Two American sparrows with similar mtDNA: Rapid speciation or hybridization?

Analyses of the nuclear genome provide the answer to this question.

An important aspect of the scientific process is considering and testing several explanations. Imagine, for example, that you sequence the mitochondrial DNA of two bird species. When you compare the genetic sequences, you notice that the two species hardly differ. What could explain this pattern? In scientific papers, you will generally come across two possible solutions: (1) the two species have only recently diverged and still share some genetic variation, or (2) the two species have hybridized and mitochondrial DNA flowed from one species into the other. In technical terms, these explanations are known as incomplete lineage sorting (see this blog post for more details on this concept) and introgressive hybridization.

The mitochondrial situation that I just described applies to the Golden-crowned Sparrow (Zonotrichia atricapilla) and White-crowned Sparrow (Z. leucophrys). In 2001, Jason Weckstein and his colleagues reported “extraordinarily low levels of sequence divergence between Z. leucophrys and Z. atricapilla, despite distinct plumage, song, and allozymes.” Moreover, several mitochondrial haplotypes were shared between these species. Although they could not rule out rapid speciation (and thus incomplete lineage sorting), they hypothesized that introgression was the most likely explanation. Recently, a study in the journal Molecular Phylogenetics and Evolution put this hypothesis to the test by exploring the nuclear DNA of these sparrows. Undifferentiated nuclear genomes would suggest rapid speciation, while distinct nuclear genomes in combination with shared mtDNA would point to introgression.

Phylogenetic Trees

Based on 73 specimens of the two species, Rebecca Taylor and her colleagues estimated phylogenetic trees for two mitochondrial genes (COI and the control region) and several thousands of nuclear genetic variants. In line with previous work, the species shared mitochondrial haplotypes and could not be separated into distinct clusters. The nuclear analyses, however, uncovered clear differences between the Golden-crowned Sparrow and White-crowned Sparrow. These contrasting patterns suggest introgression of mitochondrial DNA.

This explanation was supported by additional analyses that revealed ancient gene flow between these species. Using the software TreeMix, for instance, the researchers detected introgression from Golden-crowned into White-crowned Sparrow. And the commonly used D-statistic further corroborated this introgression event (see this blog post for an explanation how this test works). Clearly, introgressive hybridization seems to be the most likely explanation.

Phylogenetic analyses of the mitochondrial genes (left figure) could not separate the two species into distinct groups, whereas the nuclear variants uncovered clear clusters (right figure). The Golden-crowned Sparrow is depicted in red, the other colors represent subspecies of the White-crowned Sparrow. From: Taylor et al. (2021).

Adaptive or Neutral Introgression?

Answering one question mostly leads to a bunch of other mysteries. Now that we know about introgression of mitochondrial DNA, we can move on to the next aspect: was the introgression driven by neutral or selective processes? Currently, we don’t have enough information to answer this question. However, the researchers do speculate about a possible scenario:

While it is only informed speculation, populations of Z. atricapilla and Z. leucophrys could have been forced into common refugia by ice sheets and shifting vegetation during the Pleistocene, leading some individuals to seek out heterospecific mates. Several rounds of bottlenecking due to climate change might have occurred during this period and similarly caused the fixation of one haplotype in both species during periods when population sizes were small. Selection may have acted in combination with periods of small population sizes and augmented this process, if one mitochondrial haplotype conferred a fitness advantage.

This certainly sounds like a plausible scenario, but it remains to be tested with more detailed analyses. The mitochondrial mystery of the Golden-crowned Sparrow and the White-crowned Sparrow continues…

References

Taylor, R. S., Bramwell, A. C., Clemente-Carvalho, R., Cairns, N. A., Bonier, F., Dares, K., & Lougheed, S. C. (2021). Cytonuclear discordance in the crowned-sparrows, Zonotrichia atricapilla and Zonotrichia leucophrys. Molecular Phylogenetics and Evolution, 162.

Featured image: Golden-crowned Sparrow (Zonotrichia atricapilla) © V. J. Anderson | Wikimedia Commons

Quantitative genetic analyses of Song Sparrows reveal the dark side of gene flow

Genetic variation from immigrant birds contributes to lower juvenile survival.

Gene flow is always good, right? The influx of individuals from neighboring regions leads to more genetic diversity, allowing the population to cope better with environmental changes. It sounds wonderful. The reality, however, is not that straightforward. Gene flow can also have negative effects, as nicely shown by a recent paper in the journal Evolution Letters. The researchers took a quantitative genetic approach to study juvenile survival in a population of Song Sparrows (Melospiza melodia). Before we delve into the findings of this paper, we need a crash course in the mathematics behind quantitative genetics. Take a deep breath and continue reading.

Quantitative Genetics

Most phenotypic traits show a range of variation. With regard to juvenile survival, for example, some individuals will die quickly while others make it into adulthood. This phenotypic variation (V_P) is determined by genetic factors (V_G), environmental factors (V_E) and the interaction between genetics and environment (V_EG). Or if we put into a formula:

V_P = V_G + V_E + V_EG

Next, let’s focus on the genetic component (V_G). This part of the equation can be divided into three terms. The first term – additive genetic variance (V_A) – captures the effect of an allele on a particular phenotype, causing it to deviate from the population mean. For example, imagine that a gene occurs in two variants: A and B. Individuals with variant A might show survival rates above the mean, while individuals with variant B survive lower than average. This variation can be captured in the term V_A. The other two terms concern interactions between alternative alleles or different genes. The term V_D focuses on dominance effects, such as variant X being dominant over variant Y. And the term V_I captures interactions between different genes. These terms are less important for this blog post, but I mention them to provide the entire picture. Putting it all together gives this formula:

V_G = V_A + V_D + V_I

Splitting it up

Now that we have covered the basics of quantitative genetics, we can explore the findings of this study. The researchers focused on the additive genetic variance (V_A) of juvenile survival. The value of V_A can provide insights into the average fitness in a population. High values suggest plenty of genetic variation available for adaptation, while low values point to possible constraints. Scientists can calculate V_A for particular phenotypes using mathematical “animal models”. I will not go into the details of these models, but you can check this paper for more information. Most models calculate V_A for the entire population without taking into account potential population structure. Here, the new study comes into play. Jane Reid and her colleagues decided to split V_A into two parts: the genetic variance in the local population (a_i) and the genetic variance from immigrant birds (q_i). To quantify the genetic difference between local birds and several immigrant populations, the researchers added a factor g to the mix. To put it into a formula (the last one, I promise):

V_A = a_i + g.q_i

Migration-Selection Balance

With all the ingredients in place, we can finally look at the results from the analyses. When focusing on the local population, the breeding value for juvenile survival (a_i) increased over time (between 1993 and 2018). In other words, juveniles with local parents had a higher chance of surviving into adulthood. The situation was drastically different from the immigrants. The contribution of immigrant genes lead to decreased breeding values (q_i) over the years. This means that juveniles with an immigrant parent had a lower chance of reaching adulthood. The two effects – from the local population and from immigration – counteracted each other, resulting in a stable value for the total additive genetic variance (V_A). This situation can be seen as a migration-selection balance where alleles from other populations are removed from the population through the low survival of juveniles.

The breeding values for the local population increase over time (figure A), while the values from immigrants decrease (figure C). Both effects balance each other out in the total additive genetic variance (figure E). Red dots indicate the arrival immigrant birds. From: Reid et al. (2021).

Overestimation

This study nicely shows the potential negative consequences of gene flow. Genetic variation from immigrant birds leads to lower juvenile survival. This effect would not have been apparent if the researchers had not discriminated between local birds and immigrants. Indeed, analyses without the immigrant effect resulted in an overestimation (by 47%) of the additive genetic variance for juvenile survival. The exact mechanisms behind the immigrant effect remain to be determined, but could be related to local adaptation or the dispersal of low-quality individuals. Regardless of the underlying mechanism, these findings highlight the importance of taking population structure into account when running animals models. More accurate fitness estimates will help us better understand the evolutionary changes in wild bird populations.

References

Reid, J. M., Arcese, P., Nietlisbach, P., Wolak, M. E., Muff, S., Dickel, L., & Keller, L. F. (2021). Immigration counter‐acts local micro‐evolution of a major fitness component: Migration‐selection balance in free‐living song sparrows. Evolution Letters, 5(1), 48-60.

Featured image: Song Sparrows (Melospiza melodia) © Frank Schulenburg | Wikimedia Commons

Demography or selection: What determines the hybridization dynamics between Saltmarsh Sparrow and Nelson’s Sparrow?

The importance of different processes seems to vary per location.

Hybridization often leads to introgression, the exchange of genetic material between the interacting species. The resulting patterns of introgression are determined by the complex interplay between numerous factors. First, local population sizes can affect hybridization rates. When one species is rare, its members will have more difficulty finding a mate and they might settle with a partner from another species. This phenomenon – known as Hubb’s Principle – has been nicely illustrated on the Falkland Islands where a numerical imbalance between Speckled Teal (Anas flavirostris) and Yellow-billed Pintails (Anas georgica) led to hybridization. Next, selection on hybrids might play a role. The selection pressure might be endogenous, affecting the fertility or viability of the hybrids. Or hybrids might have to deal with exogenous selection when they are badly adapted to local environmental conditions. Disentangling all these different factors is a challenging endeavor. But that did not stop Logan Maxwell and colleagues from studying how neutral and selective processes determine introgression patterns between Saltmarsh Sparrow (Ammospiza caudacuta) and Nelson’s Sparrow (Ammospiza nelsoni). Their findings recently appeared in the journal BMC Ecology and Evolution.

Genotypic Distributions

The researchers genotyped more than 500 birds at two locations (coastal and inland) along the coast of New England. The genetic data allowed them to determine the number of “pure” individuals, first-generation hybrids and backcrosses. If introgression is mainly determined by neutral processes, such as local population sizes, you would expect the distribution of genotypes to follow a random pattern. This was not the case: the coastal site had less backcrossed Saltmarsh Sparrows than expected while at inland site there were more hybrids, backcrosses and “pure” Saltmarsh Sparrows than predicted. These findings suggest that neutral demographic processes are not sufficient to explain introgression rates in this hybrid zone. Selection plays a role as well.

The distribution of genotypes (in grey) differed from the expected distribution based on neutral processes (in black), indicating that selection probably plays a role in this hybrid zone. From: Maxwell et al. (2021) BMC Ecology and Evolution.

Selection Pressures

But is the selection on hybrids endogenous or exogenous? The results suggest that both types of selection are at work. The researchers detected a reduction in hybrid females among the adults. Because there was no difference in viability of females at the egg stage, it seems that hybrid females have a lower chance of survival. This pattern is in accordance with Haldane’s Rule which states that in a hybrid cross the sex with two different sex chromosomes (i.e. the female in birds) will suffer the greatest fitness reduction. In addition, there were more individuals with Saltmarsh Sparrow DNA at the coastal site, suggesting that genetic variants from this species provide an adaptive advantage in that area (see also this blog post). There is thus exogenous selection against Nelson’s Sparrows at the coast.

The distribution of genotypes for males (white) and females (grey) at the two sites reveals more individuals with Saltmarsh Sparrow DNA at the coast, suggesting selection for these genetic variants. From: Maxwell et al. (2021) BMC Ecology and Evolution.

Mating Patterns

Finally, the researchers tested whether the birds mated assortatively in the hybrid zone (i.e. finding a partner of the same species). In general, this was certainly the case: the majority of mating events (79%) occurred between the same species. However, the patterns differed by site. Assortative mating was strong at the coastal site, but not at the inland site. The inland population is smaller which could increase the frequency of hybridization there. The exact mechanisms underlying mate choice remain to be determined, but could be related to differences in song and mating behavior.

All in all, the hybridization and introgression dynamics between Saltmarsh and Nelson’s Sparrow are determined by the complex interplay of numerous factors which differ between locations. The authors nicely summarized the situation at the beginning of the discussion-section:

We found that neutral demographic factors—relative abundances of the two species—alone could not explain the observed patterns of introgression between Saltmarsh and Nelson’s Sparrows and that spatial variation in the distribution of parental and offspring genotypes was a result of both exogenous and endogenous selective forces. In addition, sexual selection played a role in maintaining species boundaries through assortative mating. However, these patterns differed on the coastal and inland site, suggesting local differences in the strength of selection.

References

Maxwell, L. M., Walsh, J., Olsen, B. J., & Kovach, A. I. (2021). Patterns of introgression vary within an avian hybrid zone. BMC Ecology and Evolution, 21(1), 1-18.

Featured image: Nelson’s Sparrow (Ammospiza nelsoni) © Andy Reago & Chrissy McClarren | Wikimedia Commons

Divergent sperm morphology as a reproductive barrier between Saltmarsh and Nelson’s Sparrow

But is the difference large enough to interfere with fertilization?

Along the east coast of North America, two small songbirds are interbreeding: the Saltmarsh Sparrow (Ammospiza caudacuta) and the Nelson’s Sparrow (A. nelsoni). Despite high levels of interspecific gene flow, these species remain largely distinct. What reproductive isolation mechanisms could prevent these sparrows from merging into one species? One possible explanation concerns adaptation to different ecological conditions: the Saltmarsh Sparrow is restricted to coastal marshes, whereas the Nelson’s Sparrow can be found in a larger variety of habitats. Hybrids might not be able to survive in certain environments. Another putative reproductive barrier could be related to differences in mating behavior. Male Nelson’s Sparrows attract females by singing and performing song flights. After a successful copulation, they tend to guard the female for a short time. Male Saltmarsh Sparrows, on the other hand, do not perform such courtship displays, but rather chase females around. Hybrids might show intermediate behaviors and will not be able to secure a mate. A recent study in the journal Ecology and Evolution examined a third reproductive barrier: sperm morphology.

Sperm Size

Female birds store sperm in specialized organs (so-called tubules). If the morphology of the sperm does not match the shape of these organs, the sperm will not be stored and can thus not be used for fertilization. If the differences in sperm morphology are small enough, this issue can be overcome and might lead to the production of hybrids. However, the resulting hybrids might experience fertility issues. The sperm cells of hybrids might be abnormally shaped and not functional, leading to sterile males (as in Flycatchers). Or hybrids can have perfectly viable sperm cells that are unable to fertilize the egg due to genetic mismatches (which is possibly the case in Long-tailed Finches). To check the situation in the Ammospiza Sparrows, Emily Cramer and her colleagues studied the sperm morphology of nine Saltmarsh Sparrows, nine Nelson’s Sparrows and four intermediate birds.

The data collection revealed that the sperm of the putative hybrids was fine. They did not show any abnormal sperm cells and are thus most likely fertile. In fact, the intermediate birds produced more sperm than both parental species (I will come back to this intriguing observation later on). But what about the sperm morphology? The analyses showed that the sperm cells of Saltmarsh Sparrows were about 4 percent longer than those of Nelson’s Sparrows. Whether this difference is sufficiently large to interfere with fertilization remains to be investigated. Indeed, the researchers indicate that a study of the female reproductive tract will be a logical next step.

There was a clear difference in the size of sperm cells between both species (top figure). Interestingly, the size of the sperm cells correlated nicely with the plumage scores of the birds (bottom figure). From: Cramer et al. (2021) Ecology and Evolution.

Plumage Scores

The researchers also noted a strong correlation between the plumage score of the birds and the size of the sperm cells. They speculate that this result could reflect some fitness consequences for the male birds. Plumage might reflect the sperm phenotype, allowing female birds to better judge males and obtain compatible sperm. This connection between a male’s phenotype and his fertility has been suggested by Ben Sheldon. He focused on variation within species, but this mechanism might extend across species boundaries. If females do indeed focus on plumage patterns to select males, the hybrids might be at a disadvantage due to their intermediate plumages. Hybrids will then copulate less, which might explain the higher sperm count in this study. The intermediate birds had not copulated recently, resulting in fuller sperm stores. The authors summarize the situation nicely in the discussion: “intermediate males suffer from reduced copulation success, but not reduced fertilization success”.

References

Cramer, E. R., Grønstøl, G., Maxwell, L., Kovach, A. I., & Lifjeld, J. T. (2021). Sperm length divergence as a potential prezygotic barrier in a passerine hybrid zone. Ecology and Evolution, 11: 9489-9497.

Featured image: Nelson’s Sparrow (Ammospiza nelsoni) © Andy Reago & Chrissy McClarren | Wikimedia Commons

A gene within a supergene: An estrogen receptor shapes the behavior of White-throated Sparrow morphs

Expression levels of the estrogen receptor determine aggressive behavior in these songbirds.

White-throated sparrows (Zonotrichia albicollis) come in two distinct morphs: the white-striped (WS) and the tan-striped morph (TS). These morphs do not only differ in their plumage patterns, but also in behavior, such as the degree of parental care that they provide (which I discussed in this blog post). The differences between these morphs have a solid genetic basis. Already in 1966, Thorneycroft identified a chromosomal rearrangement that explains the occurrence of the two white-throated sparrow morphs. Recent molecular work showed that this rearrangement is an inversion (i.e. a flipped section of DNA, more on inversions in this blog post), giving rise to a so-called supergene which links numerous genes that influence the morphology and behavior of these birds. Tan morphs have the same version of the supergene (i.e. they are homozygous) whereas white-striped morphs have two different versions (i.e. they are heterozygous).

Knowing that a supergene underlies the different morphs is only the first step. Now, we can zoom in on the contents of this supergene and determine how these linked genes work together in shaping the plumage and behavior of white-throated sparrows. A recent study in the journal PNAS performed some clever experiments to understand the role of one particular gene.

The different morphs of the White-throated Sparrow (A and B) prefer to mate with the opposite morph (see percentages in C). The differences between the morphs can be traced back to a super-gene (D). From: Campagna (2016) Current Biology.

Estrogen Receptor

As mentioned above, the white-striped and the tan-striped morphs behave differently. Studies in wild populations found that WS birds are more aggressive compared to TS birds when defending their territories. Given that territorial aggression in songbirds has been linked to steroid hormones, it makes sense to search for genes that are involved in the production or processing of these hormones. Interestingly, one of the genes (ESR1) in the supergene codes for an estrogen-receptor. Moreover, this gene comes in two different versions (ZAL2 and ZAL2m) that follow the genetic patterns underlying the two morphs. Tan morphs have the same version of gene (two times ZAL2) whereas white-striped morphs have two different versions (ZAL2 and ZAL2m). Sounds like the perfect candidate gene!

The researchers quantified the level of aggression of different birds in several behavioral trials. Next, they measured the expression levels of the different ESR1-versions in certain brain areas. They summarized their findings as follows: “the degree to which a bird engaged in territorial aggression, which was markedly higher in the WS birds than in the TS birds, was predicted by the relative expression of the ZAL2m allele.” In another experiment, the researchers knocked down the expression of the ESR1-gene in certain brain areas and assessed the aggression of the birds. This experiment revealed that the more aggressive birds became less aggressive when the ESR1-gene was turned off.

One allele (ZAL2m, in red) was more highly expressed in white-striped morphs, and correlated with aggressive behavior (measures as the number of songs per 10 minutes). These results support a central role for the estrogen receptor in shaping the behavior of the morphs. From: Merritt et al. (2020) PNAS.

Gene Network

These findings provides direct evidence that the estrogen-receptor plays a crucial role in determining the behavior of these morphs. However, it remains to be determined how it actually works. This receptor is a transcription factor that interactions with a large number of other proteins as well as with numerous regulatory elements. A previous study reported that ESR1 lies within an interconnected module of 157 genes that are differentially expressed between the morphs. Of these 157 genes, 115 are located in the supergene. More experimental work is needed to disentangle this complex web. Slowly but steadily we are getting closer to the genetic underpinnings of these intriguing morphs.

References

Merritt, J. R., Grogan, K. E., Zinzow-Kramer, W. M., Sun, D., Ortlund, E. A., Soojin, V. Y., & Maney, D. L. (2020). A supergene-linked estrogen receptor drives alternative phenotypes in a polymorphic songbird. Proceedings of the National Academy of Sciences, 117(35), 21673-21680.

Featured image: White-throated sparrows (Zonotrichia albicollis) © Cephas | Wikimedia Commons

Admixture in Amazonia: Reconstructing the evolutionary history of the Pectoral Sparrow

Genomic data tell the story of how this passerine spread across South America.

Apart from managing the Avian Hybrids Project, I regularly contribute to the blog of the British Ornithologists’ Union (the BOUblog, you can find an overview of my blog posts here). A few weeks ago, I published my 50th story for the BOUblog, which focused on the phylogenetics of the Pectoral Sparrow (Arremon taciturnus). A recent study used mitochondrial DNA to unravel the evolutionary history of this neotropical species. The researchers uncovered six distinct lineages and speculated about the factors responsible for their origins. Here is my summary:

Could it be that the origin of these rivers drove the diversification of the Pectoral Sparrow? Not exactly, because these rivers started crisscrossing the South American landscape between 9 and 2.5 million years ago. The rivers certainly prevent neighboring populations from mixing extensively, but they are not the main cause for the origin of these lineages. Rather, the researchers suspect that ‘past ecological barriers must have played a role in accounting for the observed phylogeographical structure.’ During the glacial cycles of the Pleistocene, forests contracted and expanded. The Pectoral Sparrows became isolated in forest fragments during contraction phases and followed the spreading forests during the expansions. At rivers, however, the birds could not disperse further, giving rise to the geographical boundaries between the six lineages. Amazing what you can deduce from a string of A, T, G and Cs.

Although this scenario seems plausible, it remains largely speculative. Indeed, the authors indicated the uncertainties in their proposed model and wrote that “alternative scenarios could be tested with more powerful genomic datasets.” Luckily, another recent paper did just that. Using genomic data, Nelson Buainain and his colleagues provided a more fine-grained picture of the evolution of the Pectoral Sparrow.

Analyses of mitochondrial DNA indicated six distinct lineages within the Pectoral Sparrow. From: Carneiro de Melo Moura et al. (2020) Ibis.

Genotypes and Phenotypes

In contrast to the six mitochondrial lineages, the genomic data suggested four main genetic clusters. The genetic make-up of these four groups reveals an interesting pattern. Individuals from the Guyana Shield (region A in the figure above), southwestern Amazonia (region F) and the Atlantic Forest (region D) generally have “pure” genotypes. In other words, they do not share genetic variation with other regions. In central Amazonia (regions B, C and E), however, individuals have admixed genotypes.

The genetic patterns are mirrored in the plumage of the birds. In the genetically “pure” populations, pectoral band patterns are mostly homogenous, whereas the admixed populations show a variety of shapes and sizes in the pectoral bands. The most likely scenario is that populations became isolated in different forest patches across central Amazonia and established secondary contact, giving rise to the genetic and morphological variety we see today. This interpretation was further supported by ecological niche modelling.

Genetic and morphological variation across the range of the Pectoral Sparrow. Notice the admixed nature of the populations in central Amazonia. From: Buainain et al. (2020) Molecular Ecology.

North to South

The genetic analyses indicated that the populations north and south of the Amazon separated about 160,000 to 380,000 years ago. The higher genetic diversity in the northern populations of the Guyana Shield suggest that the Pectoral Sparrow started its journey across South America here. As birds spread to the south, they separated into distinct populations, settling in the patches of suitable habitat. These populations were probably separated in at least three regions south of the Amazon River, namely southwestern Amazonia and south‐central Amazonia and the Atlantic Forest. These regions functioned as refugia during harsh climatic periods when forest fragments were isolated. Occasionally, climatic changes would cause these forests to expand, resulting in the secondary contact that I described above. This scenario nicely builds upon the patterns that arose from the mitochondrial study. Step by step, we are unraveling the complex evolutionary history of South American birds.

References

Buainain, N., Canton, R., Zuquim, G., Tuomisto, H., Hrbek, T., Sato, H., & Ribas, C. C. (2020). Paleoclimatic evolution as the main driver of current genomic diversity in the widespread and polymorphic Neotropical songbird Arremon taciturnus. Molecular Ecology, 29(15), 2922-2939.

Featured image: Pectoral Sparrow (Arremon taciturnus) © Caio Brito | eBird