Mismatches between mtDNA, nuclear markers and morphology in the Canada Jay

Extensive hybridization in contact zones leads to mitonuclear discordance.

Different genes tend to tell different stories. This phenomenon – known as gene tree discordance – can be particularly obvious when comparing mitochondrial DNA with nuclear markers. Mismatches between these molecular markers can be the outcome of different processes, such as hybridization, incomplete lineage sorting, sex-biased dispersal or natural selection. Regardless of the underlying mechanism, mitonuclear discordance can be problematic in species delimitation, especially when morphological information is insufficient to delineate species. How can you discriminate between two or more species when different molecular markers give you different answers? This issue is relevant for the Canada Jay (Perisoreus canadensis) which consists of three morphotypes that might even interbreed. A recent study in the Biological Journal of the Linnean Society attempted to unravel this complex situation.

Crown Patch

The most widespread morphotype of the Canada Jay – the Boreal morphotype – can be found from Alaska to Newfoundland. The other two morphotypes are more restricted: the Pacific form resides along the coast from California to British Columbia, and the Rocky Mountains form occurs at high elevation in the Rocky Mountains from New Mexico to British Columbia. Apart from their distinct – but overlapping – distributions, these morphotypes show variation in their plumage patterns. Specifically, the Pacific birds have a darker crown patch compared to the Boreal birds, whereas the Rocky Mountain birds have a conspicuous white head. Despite these subtle differences, some intermediate birds have been observed, suggesting the occurrence of hybridization.

An overview of morphological differences between the three morphotypes: (a,d) Pacific, (c,e) Rocky Mountains, and (c,f) Boreal. And their distribution in the west of North America. From: Graham et al. (2021).

Mitonuclear Discordance

Between 1990 and 2018, Brendan Graham and his colleagues collected blood samples from over 600 individuals. Based on several morphological traits, each of these individuals was assigned to one morph. However, 15 individuals showed traits of at least two morphotypes and were classified as intermediates. Next, the researchers took a closer look at the genetic make-up of all birds, using 12 microsatellites and the mitochondrial control region. Both marker types were reasonable successful in discriminating between the three morphotypes. The geographic distribution of the morphotypes was mirrored by the mitochondrial lineages, although some populations contained individuals from multiple lineages. And analyses of the nuclear data pointed to three distinct clusters, corresponding to the three morphotypes. However, in regions of overlap, a large number of individuals (27%) were assigned to a genetic cluster that did not match their morphology.

Moreover, no less than 222 individuals showed mismatches between the mitochondrial and nuclear assignments. Mitonuclear discordance was most prominent in sympatric areas, such as southern British Columbia and northern Washington, Wyoming and Utah. The most likely explanation for these patterns seems extensive hybridization after a period of isolation.

Distribution of mtDNA, microsatellite genetic cluster and combined mtDNA and microsatellite genotypes among the three morphotypes and intermediate morphotypes. The first four categories (Pacific, Intermountain West-IMW, Boreal and Southeast) represent individuals that showed congruent patterns between mtDNA and microsatellites. The fifth category (Cyto-ND) represents individuals that exhibited mitonuclear discordance. For each of the main morphotypes, the expected genetic group is marked with an asterisk. From: Graham et al. (2021).

Plumage Genes

The mismatches between morphology and the genetic markers in this study suggest that the plumage patterns in the morphotypes are controlled by a few genetic loci. Similar patterns have been uncovered in several other bird groups, such as wagtails, woodpeckers and warblers. The authors note that “analysis with next generation sequencing techniques may help uncover the genes associated with plumage variation in Canada Jays.” And when they manage to perform these analyses, you will definitely read about it on the Avian Hybrids blog.


Graham, B. A., Cicero, C., Strickland, D., Woods, J. G., Coneybeare, H., Dohms, K. M., Szabo, I. & Burg, T. M. (2021). Cryptic genetic diversity and cytonuclear discordance characterize contact among Canada jay (Perisoreus canadensis) morphotypes in western North America. Biological Journal of the Linnean Society132(4), 725-740.

Featured image: Canada Jay (Perisoreus canadensis) © Mdf | Wikimedia Commons

Isolated in Iberia: Unraveling the origin of an Iberian Rook population

Is it an ancestral population or the result of a recent expansion?

During my postdoc in the Swedish city Uppsala, I would regularly visit a colony of Rooks (Corvus frugilegus) close to the city center. Their heavy, greyish beaks always fascinated me and I enjoyed observing their social interactions. If you want to find Rooks, you do not have to travel specifically to Sweden. This corvid species has a wide distribution, stretching from western Europe into eastern Siberia. Interestingly, there is a small, isolated population of Rooks on the Iberian peninsula (i.e. Spain and Portugal). The origin of this population is still largely unknown. In general, there are two possible explanations. Perhaps the Iberian birds are the descendants from a larger population that survived in the south of Europe during the glacial cycles of the Pleistocene. As Europe was covered by thick ice sheets, Iberia functioned as a glacial refugium. When the ice retreated, Rooks recolonized Europe from this refugium. Another possibility is unrelated to the ice ages, but entails habitat fragmentation from a much wider distribution. The Iberian population might have been split off from a bigger population in Europe. A recent study in the Journal of Avian Biology relied on genetic data to discriminate between these scenarios.


Both explanations – glacial refugium or fragmented population – lead to specific predictions about the expected genetic patterns. From a phylogenetic point of view, the glacial refugium scenario would give rise to a star-like phylogeny with the Iberian individuals in the center. Rook populations in western Europe would then represent subsamples from the ancestral population that expanded out of Iberia, giving rise to different rays of the star-like pattern. In addition, the European Rook populations would show genetic signatures of recent population expansion (which can be captured with several statistical tests). Finally, genetic diversity should decrease from south to north, as each expansion involves a genetic bottleneck. The expectations for the fragmented population scenario are drastically different. On a phylogenetic tree, the Iberian individuals would represent a small cluster within a larger European clade. There would be no signs of population expansion in Europe, and a significant decrease in genetic diversity in the Iberian population.

The wide distribution of Rooks, including the isolated population in Iberia. From: Salinas et al. (2021).

Population Expansion

Pablo Salinas and his colleagues tested these predictions using mtDNA and several nuclear markers. Let’s have a look at the patterns that emerged from their analyses. First, the haplotype network of the mitochondrial markers consisted of a central core of Iberian samples, surrounded by several rare haplotypes from European populations. This star-like pattern points to population expansions from an ancestral population in Iberia. This interpretation was supported by Fu’s and Tajima’s neutrality test, which was consistent with a recent population expansion. To remove all doubt, explicit scenario testing with coalescent modelling also indicated that the European population diverged from an ancestral Iberian population. Hence, the glacial refugium scenario seems most likely.

Haplotype networks of the two mitochondrial markers (B = ND2 and C = control region) show a clear star-like pattern with the Iberian samples (dark purple) in the center. From: Salinas et al. (2021).

Genetic Gradient

However, the pattern of genetic diversity did not fit with the glacial refugium scenario. In this scenario, we expected that the level of genetic diversity would decrease from south to north, but in reality there was no such gradient. This result can be explained by the isolated nature of the Iberian population and the vastness of the European populations. Indeed, the authors indicate that “Isolated populations are also expected to have low genetic diversity because of limited gene flow with other populations, as previously argued for the small breeding rook population in the Iberian Peninsula. This is in contrast to the widespread populations in northern Europe, which have been able to maintain large effective population sizes and thus increase genetic diversity through the persistence of recent mutations.”

All in all, we now have several lines of genetic evidence to argue that the Iberian population represents the remnant of an ancient ancestral population. Moreover, the glacial refugium scenario is further supported by fossil data. Paleontologists have found Rook fossils in early Pleistocene rocks from Iberia, indicating that this species was present there before the last glacial maximum. Isn’t it wonderful when everything converges upon the same explanation?


Salinas, P., Morinha, F., Literak, I., García, J., Milá, B., & Blanco, G. (2021). Genetic diversity, differentiation and historical origin of the isolated population of rooks Corvus frugilegus in Iberia. Journal of Avian Biology52(3).

Featured image: rook (Corvus frugilegus) © Hobbyfotowiki | Wikimedia Commons

The genetic basis of tool use in New Caledonian and Hawaiian Crows

Genomic analyses uncover subtle genetic changes.

On the Pacific archipelago of New Caledonia, you can observe an excellent example of tool use in birds. The local crow species – the New Caledonian Crow (Corvus moneduloides) – collects and manufactures tools for foraging. This behavior has resulted in several morphological adaptations, such as a straight bill to better handle the tools and large eyes to facilitate coordination during tool use. In addition, some studies reported unusually large brains in these birds, probably associated with increased cognitive abilities (although other studies could not replicate these findings). For a long time, it was thought that New Caledonian Crows were the only natural tool users in the genus Corvus. Until the Hawaiian Crow (C. hawaiiensis) showed its tool-using skills to the scientific community. These two species are not closely related, suggesting that tool use evolved convergently. An excellent opportunity to unravel the genetic basis of this behavior. A recent study in the journal Molecular Ecology sequenced the genomes of these species (along with 10 other crow species) to tackle this challenge.

Positive Selection

A large team of international researchers joined forces and used several approaches to detect genes under positive selection in the New Caledonian Crow and the Hawaiian Crow. One approach (the dN/dS ratio) relies on the ratio between nonsynonymous and synonymous substitutions. As I explained in a previous blog post, nonsynonymous substitutions lead to a change in the protein sequence (i.e. another amino acid) whereas synonymous substitutions do not due to the redundancy in the genetic code. A higher number of nonsynonymous substitutions suggests positive selection and it apparent in a dN/dS ratio larger than one. The second approach – the McDonald-Kreitman test – uses a similar calculation by comparing the amount of variation within a species to the divergence between species at sites with synonymous and nonsynonymous substitutions. Finally, the third approach focuses on selective sweeps, i.e. the situation in which a genetic variant is beneficial and increases in frequency. The resulting selection event leads to a reduction in genetic diversity in the genomic region where this beneficial variant resides. Several summary statistics, such as Tajima’s D and Fay & Wu’s H, can pick up these signatures of selection.

The first two approaches uncovered 26 genes under positive selection (12 in the dN/dS ratio test and 14 in the McDonald-Kreitman test). These genes play a role in the development of the brain, the nervous system and the eye. The selective sweep search pointed to 11 genomic regions that contained 350 genes, some of which are known to be involved in the evolution of bill morphology (e.g., CALM1). A nice set of candidate genes that require further research.

An overview of the phylogeny and distributions of the Corvus species in the study. The tool using species are highlighted in yellow. From: Dussex et al. (2021) Molecular Ecology.

Reference Genomes

Despite the detection of several genes under putative positive selection, the researchers remain careful and write that “genetic changes associated with tool use in crows appear subtle.” Most positively selected genes seem to be involved in morphological changes in the beak and the eye (as indicated above). It is possible that other important adaptations – such as cognition – are not due to changes in protein-coding genes, but rather related to differences in gene expression (see for example this recent study). Moreover, some adaptive changes might be underpinned by several genes with small effects, making them harder to detect.

The analyses also revealed an important methodological issue. The use of different reference genomes – either the New Caledonian Crow or the Hooded Crow (C. cornix) – resulted in the detection of different candidate genes under selection. This finding highlights the danger of using a single reference genome in population genomic analyses. Indeed, another recent paper in Molecular Ecology showed how the choice of a reference genome can significantly impact analyses on demography and genetic diversity. Hence, the researchers of the crow study provide some important advice: “Ideally, reference genomes should be assembled for each species under consideration along with population genomic data to also account for within-species variation.” A few years ago, such an approach would seem unaffordable. However, the genomic resources for avian studies are accumulating rapidly. And so are the tools to use them.


Dussex, N., Kutschera, V. E., Wiberg, R. A. W., Parker, D. J., Hunt, G. R., Gray, R. D., Rutherford, K., Abe, H., Fleischer, R. C., Ritchie, M. G., Rutz, C., Wolf, J. B. W. & Gemmell, N. J. (2021). A genome‐wide investigation of adaptive signatures in protein‐coding genes related to tool behaviour in New Caledonian and Hawaiian crows. Molecular Ecology30(4), 973-986.

Featured image: New Caledonian Crow (Corvus moneduloides) © Natalie Uomini | Flickr

Ghost populations explain how the Red-billed Chough reached the Canary Islands

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.

Colonization Scenarios

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.

Different island colonization scenarios for the Red-billed Chough (a) Current distribution of Red-billed Choughs in Iberia, inland Morocco and La Palma (Canary Islands), with sampling sites indicated by star symbols. (b) The long-distance colonization hypothesis proposes that choughs colonized La Palma through a transoceanic flight from Iberia or from current populations in inland Morocco. (c-d) The ghost population hypothesis proposes that colonization of La Palma Colonization from a coastal Morocco (ghost) population that has since gone extinct as habitat desertified and became unsuitable for choughs. From: Morinha et al. (2020) Journal of Biogeography.

Genetic Evidence

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:

  1. Red-billed Choughs are non-migratory and do not disperse far (a few 100 kilometers at most)
  2. These is no fossil evidence of choughs on other islands, such as the Azores or Madeira, that lie between Iberia and the Canary Islands.
  3. 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.

An overview of the different models to explain the colonization history of La Palma by the Red-billed Chough. The scenario with the highest probability (figure d) suggests a ghost population (black) connecting Iberia (orange) with La Palma (blue). From: Morinha et al. (2020) Journal of Biogeography.


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 Biogeography47(12), 2567-2583.

Featured image: Red-billed Cough (Pyrrhocorax pyrrhocorax) © Malte Uhl | Wikimedia Commons

How the Hooded Crow got its hood: A tale of two crows and a transposable element

Structural variants provide another clue to the genetic basis of plumage color in crows.

Turning an all-black Carrion Crow (Cornix c. corone) into a grey Hooded Crow (C. c. cornix) might be as easy as flipping a genetic switch. Extensive studies of a European hybrid zone between these species have uncovered many details about the genetic underpinnings of these plumage patterns. Let me quickly recap our understanding so far. A genome-wide association study found three genomic regions associated with plumage color: a big region on chromosome 18 and two smaller regions on chromosomes 1 and 1A. The region on chromosome 1 contains the candidate gene NDP, which also regulates plumage patterns in pigeons. Differential expression of this gene in developing feathers could thus explain the evolution of different plumage patterns in both pigeons and crows. A recent study in the journal Nature Communications might have found the mechanism that explains this differential gene expression in crows.


Structural Variants

Matthias Weissensteiner and his colleagues decided to take a closer look at structural variation in the genomes of several crow species. Structural variation refers to a panoply of mutations, such as deletions, insertions, duplications and inversions (you check out this blog post on the role of inversions in avian evolution). These types of mutations have been very difficult to characterize because you need highly contiguous genome assemblies that have only recently became available. Indeed, most bird genome assemblies are far from complete. Using the latest technologies in genome sequencing, the researchers managed to generate high-quality, contiguous assemblies for several crow species. The search for structural variants can begin.

An overview of the different crow species in the study. The numbers indicate the technologies used to generate the sequences: short read (SR), long read (LR) and optical mapping (OM). From: Weissensteiner et al. (2020) Nature Communications.



The analyses resulted in a total of of 220,452 insertions, deletions and inversions. I will not discuss them all. That would result in a very long and boring blog post. Instead, I will focus on one particular insertion: a LTR retrotransposon on chromosome 1. Retrotransposons are a type of genetic parasites that jump through the genome using a copy-and-paste mechanism. The LTR in their name stands for “Long Terminal Repeats” because these genetic sequences are flanked by long stretches of repetitive DNA. This particular LTR retrotransposon inserted itself about 20,000 nucleotides from the gene NDP (you can feel where this is going).

It turned out that all Hooded Crows in the study were homozygous for the LTR retrotransposon (i.e. they carried the insertion on both copies of chromosome 1). This observation suggests that there has been strong selection for this insertion in the Hooded Crow population. Could it be related to the activity of NDP? Further analyses confirmed this hunch: the expression of NDP was significantly lower in birds that were homozygous for the insertion. It thus seems that the insertion of the LTR retrotransposon affected the expression of NDP, giving rise to the hooded phenotype. This plumage pattern consequently came under strong sexual selection because crows prefer to mate with birds of the same plumage type. Another piece in the plumage pattern puzzle.

The insertion of the LTR retrotransposon (figure a) is homozygous in hooded crows (figure b) and affects the expression of the NDP-gene (figure c). From: Weissensteiner et al. (2020) Nature Communications.



Weissensteiner et al. (2020). Discovery and population genomics of structural variation in a songbird genus. Nature communications11(1), 1-11.

Featured image: Hooded Crow in Berlin © Pelican | Wikimedia Commons


This paper has been added to the Corvidae page.