Do conservation units correspond to the six recognized subspecies?
A few months ago, I wrote about the unusually low genetic diversity of the Red-billed Tropicbird (Phaethon aethereus), which renders this species vulnerable to extinction (see this blog post for the whole story). Another tropicbird species of conservation concern is the White-tailed Tropicbird (P. lepturus). Despite its wide distribution across the Atlantic, Pacific, and Indian Oceans, several breeding colonies are threatened due to predation by invasive species and habitat destruction from human activities or tropical storms. To implement proper conservation measures it is important to identify the number of conservation units within the White-tailed Tropicbird. Some colonies might be part of a larger metapopulation that needs to be protected as a whole, whereas other colonies are isolated and require specific conservation efforts.
Currently, the White-tailed Tropicbird is classified into six subspecies. Three large subspecies breed in the western (lepturus) and eastern (fulvus) Indian Ocean and in the northwestern Atlantic Ocean (catesbyi). Three small subspecies breed in the Pacific (dorotheae), Indian (europae), and south Atlantic Oceans (ascensionis). To determine whether all these subspecies represent distinct conservation units, a recent study in the journal Ecology and Evolution studied the morphological and genetic patterns of 13 breeding colonies.
The genetic analyses – based on microsatellites and mtDNA – uncovered four separate clusters. Three of these clusters correspond to specific subspecies (catesbyi, europae and ascensionis), while the fourth genetic cluster collects the remaining three subspecies from the Indo-Pacific region (lepturus, fulvus and dorotheae). The morphological data did not add much in terms of differentiation, since it only discriminated between the largest (catesbyi) and smallest (europae) subspecies, with all the other subspecies forming an intermediate group.
More detailed analyses of the Indo-Pacific subspecies revealed that the population on Christmas Island might represent a differentiated genetic pool and could be considered as a distinct conservation unit. All in all, the researchers suggest the recognition of five conservation units: (1) Bermuda (and all populations of the northwest Atlantic Ocean); (2) Ascension/Fernando de Noronha (and all populations of the southern tropical Atlantic Ocean); (3) Europa; (4) Christmas Island; and (5) the other Indo-Pacific colonies. Patterns of genetic diversity indicated that the first three conservation units are clearly vulnerable (catesbyi), endangered (ascensionis) or critically endangered (europae). They are thus in need of urgent conservation measures.
Humeau et al. (2020). Genetic structuring among colonies of a pantropical seabird: Implication for subspecies validation and conservation. Ecology and Evolution, 10(21), 11886-11905.
But does this red plumage also honestly signal male quality?
Despite four million years of divergence, Red-fronted Tinkerbird (Pogoniulus pusillus) and Yellow-fronted Tinkerbird still hybridize. Why has reproductive isolation not solidified between these two African species? The answer might be connected with female choice. In a 2019 study, Emmanuel Nwankwo and his colleagues documented extensive introgression between Red-fronted and Yellow-fronted Tinkerbird. Their analyses revealed that hybridization was asymmetrical with a high proportion of individuals with Yellow-fronted genotypes that sported a red forecrown. This finding suggests that the red plumage trait is spreading in the Yellow-fronted Tinkerbirds, possibly because females prefer males with a red forecrown.
However, for a trait to be involved in mate choice, it needs to send out an honest signal. This means that females should be able to judge the quality of a male by that trait. Red plumage tends to be the product of carotenoids which individuals acquire in their diet. Some species use carotenoids unchanged from dietary components while others biochemically convert them before deposition in feathers. A large-scale analysis on feather coloration suggested that converted carotenoids better reflect individual quality because of the physiological links between cellular function and carotenoid metabolism. So, understanding the genetic basis of the red forecrown in tinkerbirds can show if this trait is an honest signal.
In a recent Molecular Ecologypaper, Alexander Kirschel and his colleagues analyzed the genetic make-up of 85 individuals that were collected across a hybrid zone in South Africa. A genome-wide association study (GWAS) pointed to a significant connection between the red forecrown and a genomic region on chromosome 8. Close inspection of this region revealed the gene CYP2J19 which is known to be involved in red coloration in other bird species, such as canaries and zebra finches. In the tinkerbirds, the different variants of this gene nicely explained the color of the forecrown. All individuals with two copies of the yellow-variant had a yellow forecrown and all individuals with two copies of the red-variant showed a red forecrown. Moreover, birds with a yellow-variant and a red-variant had either red, reddish or orange crowns.
But what about the function of CYP2J19? This gene codes for a ketolase, an enzyme that convert dietary yellow carotenoids into red ketocarotenoids. As I explained above, converted carotenoids might reflect individual quality better and can thus be used as an honest signal in mate choice. However, the exact mechanism underlying the workings of CYP2J19 remain to be unraveled. The GWAS uncovered other genomic regions that might contain the regulatory switches that control the expression of CYP2J19 (perhaps similar to the situation in Hooded Crows). More research is thus needed to understand how evolution is tinkering with the tinkerbirds.
Kirschel, A. N., Nwankwo, E. C., Pierce, D. K., Lukhele, S. M., Moysi, M., Ogolowa, B. O., Hayes, S. C., Monadjem, A. & Brelsford, A. (2020). CYP2J19 mediates carotenoid colour introgression across a natural avian hybrid zone. Molecular Ecology, 29(24), 4970-4984.
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.
Genetic analyses point to a ghost population on the African coast.
Island populations originate when small sections of the mainland population colonize remote archipelagos. So, just look for the closest mainland population and you have identified the source population. This reasoning sounds logical, but it ignores one important issue: species distributions change over time. The current range of a species does not necessarily represent the situation when the islands were colonized. The populations that fueled the island colonization might have disappeared. It is thus important to consider the possibility of these “ghost populations”. A recent study in the Journal of Biogeography investigated whether ghost populations played a role in the establishment of the Red-billed Chough (Pyrrhocorax pyrrhocorax) on the Canary Islands.
I have always associated the Red-billed Chough with alpine environments, so I was surprised to learn that this corvid also occurs on the Canary Islands. The population on the island of La Palma is even one of its strongholds in the western Palearctic with an estimated 2800 individuals. But how did the Red-billed Chough reach La Palma? When we look at its current distribution on the mainland, we can narrow it down to two source populations: Iberia (Spain and Portugal) or the Atlas Mountains in Morocco. Both populations are quite far from the Canary Islands: a trip from Iberia covers about 1200 kilometers, while the distance between Morocco and La Palma amounts to 800 kilometers. Red-billed Choughs are not known to travel such large distances, so long-distance dispersal seems unlikely.
Another possibility is that there has been suitable habitat for choughs along the North African coast. This scenario is supported by paleoclimatic studies, revealing that the Sahara has experienced periods of a wet, subtropical climate. Today, the nearest distance between the coast and the closest island (Fuerteventura) is 96 km, and during ice ages this distance would be even shorter due to drops in sea level. Francisco Morinha, Borja Milá and their colleagues used genetic data to test these scenarios (long-distance dispersal vs. ghost populations) and reconstruct the colonization history of the Red-billed Chough.
The genetic analyses of mitochondrial DNA and ten microsatellites indicated that Red-billed Choughs from La Palma are most closely related to the Iberian population. That still leaves the question whether these birds flew all the way from Iberia or if they originate from ghost populations that were connected to Iberia. The researchers discard the long-distance dispersal scenario for several reasons:
Red-billed Choughs are non-migratory and do not disperse far (a few 100 kilometers at most)
These is no fossil evidence of choughs on other islands, such as the Azores or Madeira, that lie between Iberia and the Canary Islands.
The mtDNA shows no signs of a genetic bottleneck which would be expected if a small population from Iberia colonized the Canary Islands.
These are all reasonable arguments, but disproving the long-distance dispersal scenario does not automatically validate the ghost population scenario (that would be a black-or-white fallacy). So, what about the evidence in favor of the scenario involving a ghost population? The researchers tested this hypothesis using Approximate Bayesian Computation in which they compared different biogeographic models. The results revealed that “the model including a ghost population connecting Iberia and La Palma was more likely than alternative models.” However, the researchers warn that this modelling approach is based on just ten microsatellites, and will need to be validated with genomic data. Nonetheless, based on the current evidence, it seems likely that the Red-billed Chough reached La Palma through a ghost population on the African coast.
Morinha, F., Milá, B., Dávila, J. A., Fargallo, J. A., Potti, J., & Blanco, G. (2020). The ghost of connections past: A role for mainland vicariance in the isolation of an insular population of the red‐billed chough (Aves: Corvidae). Journal of Biogeography, 47(12), 2567-2583.
Several lines of evidence indicate strong selection against hybrids.
An interesting debate in avian speciation concerns the relative importance of prezygotic versus postzygotic isolation mechanisms. Prezygotic isolation mechanisms operate before the fertilization of the egg. They can be behavioral, for example when members from different species don’t see each other as potential mates because they look or sound too different. When such behavioral isolation is imperfect and copulation does occur, fertilization might still fail if sperm and egg are incompatible (see this review paper for more on these so-called postcopulatory prezygotic barriers). Postzygotic isolation mechanisms act after fertilization and can be either intrinsic or extrinsic. Intrinsic mechanisms lead to sterility or unviability of the offspring, while extrinsic mechanisms encompass lower fitness of the hybrid offspring due to ecological or behavioral issues. For example, some hybrid hummingbirds display aberrant courtship behavior and cannot attract a mate (see this blog post). But which of these mechanisms contributes most to reproductive isolation during the formation of new bird species?
In some cases, the prezygotic isolation mechanisms are crystal-clear (although they can still be incomplete). Think of the distinct plumage patterns of the Golden-winged (Vermivora chrysoptera) and Blue-winged Warbler (V. cyanoptera, you can read more on these species in this blog post). Or just listen to the drastically different songs of the Common Chiffchaff (Phylloscopus collybita) and the Willow Warbler (Phylloscopus trochilus). However, the situation becomes more complicated when you venture into the Amazonian rainforest. Here, some bird species show minor differences in morphology or song despite millions of years of evolution. The Rondonia Warbling-antbird (Hypocnemis ochrogyna) and the Spix’s Warbling-antbird (H. striata), for example, look and sound almost similar, but have been on separate evolutionary trajectories for more than one million years. What keeps these species distinct? Could it be postzygotic isolation mechanisms? A recent study in the journal Evolution tried to find out.
Hybrid Trianglesand Clines
Áurea Cronemberger and her colleagues took a genetic approach to study a hybrid zone between the two warbling-antbird species. They applied several methods to quantify the strength of selection against hybrids. First, they constructed “hybrid triangles” to determine the frequency of first-generation hybrids and backcrosses in the hybrid zone. These triangles combine information from a hybrid index (i.e. genetic ancestry of an individual) and heterozygosity to discriminate between different hybrid classes. In general, “pure” individuals are located in the lower corners, while first generation hybrids are at the top. The sides of the triangles indicate backcrosses. This analysis revealed six first-generation hybrids and a series of backcrossed individuals. One individual (in the center of the plot) looks like a second-generation hybrid (i.e. the offspring of two hybrids).
The presence of several backcrosses seems to suggest that there is no strong selection against hybrids. However, the production of backcrosses does not mean that the species are exchanging genetic material (i.e. introgression). It is possible that these backcrosses do not reproduce themselves due to genetic incompatibilities in their genomes. To check whether the production of backcrosses leads to introgression, the researchers turned to cline analyses. I have covered this approach in a previous blog post, but I will quickly recap the most important lessons here: “a steep cline suggests strong reproductive isolation between hybridizing species, while a wide cline points to weak isolation. And a displaced cline suggests gene flow from one species into the other.”
The researchers calculated the expected cline width under neutral conditions (no selection against hybrids), which amounted to 211 kilometers. The genetic analyses revealed that only one locus out of 5387 exceeded this threshold and 95% of the loci even showed cline widths less than 45 kilometers. In other words, a lot of steep clines. In addition, the researchers reported little variability in the geographic positions of the clines. The lack of displaced clines suggests that there is no introgression between the hybridizing species. In summary, the shape and position of the clines point to strong selection against hybrids and backcrosses.
This study provides strong evidence for postzygotic isolation between the Rondonia Warbling-antbird and the Spix’s Warbling-antbird. Despite minor differences in morphology and vocalization, these birds can thus be considered distinct species. Similar results have been reported for other Amazonian birds, such as antbirds and woodcreepers (see this blog post), suggesting that postzygotic isolation mechanisms are the main driver of avian speciation in Amazonia. This finding might have important consequences for the species richness in this area. If postzygotic isolation evolves between morphologically cryptic species, how many species are still waiting to be discovered with genetic analyses. Indeed, the researchers conclude that “Our results thus suggest the strong possibility that species richness could be dramatically underestimated in Amazonia, especially for antbirds and other understory specialists.” Time for the next expedition!
Cronemberger, Á. A., Aleixo, A., Mikkelsen, E. K., & Weir, J. T. (2020). Postzygotic isolation drives genomic speciation between highly cryptic Hypocnemis antbirds from Amazonia. Evolution, 74(11), 2512-2525.
A genetic study detected only one hybrid individual.
Estimating the incidence of hybridization on an individual level is extremely challenging. Recently, Nicholas Justyn and his colleagues used data from the citizen science database eBird to investigate how often birds hybridize in North America. They found that 0.064% of the reported sightings were hybrids. This estimate can probably be regarded as a lower bound, because birdwatchers tend to under-report common hybrids (as I argued together with David Slager in a response to this study, and see also this paper by Hannah Justen and her colleagues). These papers highlight the difficulty of estimating hybridization rates in wild birds, even if you are focusing on just two species. In some cases, hybrids might be difficult to identify morphologically or the study species live in remote areas. Here, genetic data can be a valuable asset (see for example this blog post on penguin hybrids).
A recent study in the Journal of Avian Biology attempted to estimate the incidence of hybridization between Barrow’s Goldeneye (Bucephala islandica) and Common Goldeneye (B. clangula). Field observations suggest that these sea ducks occasionally interbreed (see the Anseriformes page for an overview), but the exact proportion of hybrids in their populations remains unknown.
Joshua Brown and his colleagues followed a genetic approach and took a closer look at the DNA of 61 individuals. Using two different genetic markers (microsatellites and ddRAD-seq), they found evidence for one hybrid individual. Additional demographic analyses pointed to an evolutionary model of allopatric speciation with secondary contact. The migration rate, however, amounted to less than one migrant per generation in both directions. In other words, an extremely low estimate of gene flow due to hybridization. These findings indicate that hybrid goldeneyes are a rare sighting. The authors attribute this low occurrence of hybridization to “assortative mating, differences in habitat preferences and territorial behaviors exhibited during mate pairing.”
In addition to quantifying hybridization, the researchers also investigated population structure in both species. Previous work reported clear population structure in terms of mitochondrial DNA, suggesting that females rarely disperse between breeding grounds (mtDNA is inherited through the female line). The same study also found no overlap in winter band recoveries among individuals marked in Alaska and British Columbia. Based on these patterns, the researchers expected to find some population structure in the nuclear DNA as well.
Surprisingly, there was no discernable population structure in the microsatellites or the ddRAD-seq data. This lack of nuclear population structure might be explained by dispersal of males between colonies. However, Barrow’s Goldeneye shows a high level of breeding site fidelity in both sexes, with the average yearly return rate of males (67%) roughly identical to that of females (63%). The situation in Common Goldeneyes is unknown due to lack of data. But not all males are equal. It is known that subadult males return to natal nesting grounds significantly less often than subadult females and are thus much more likely to disperse between colonies. Hence, the authors argue “that homogeneity across the nuclear genome most likely results from high levels of juvenile male dispersal despite high mtDNA structure.”
Brown et al. (2020). High site fidelity does not equate to population genetic structure for common goldeneye and Barrow’s goldeneye in North America. Journal of Avian Biology, 51(12).
Genomic analyses test whether alternative splicing contributes to sexual dimorphism.
It is no secret that male and female birds can look drastically different. Just think of the extravagant male birds-of-paradise compared to the dull females. It is interesting to contemplate how this extreme sexual dimorphism can develop given that males and females largely have the same genomes. One possible solution to this genomic conundrum is differential gene expression where males and females produce different amounts of certain genes that underlie particular traits. So, it is not about what you have, but how you use it. There has been a lot of research on the relationship between sex-specific gene expression and sexual dimorphism (see for example this review paper by Judith Mank), but other regulatory mechanisms have been largely overlooked. A recent paper in the journal Molecular Biology and Evolution took a closer look at one of these mechanisms: alternative splicing.
Mixing it up
Alternative splicing is a molecular mechanism that allows a single gene to code for multiple proteins. To understand this mechanism, we need to quickly recap the structure of a gene. As you might remember from high school biology, most genes are made up of exons and introns. An exon is a section in a gene that encodes part of the final messenger RNA (mRNA). Introns, on the other hand, are removed from the mRNA molecule and will not be expressed. In alternative splicing, these exons and introns are used in different combinations, giving rise to several protein variants (known as isoforms) that might have different functions.
Thea Rogers and her colleagues investigated whether alternative splicing might contribute to sexual dimorphism in birds. They studied gene expression patterns in three species: the Mallard (Anas platyrhynchos), the Turkey (Meleagris gallopavo) and the Helmeted Guineafowl (Numida meleagris). The results indicated that alternative splicing was common in all species, with an average of 21%, 17%, and 24% of the autosomal genes undergoing at least one splice event in the Mallard, Turkey, and Helmeted Guineafowl, respectively. But were there any sex-specific differences in splicing?
More detailed analyses revealed that the patterns of alternative splicing varied between the sexes. Interestingly, the most genes with sex-specific splicing were found in the reproductive tissues (i.e. the testes and ovaries). The authors write that “this suggests that ovaries and testes are regulated by distinct sex-specific gene regulatory networks, and that sex-specific splice variants play a role in the construction of sex-specific genetic architecture.” However, these patterns only suggest a role for alternative splicing in sexual dimorphism. Therefore, the researchers took it one step further and compared splicing patterns in Turkeys with different degrees of male characteristics.
Turkeys come in two male phenotypes: dominant and subordinate birds. Dominant males are bigger and show more elaborate male features compared to the subordinate males. This results in a gradient of sexual dimorphism from females over subordinate males to dominant males. The researchers took advantage of this variation for some clever analyses. First, they classified the alternative isoforms as either male- or female-biased depending on whether they were expressed more highly in dominant males or females. Next, they looked at the expression of these isoforms in the subordinate males. This approach revealed that subordinate males express male-biased isoforms at lower levels and female-based isoforms at higher levels compared to dominant males. These patterns clearly support a role for alternative splicing in the development of sexual dimorphism.
This study nicely shows that alternative splicing of certain genes plays a role in sexual dimorphism. The analyses focused on the big patterns, comparing hundreds of genes across species and tissues. A logical next step would be to zoom in on particular genes (or genetic networks) and unravel how they contribute to the differences between males and females. One thing is certain though, the genetic underpinnings of sexual dimorphism in birds are more complicated that we anticipated. Geneticist Steve Jones put it succintly: “Genetics has always turned out to be much more complicated than it seemed reasonable to imagine. Biology is not like physics. The more we know, the less it seems that there is one final explanation waiting to be discovered.” Exciting times to be a biologist!
Rogers, T. F., Palmer, D. H., & Wright, A. E. (2021). Sex-specific selection drives the evolution of alternative splicing in birds. Molecular Biology and Evolution, 38(2), 519-530.
Reduced genetic diversity in neutral and functional markers raises a red flag.
In a recent perspective paper, João Teixeira and Christian Huber argued that “neutral genetic diversity has only very limited relevance for conservation genetics.” They indicated that there is no simple general relationship between neutral genetic diversity and the risk of species extinction. We should thus be careful when using this metric to assess the long-term survival of a species. However, conservation biologists should not completely discard neutral genetic diversity in their work (as explained in this paper). Neutral variation certainly plays a role in adaptation (see for example this blog post) and can be a sign of past genetic drift and inbreeding due to a low effective population size. Nonetheless, it only covers a small section of the total genetic diversity in a species. It is thus advisable to also quantify functional genetic diversity.
One group of genetic markers that can be used to probe functional genetic diversity are immune genes. These genes are expected to experience strong selective pressures due a variety of pathogens. They might thus provide insights into the adaptive potential of a species and its capacity to deal with changing conditions. A recent study in the journal Conservation Genetics focused on one class of immune genes – the Toll-like Receptors (TLRs) – to assess the level of functional genetic diversity in the endangered Antioquia Wren (Thryophilus sernai). The researchers also looked at several neutral genetic markers – microsatellites and the mitochondrial control region – to cover the different aspects of genetic diversity in this species.
Low Genetic Diversity
The Antioquia Wren is a recently described species that can be found in the Cauca river Canyon in Colombia. In recent years, this small passerine species has lost nearly 90% of its habitat, leading to a drastic population contraction to less than 1000 individuals (probably even below 250). The declining population is further threatened by the construction of the Ituango Hydroelectric project which flooded a significant section of its habitat. Conservation efforts might be warranted and it is thus important to understand the adaptive potential of this species in terms of genetic diversity.
Danny Zapata and his colleagues sequenced three genetic markers (microsatellites, mitochondrial control region and TLRs) for 31 individuals. The genetic analyses revealed low levels of genetic diversity in all markers. Hence, the researchers concluded that “these results suggest current low evolutionary potential for the species, as its reduced genetic diversity is expected to increase extinction risk by limiting the ability to cope with environmental changes.” Bad news for the Antioquia Wren.
However, the low genetic diversity of the immune genes might not be a big problem. A previous study on variation in Toll-like Receptors of the endangered Pale-headed Brushfinch (Atlapetes pallidiceps) found that individuals with low genetic diversity at these immune genes had higher survival rates. This finding suggests that this low diversity might be adaptive for the selection regime in a restricted habitat where the birds are exposed to few pathogen species. The current diversity in Toll-like Receptors of the Antioquia Wren could thus be beneficial in its limited distribution.
However, this potential benefit will probably not hold in the long run. The low genetic diversity at these immune genes – and at the neutral markers – diminishes the adaptive potential of this species, making it extremely vulnerable to changing conditions (which can be expected due to further habitat loss and climate change). Conservation efforts will need to be implemented soon.
Zapata, D., Rivera-Gutierrez, H. F., Parra, J. L., & Gonzalez-Quevedo, C. (2020). Low adaptive and neutral genetic diversity in the endangered Antioquia wren (Thryophilus sernai). Conservation Genetics, 21(6), 1051-1065.