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.
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).
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 Biology, 31(3), 643-649.
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?
Before we delve into the possibility of a hybrid zone, we need to clarify the distribution of the M. ornatus–M. 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.
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.
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. Ornithology, 138(1), ukaa065.
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.
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).
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.
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 Letters, 4(6), 502-515.
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).
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.
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 Ornithology, 131(1), 161-165.
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.
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.”
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.”
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 Evolution, 10(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.
Are they first generation hybrids, backcrosses or something else?
Some bird hybrids were initially described as distinct species. I have covered some notable examples on this blog, such as Rawnsley’s Bowerbird (Ptilonorhynchus rawnsleyi) and Argus Bare-eye (Phlegopsis barringeri). In most cases, the species name disappears when the hybrid identity of the bird has been revealed, but sometimes the name stays around. In papers on hybridization dynamics between Golden-winged Warbler (Vermivora chrysoptera) and Blue-winged Warbler (V. pinus), you often come across Brewster’s Warblers and Lawrence’s Warblers. The latter two “species” turned out to be hybrids. In 1893, Sage already expressed his doubt by stating that ‘I am not inclined to believe leucobronchialis [i.e. Brewster’s Warbler] a hybrid, but hope to have more to say on this subject at another time.” However, the names are still used to indicate the characteristics of these birds.
“Lawrence’s” hybrids are similar to Blue-winged Warblers (i.e. yellow overall, with 2 narrow white wing bars) but have the black throat patch and face mask, similar to Golden-winged Warblers. “Brewster’s” hybrids, by contrast, lack a black throat patch, have little to no yellow on the underparts, and commonly have partially separated yellowish wing bars.
Based on these traits, Nichols (1908) and Parkes (1951) speculated that first generation hybrids would look like Brewster’s Warblers, while second generation hybrids and backcrosses would resemble Lawrence’s Warblers. With the advent of genomic data, we can put these hypotheses to the test. In a recent study in the journal The Auk, Marcella Baiz and her colleagues examined the genetic make-up of nine Vermivora warblers.
The different species and hybrids of Vermivora Warblers. From: Baiz et al. (2020) The Auk.
To figure out whether Nichols and Parkes were right, the researchers used triangle plots. Based on two statistics – heterozygosity and hybrid index – you can deduce what kind of hybrid or backcross you are dealing with. Pure individuals are located in the lower corners, while first generation hybrids are at the top. The sides of the triangles (D1 and D2) indicate backcrosses. You would thus expect that Brewster’s Warblers (F1) end up at the top and Lawrence’s Warblers at the sides (backcrosses) of these triangles.
This was, however, not the case. The sequenced individuals were scattered across the triangle and did not follow the predictions by Nichols and Parkes. The Lawrence’s Warbler in this study is not a backcross, but probably a multigenerational hybrid with mostly Blue-winged Warbler ancestry. Similarly, the Brewster’s hybrids are not F1 hybrid, but can trace the majority of their ancestry to either parental species. It thus seems that these hybrid types are quite variable and that F1 hybrids and backcrosses are not easy to distinguish based on the coloration of their underparts.
An example of a triangle plot (left, adapted from Pulido-Santacruz et al. 2018). In this case, you would expect Brewster’s Warblers (F1) at the top and Lawrence’s Warblers at the sides (backcrosses) of these triangles. The results show a different picture, indicating that the hybrids are quite variable (right, from Baiz et al. 2020).
Black Throat Patch
The story of the black throat patch is very different. Previous work by David Toews and his colleagues uncovered high genetic differentiation between Golden-winged and Blue-winged Warblers near the gene ASIP. This candidate gene has been linked to plumage differences in other bird species, such as Sporophila Seedeaters, Setophaga Warblers and Lonchura Munias. In this study, the researchers could zoom in on the genomic region where this gene resides. They found genetic variants in front of ASIP, suggesting that mutations in the regulatory sequences – the on-and-off switches – are responsible for the presence or absence of a black throat patch. Gene expression studies are needed to confirm this prediction. So, we moved on from one set of predictions (by Nichols and Parkes) to the next one. In science, we call that progress!
A clear signal of genetic differentiation at the ASIP gene (highlighted in grey). From: Baiz et al. (2020) The Auk.
Baiz, M. D., Kramer, G. R., Streby, H. M., Taylor, S. A., Lovette, I. J., & Toews, D. P. (2020). Genomic and plumage variation in Vermivora hybrids. The Auk, 137(3), ukaa027.