birds to find high-quality stop-over sites, avoid predators, and reduce the chance of getting lost during migration. The researchers suspect that selection for similar flight calls was quite strong during the last 50,000 years of glacial cycles when “shifting migratory routes may have driven the need for increased communication.” However, the link between flight calls and migratory strategies remains to be tested in other bird species.

Finally, I cannot help but think of a possible role for flight calls in hybridization. Wood warblers are known for their propensity to hybridize (see the Parulidae page for an overview). Could this be related to the similarity in flight calls or migration strategies? Disentangling these two factors will be tricky, but worthwhile to explore.


Gayk, Z. G., Simpson, R. K., & Mennill, D. J. (2021). The evolution of wood warbler flight calls: Species with similar migrations produce acoustically similar calls. Evolution75(3), 719-730.

Featured image: Blackburnian warbler (Setophaga fusca) © Mdf | Wikimedia Commons

Swapping pigmentation genes across the Parulidae phylogeny

Genomic analyses reveal repeated exchange of pigmentation genes among these warblers.

The bird family Parulidae is known for its diversity in plumage patterns, primarily related to the pigments melanin (brown and black colors) and carotenoids (yellow, red and orange colors). Several studies have unraveled the genetic basis of these color differences, usually taking advantage of natural hybrids. Because genetic material gets shuffled around in hybrids, it can be easier to pinpoint particular genomic regions and identify candidate genes for further research. Most of these studies focused on hybridization between two species, such as the hybrid zone between Townsend’s Warbler (Setophaga townsendi) and Hermit Warbler (S. occidentalis). In a recent Current Biology study, Marcella Baiz and her colleagues took a broader perspective and compared the genomes of all 34 species in the genus Setophaga.

Gene Trees

Probing the genomes of these warblers revealed several divergent genomic regions that were shared by multiple species pairs. Some smaller regions contained the pigmentation genes ASIP (agouti signaling protein) and BCO2 (beta-carotene oxygenase 2). These genes also popped up in other studies on plumage coloration (see for example here and here) and are thus excellent candidates for more detailed analyses. Because different species have independently evolved similar plumage patterns, it is possible that these genes have been exchanged across the phylogeny of the Parulidae. To unravel the evolution of these genes, the researchers constructed gene trees for ASIP and BCO2, and compared these with the species tree.

The gene tree of ASIP was largely concordant with the expected phylogenetic relationships, suggesting that it has not been exchanged between species. The convergent evolution of black plumage patterns (in which ASIP is involved) might thus be due to repeated mutations. The situation for BCO2 is drastically different. Its gene tree deviated from the species tree, revealing several instances of introgression between distantly related species. These patterns were confirmed with the D-statistic, a commonly used test to detect introgression. The BCO2-gene has been exchanged between the Magnolia Warbler (S. magnolia) and the Yellow Warbler (S. petechia), and there was probably an introgression event involving the ancestor of the Prairie Warbler (S. discolor) and the Vitelline Warbler (S. vitellina).

The discordance between the species tree (left) and the BCO2 gene tree (right) point to repeated introgression of this gene. From: Baiz et al. (2021) Current Biology.

Tip of the Iceberg

These patterns show how the plumage patterns in the Parulidae evolved through the interplay of repeated mutations in some genes and extensive introgression of other genes. This study focused on just two genes (ASIP and BCO2), but many more genomic regions and candidate genes are waiting to be studied in more detail. The authors wrote that the parulid warblers have a “rich legacy of study, including cornerstones of community ecology and phylogenetic diversification”. I am confident that this bird family will continue to be a focal point of much more scientific research.


Baiz, M. D., Wood, A. W., Brelsford, A., Lovette, I. J., & Toews, D. P. (2021). Pigmentation genes show evidence of repeated divergence and multiple bouts of introgression in Setophaga warblers. Current Biology31(3), 643-649.

Featured image: Magnolia Warbler (Setophaga magnolia) © Cephas | Wikimedia Commons

Searching for a hybrid zone between two Andean warblers

Extensive phenotypic variation across a transect between Ecuador and Colombia.

From the 1930s into 1950s, John Zimmer published numerous extensive monographs about “Studies on Peruvian Birds”. In number 54 of the series, he focused on the bird families Catamblyrhynchidae (now part of the Thraupidae) and Parulidae. While describing specimens of the genus Myioborus, he commented on some “puzzling specimens” that showed characteristics of two subspecies of the Golden-fronted Redstart (M. ornatus chrysops) and the Spectacled Redstart (M. melanocephalus ruficoronatus).

The extreme characters of ruficoronatus strongly suggest those of the ornatus group, and an occasional specimen of o. chrysops shows a noticeable patch of rufous in the center of the crown, strongly suggesting ruficoronatus. It is not impossible, therefore, that the puzzling specimens of one sort and another may represent intergrades or even hybrids between the two groups, but much more material will be necessary before an adequate solution is reached.

Several researchers followed the advice of Zimmer and collected more material on these birds. A recent study in the journal Ornithology reported on their findings. Is there a hybrid zone or not?

Two Subspecies

Before we delve into the possibility of a hybrid zone, we need to clarify the distribution of the M. ornatusM. melanocephalus species complex. First, the Spectacled Redstart (M. melanocephalus) is divided into five subspecies that replace each other when you travel along the Andes from Bolivia to Ecuador (see dots in the map below). The southern subspecies (malaris, melanocephalus and bolivianus) lack a rufous crown, which is present in the two northern subspecies (griseonuchus and ruficoronatus). Second, the Golden-fronted Redstart (M. ornatus) comprises two subspecies (ornatus and chrysops) that occur in different cordilleras in Colombia and Venezuela (see triangles in the map below). The putative hybrid zone concerns interactions between the most northern subspecies of the Spectacled Redstart (ruficoronatus) and the western subspecies of the Golden-fronted Redstart (chrysops). Laura Céspedes-Arias and her colleagues collected samples of these subspecies across a transect running from Ecuador into Colombia.

(A) Map showing the distribution of the subspecies in the M. ornatus-M. melanocephalus species complex. (B) Overview of the sampling transect in the study. From: Céspedes-Arias et al. (2021) Ornithology.

Clinal Patterns

Plumage analyses of over 300 specimens revealed a wide variety of phenotypes, representing different trait combination of both subspecies. It quickly became clear that individuals with intermediate phenotypes were most common along the transect. Some traits, such as head and chest coloration, showed a clear clinal transition from one subspecies into the other (see this blog post for more information on cline theory). All in all, these morphological patterns pointed to a roughly 200 kilometer wide hybrid zone, confirming the suspicion of John Zimmer.

Next, the researchers turned to genetic data by sequencing the mitochondrial gene ND2. In contrast to the plumage traits, this gene did not show a smooth transition. Instead the researchers found extensive haplotype sharing between the subspecies. This pattern can be explained by the recent origin of these subspecies (i.e. incomplete lineage sorting) or by extensive introgression due to hybridization. Genomic analyses will be needed to discriminate between these possibilities. Nonetheless, the most likely scenario seems to entail allopatric divergence leading to differences in plumage traits, followed by secondary contact and extensive hybridization. Another exciting avian hybrid zone to study in more detail.

A sample of the diversity of plumage phenotypes across the hybrid zone, from a typical chrysops (top) over intermediates to a typical ruficoronatus (bottom). From: Céspedes-Arias et al. (2021) Ornithology.


Céspedes-Arias, L. N., Cuervo, A. M., Bonaccorso, E., Castro-Farias, M., Mendoza-Santacruz, A., Pérez-Emán, J. L., Witt, C. C. & Cadena, C. D. (2021). Extensive hybridization between two Andean warbler species with shallow divergence in mtDNA. Ornithology138(1), ukaa065.

Featured image: Spectacled Redstart (Myioborus melanocephalus) © Francesco Veronesi | Wikimedia Commons

A small genomic region explains the plumage differences between Townsend’s and Hermit Warbler

Three pigmentation genes might contribute to reproductive isolation.

If I had a dollar (or euro) for every time I read “hybrid zones are natural laboratories” in a paper, I could probably sequence a fair number of bird genomes. This popular phrase can be traced back to a classic paper by Godfrey Hewitt: “Hybrid zones-natural laboratories for evolutionary studies“. And it is certainly true. Hybrid zones are extremely useful settings to learn more about the evolutionary process. Moreover, because of the recombination of different genomic regions in hybrids, it is sometimes possible to uncover the genes underlying certain traits. This approach has been successful in finding “migration genes” in the Willow Warbler (Phylloscopus trochilus) and “plumage genes” in Vermivora warblers. A recent study in the journal Evolution Letters relied on a hybrid zone between Townsend’s Warbler (Setophaga townsendi) and Hermit Warbler (S. occidentalis) to identify the genetic underpinnings of several plumage traits.

Three Genes

Silu Wang and her colleagues quantified plumage patterns in 265 individuals. They focused on seven traits: (1) cheek coloration, (2) crown coloration, (3) throat bib darkening, (4) throat bib intensity, (5) extent of breast yellow, (6) presence of black streaks on the flank, and (7) intensity of green chroma on the back. Next, the researchers performed a genome-wide association study (GWAS) to determine which genetic variants correspond to particular traits. The analyses revealed that a single variant was significantly associated with the colors of the cheek, crown and flank. This variant is located in an intron of the RALY-gene, which is known to be involved in the yellow versus black pigmentation of mice and quail. In addition, two other pigmentation genes can be found in the same region: ASIP (influences skin pigmentation in vertebrates) and EIF2S2 (associated with human skin pigmentation). How these three genes work together is still unclear, but they might function as a “super-gene” (see this blog post for more on this topic).

Location of the hybrid zone (yellow) between Townsend’s Warbler (blue) and Hermit Warbler (purple), and an overview of the different plumage traits investigated in this study. From: Wang et al. (2020) Evolution Letters.

Reproductive Isolation

Next, the researchers used the genomic data to pinpoint differentiated sections in the genome that might be involved in reproductive isolation between these warblers. This search indicated four highly differentiated genomic regions, located on chromosomes 1A, 5, 20 and the Z-chromosome. Interestingly, the region on chromosome 20 corresponds to the location of the three pigmentation genes from the GWAS. This finding suggests that the ASIP-RALY region is involved in maintaining species-specific differences and preventing these warblers from merging into one species.

The exact mechanism of reproductive isolation remains to be determined. It could be that the ASIP-RALY region facilitates assortative mating (i.e. choosing a partner that looks like you). However, a recent simulation study suggested that assortative mating alone is insufficient to stabilize hybrid zones, some degree of postzygotic selection is needed. Another possibility is that the ASIP-RALY region contributes to lower fitness in hybrids. The patchy plumage patterns of hybrids might be a disadvantage in territorial disputes, complicating a hybrids’ attempt to secure a good territory. Exciting avenues for further research, showing how genomic analyses can generate hypotheses to be tested in the field.

Exploring genomic landscape of differentiation with different genomic datasets revealed several highly differentiated regions (highlighted with red dots), including the section with the three pigmentation genes. From: Wang et al. (2020) Evolution Letters.


Wang, S., Rohwer, S., de Zwaan, D. R., Toews, D. P., Lovette, I. J., Mackenzie, J., & Irwin, D. (2020). Selection on a small genomic region underpins differentiation in multiple color traits between two warbler species. Evolution Letters4(6), 502-515.

Featured image: Townsend’s Warbler (Setophaga townsendi) © Alan Vernon | Wikimedia Commons

This paper has been added to the Parulidae page.

Hybrid pairing between a Cerulean Warbler and a Black-throated Blue Warbler

Detailed nest observations confirm the first case of this hybrid combination.

When it comes to hybridization, wood-warblers are the bird family to study (see the Parulidae page for an overview). In 2014, Pamela Willis and her colleagues counted 24 species (out of 45) that are known to hybridize. With all these crosses, it is no surprise that some species hybridize with several other species. This became clear when I visualized the hybridization patterns in my review paper on multispecies hybridization in birds (see figure below). However, the resulting network is already outdated. A recent paper in the Wilson Journal of Ornithology reported on a new hybrid cross between two wood-warblers: the Cerulean Warbler (Setophaga cerulea) and the Black-throated Blue Warbler (S. caerulescens).

A hybrid network displaying the incidence of hybridization between different members—genera Geothlypis, Mniotilta, Oreothlypis, Setophaga, and Vermivora—of the Parulidae family. Thin, black edges indicate uncommon hybridization, while thick, red edges indicate extensive hybridization. From: Ottenburghs (2019) Avian Research.

Nest Observations

On 7 July 2017, birdwatcher Matt Wistrand discovered a nest that was being visited by a male Cerulean Warbler and a female Black-throated Blue Warbler in Brown County, Indiana. He took several pictures and made some audio recordings. This observation caught the attention of Clayton Delancey, Garrett MacDonald and Kamal Islam, who were allowed access to the nesting site on 12 and 13 July 2017. They monitored the nest and collected extra information on the behavior of the birds. The nest contained four nestlings that were fed by both parents. The researchers noted that they “did not observe any aggressive interaction between the male Cerulean Warbler and the female Black-throated Blue Warbler, and both individuals were observed multiple times at the nest simultaneously.”

All in all, these observations suggest that we are dealing with a hybrid pairing between these two species. To remove all doubt, the researchers returned to the nest on 13 July 2017 to collect the nestlings and take blood samples for genetic analyses. Unfortunately, they found the nest on the ground with no sign of the nestlings. It seems that it had been predated.

Pictures of the hybrid nest. (a) View of the Cerulean Warbler–Black-throated Blue Warbler nest in mixed live and dead foliage. (b) Closeup of 4 nestlings. (c) Male Cerulean Warbler with a beak full of green caterpillars. (d) Male Cerulean Warbler feeding nestlings. (e) Female Black throated Blue Warbler at the nest. (f) Female Black-throated Blue Warbler feeding nestlings. © Clayton Delancey

Rare Occasion

The final piece of (genetic) evidence to confirm this hybrid mating is thus missing, but it is likely that these birds paired up due to a scarcity of partners. The male Cerulean Warbler might have been unsuccessful in attracting a female, causing it to settle with a Black-throated Blue Warbler that happened to be around. Black-throated Blue Warblers are not known to breed in Indiana, making this hybrid pairing even more special. We will probably not see this species combination in the near future, but you never know… A perceptive birdwatcher might discover an unusual nesting situation.


Delancey, C. D., MacDonald, G. J., & Islam, K. (2019). First confirmed hybrid pairing between a Cerulean Warbler (Setophaga cerulea) and a Black-throated Blue Warbler (Setophaga caerulescens). The Wilson Journal of Ornithology131(1), 161-165.

Featured image: Cerulean Warbler (Setophaga cerulea) © Mdf | Wikimedia Commons

This paper has been added to the Parulidae page.

How strong is reproductive isolation between Golden-winged and Blue-winged warbler?

A summary of a recent debate in the journal Ecology and Evolution.

The future of the Golden-winged Warbler (Vermivora chrysoptera) is threatened by habitat loss. In addition, it runs the risk of being outcompeted and “out-hybridized” by the invading Blue-winged Warbler (V. cyanoptera). The interactions between these two closely related species have a long history of scientific research (summarized on the Parulidae page). However, it is still unclear how strong the level of reproductive isolation between these warblers is. Recent work by David Toews and his colleagues pointed to six genomic regions that are highly divergent between Golden-winged and Blue-winged Warblers, of which four are likely involved in feather development or pigmentation. These findings suggest that differences in plumage patterns could act as a strong reproductive barrier. The strength of this potential barrier can be tested by quantifying the frequency of mixed pairings between different plumage types, and following the reproductive success of hybrids (if there are any).

A recent study in the journal Ecology and Evolution performed these measurements and reported strong reproductive isolation between Golden-winged and Blue-winged Warblers. However, another team of researchers questioned these results and indicated potential pitfalls in the analyses, to which the original authors responded. In this blog post, I will try to summarize the main arguments in this interesting debate.

Strong Reproductive Isolation?

Let’s start with the first study. To determine the degree of reproductive isolation between Golden-winged Warbler and Blue-winged Warbler, John Confer and his colleagues aggregated data on social pairing from nine studies. Apart from the two pure phenotypes, the researchers also considered two hybrid phenotypes: the “Brewster’s Warbler” and the “Lawrence’s Warbler”. These phenotypes were initially described as distinct species before they were recognized as hybrids. According to the model of Kenneth Parkes (1951), “Brewster’s Warblers” are first-generation hybrids between genetically pure Golden-winged and Blue-winged Warblers, while the “Lawrence’s Warbler” can be produced by crossing two first-generation hybrids.

The analyses revealed a low level of hybridization. Only 14 out of 1680 (0.9%) Golden-winged Warblers and 14 out of 583 (2.4%) Blue-winged Warblers formed a social pair with a pure-looking bird of the alternative phenotype. These patterns indicate high levels of behavioral isolation between the different plumage phenotypes. Next, the researchers turned to the breeding success of the hybrid phenotypes. The pairing success of “Brewster’s Warblers” (54%) was significantly smaller compared to the pure Golden-winged (83%) and Blue-winged Warblers (77%). These percentages suggest some degree of sexual selection against hybrids. Putting it all together, the researchers calculated a reproductive isolation score of 0.96. Given that a score of 1 corresponds to complete reproductive isolation, this number indicates strong reproductive isolation.

The different phenotypes considered in the study: Golden-winged Warbler (V. chrysoptera; GWWA), Blue-winged Warbler (V. cyanoptera; BWWA), “Brewster’s” Warbler (hybrid; BRWA) and “Lawrence’s” Warbler (hybrid LAWA). From: Confer et al. (2020) Ecology and Evolution.

Three Points of Critique

A few months later, David Toews and his colleagues published a critique on this conclusion of strong reproductive isolation, raising three main issues. First, the plumage classification scheme in the original study is not suitable to determine hybrid ancestry in these warblers. Recent genetic work by Marcella Baiz and her colleagues showed that none of the six “Brewster’s Warblers” that they analyzed were first-generation hybrids (see this blog post for the details). Moreover, many warblers that look like pure phenotypes might actually contain some genetic ancestry from past hybridization. The original study did not take these “cryptic hybrids” into account.

A second issue that was not considered in the analyses concerns extra-pair copulations in which birds mate with other individuals besides their social partner. This phenomenon has been well-documented in Vermivora warblers and could significantly contribute to hybridization between Golden-winged and Blue-winged Warblers.

Finally, Toews et al. (2021) pointed out that behavioral isolation is not always sufficient to maintain complete reproductive isolation. For example, recent simulations by Darren Irwin showed that assortative mating on its own cannot prevent populations from merging, some form of postzygotic isolation is needed (see this blog post for the whole story). Although “Brewster’s Warblers” have lower pairing success compared to pure phenotypes, their reproductive output might still be too high to prevent genetic exchange. Hence, the authors of the critique argue that “extensive mixing in areas of sympatry is more consistent with low levels of total reproductive isolation—that is, both low pre-and postmating isolation—and results in high gene flow.”

The range of Golden-winged (orange) and Blue-winged (blue) Warblers. Areas of overlap are highlighted in light blue. From: Toews et al. (2021).


Recently, the authors of the original study – this time led by Cody Porter – replied to the critique by Toews et al. (2021). First, with regard to the unsuitability of the plumage classification scheme, they explain that the complex genetic ancestry of the warblers (including cryptic hybrids) is actually not that relevant for their question. The focus of their study concerns different plumage phenotypes, not the whole genomic context. They write: “In essence, our study could be viewed as testing whether the six major genomic differences between V. chrysoptera and V. cyanoptera (which largely correspond to plumage differences; Toews et al., 2016) promote reproductive isolation.”

Second, they argue that extra-pair copulations were unlikely to bias their results, referring to the findings of Vallender et al. (2007). This study found only 3 cases of extra-pair copulations (ca. 1.5%) between different phenotypes. In two cases a hybrid female mated with a Golden-winged Warbler and in one case a Golden-winged Warbler female mated with a hybrid.

The third point of critique focuses on the contribution of behavioral isolation to the level of reproductive isolation. You need some degree of postzygotic isolation to prevent species from merging. Toews et al. (2021) argued that the reproductive success of the hybrids is still too high, facilitating gene flow between the species. The authors counter this argument by highlighting the 26% reduction in the pairing success of phenotypic hybrids compared to both parental forms and the fact that only 1.2% of birds with a “pure” phenotype paired with an individual of the alternative phenotype. These numbers “appear to fall well within the parameters for a stable hybrid zone according to Irwin’s (2020) simulations.”

The Verdict

You might be wondering who won this debate? I don’t think this is the right question to ask here. Both groups of authors approached the scientific conundrum of reproductive isolation from a different perspective. The original study focused on behavioral isolation on the phenotypic level, whereas the critique used the genomic patterns of introgression as a starting point. At first sight, the strong reproductive isolation between plumage phenotypes seems incompatible with the largely homogeneous genomes of these warblers. However, reproductive isolation is not complete (remember the score of 0.96), which seems to allow for enough gene flow to homogenize the majority of the genome. Only the genomic regions containing “plumage genes” are able to withstand this homogenizing force.

Similar patterns have been described in other avian systems, such as Hooded Crow (Corvus cornix) and Carion Crow (C. corone) or Taiga Bean Goose (Anser fabalis) and Tundra Bean Goose (A. serrirostris). A few divergent genomic regions seem to be sufficient for a high level of reproductive isolation. We need more studies that quantify reproductive isolation at the phenotypic level and provide a link with the genomic underpinnings of the isolation barriers. Studying the evolution of reproductive isolation from different perspectives – behavioral, morphological and genetic – will fuel healthy debates and will provide more insights into the origin of species.


Confer, J. L., Porter, C., Aldinger, K. R., Canterbury, R. A., Larkin, J. L., & Mcneil Jr, D. J. (2020). Implications for evolutionary trends from the pairing frequencies among golden‐winged and blue‐winged warblers and their hybrids. Ecology and Evolution10(19), 10633-10644.

Toews, D. P., Baiz, M. D., Kramer, G. R., Lovette, I. J., Streby, H. M., & Taylor, S. A. (2021). Extensive historical and contemporary hybridization suggests premating isolation in Vermivora warblers is not strong: A reply to Confer et al. Ecology and Evolution.

Porter, C. K., Confer, J. L., Aldinger, K. R., Canterbury, R. A., Larkin, J. L., & McNeil Jr, D. J. (2021) Strong yet incomplete reproductive isolation in Vermivora is not contradicted by other lines of evidence: A reply to Toews et al. Ecology and Evolution.

Featured image: Golden-winged warbler (Vermivora chrysoptera) © Bettina Arrigoni | Wikimedia Commons

These papers have been added to the Parulidae page.