The role of interspecific gene flow in species diversification has long been debated and is increasingly appreciated. However, the effect of gene introgression on phenotypic divergences and genome heterogeneity remain unclear in case of early speciation. To investigate these questions Poelstra and colleagues studied the hybrid zone between the all-black carrion crows (Corvus corone) and the gray-coated hooded crows (C. cornix). Indeed, the absence of neutral genetic diversity between these two species and successful back-crossing of hybrids strongly contrast with the plumage coloration polymorphism that remained stable in natural populations. Moreover, colour assortative mating has been observed suggesting a prezygotic isolation and ongoing speciation. To investigate the causes of this stable phenotypes the authors first analysed the effect of gene flow on genome heterogeneity and then tried to link the observed gene flow heterogeneity with gene expression and phenotypes.
Genetic differentiation between hooded and carrion crows
First, they assembled and annotated a high-quality reference genome of one hooded male crow and identified 20’794 protein-coding genes. This reference genome was then used to aligned 60 genomes of unrelated individuals from two populations of carrion and two populations of hooded crows (Fig. 1). They identified 8.44 million of SNPs among which 5.27 millions were shared between carrion and hooded crows. Although the major axes of genetic variation is consistent with the hypothesised expansion out of Spain after the last glaciation maxima the German carrion crows clustered closer with local German hooded crows than Spanish carrion crows (Fig. 1, on which the points represent the genetic distances between population according to the axes 1 & 2 of the principal component analysis and not the geographical location of sampled populations). Gene flow between hooded and carrion crows was also supported by complementary analyses.
Gene expression divergence
Further more by estimating gene expression through mRNA sequencing (19 individuals, 5 tissues) they found that only between 0.03% and 0.41% of the genes were differentially expressed between the two species. Interestingly they observed that lots of these genes were implicated in plumage coloration. Especially expression bias in the growing feather follicles from the torso where hooded crows are grey and carrion crows black was predominated by genes implicated in the melanogenesis pigmentation pathway and under-expressed in hooded crows (19 of the 20 identified genes). They confirmed that this expression bias was not due to different melanocyte density (Fig. 4) but rather to a broad scale down-regulation of genes implicated in melanogenesis.
Genomic divergence and gene expression between the two species
The authors investigated the landscape of genomic divergence with a 50-kb window-based approach (Fig. 2A) and a free clustering phylogenetic reconstruction for each window (Fig 2C). Out of the phylogenetic trees they inferred that only 0.28% of the genome strongly differ between the two species. Both methods revealed a 1.95 MB region exhibiting extreme genetic differentiation and regrouping 81 of all 82 fixed sites between the two species. Moreover, this region showed reduced nucleotide diversity and linkage disequilibrium with two local FST peaks connected by a saddle, revealing a possible inversion.
In the centre of one of these two FST peaks one region showed evidence for recent positive selection in hooded crows (enriched for fixed hooded crow specific variants and reduced values of Fu and Li’s D statistic (P < 0.05)). This region contains some CACNG genes that code for regulators influencing the transcription factor gene MITF. This transcription factor is a central regulatory element of the melanogenesis pathway (Fig. 3C) and regulates at least 11 melanogenesis genes under-expressed in the hooded crows. Therefore, the authors suggest a link between gene expression, colour phenotype and the signature of local divergent selection. Two others differentially expressed melanogenesis genes were located in divergent genomic regions. Yet, multigenic architecture of the colour trait is consistent with the colour polymorphism observed in the hybrids.
One gene stepped aside of the other one; the gene RGS9 which play a role in visual perception in vertebrates. This gene was underexpressed in hooded crows (including expression in eye). Besides its implication in visual perception alternative splicing forms (present in crows) of RGS9 play a role in dopamine regulation and opioid signalling in the brain and therefore may influence the observed assortative mating.
To conclude this paper showed that small local peaks of divergence (less than 1% of the genome, also called “speciation island”) is sufficient to maintain strong phenotypic differences between both species despite considerable gene flow. Moreover, they showed that assortative mating and sexual selection can exclusively cause phenotypic differentiation and speciation as there is apparently no ecological selection between the two species.
This paper nicely linked local genetic differences, regulatory pathway, gene expression and finally phenotypic differentiation between two closely related species. Clearly the authors had to produced an important and complete work and were somehow lucky to find a straightforward explanation. Yet, they provided evidences for several debated questions, as speciation without ecological selection, gene flow heterogeneity and “speciation island”, species identity even under important gene flow and the probable role of inversions in evolution.
Poelstra, J., Vijay, N., Bossu, C., Lantz, H., Ryll, B., Muller, I., Baglione, V., Unneberg, P., Wikelski, M., Grabherr, M., & Wolf, J. (2014). The genomic landscape underlying phenotypic integrity in the face of gene flow in crows Science, 344 (6190), 1410-1414 DOI: 10.1126/science.1253226