Genetic divergence, reproductive isolation and the early stages of speciation

The process of speciation is the splitting of single populations into two or more distinct, reproductively isolated taxa. Common modes of speciation are sympatric, allopatric and parapatric speciation, with speciation in allopatry being the most frequently documented mode to date. In allopatric speciation, geographical barriers physically separate populations, allowing these now isolated groups to evolve reproductive barriers, i.e. barriers to successful reproduction, which can take the form of premating, postmating prezygotic or postzygotic barriers. Species level phylogenies derived from molecular data may provide an indirect record of speciation events, and can, when combined with morphological traits, be used to investigate at what stage in the speciation process (e.g. early speciation, recent speciation, reversed speciation) taxa currently are. In this thesis, I used a range of molecular methods and morphological analysis to investigate different stages in the speciation process. More specifically, I investigated four different species/species complexes exhibiting varying degrees of genetic and morphological divergence in order to investigate where in the speciation process taxa are and to discuss the evolutionary processes involved in the speciation events. First, the phylogeographic pattern of the common redstart (Phoenicurus phoenicurus) was described and the level of genetic divergence quantified. In this system, high divergence within the mitochondrial DNA (5% K2P distance, COI) combined with low morphological divergence appears to reflect reversed speciation. Second, I found a similar pattern of high genetic divergence (1.5-4.1% K2P distance, COI) in the autumnal moth (Epirrita autumnata), for which low morphological divergences have previously been found. Moreover, an association between the moths’ mtDNA divergence and infection by different Wolbachia strains was found, and I suggest that this association maintains the mitochondrial variation. In contrast to these two studies, the bluethroat (Luscinia svecica) subspecies complex was characterized by exhibiting low genetic divergence (mean genetic distance 0.7%, K2P distance, COI) and high morphological differences and, as such, appears to exhibit signs of early speciation. Importantly, these contrasting patterns may be explained by differences in both ecology and sexual selection pressures experienced by each of the species/populations, with the bluethroats being subject to strong diversifying sexual selection for male primary and secondary sexual characters. A third goal of this thesis was to investigate whether sperm characters and genetic markers evolve at different speeds. In the bluethroat subspecies complex, where mitochondrial divergence was low, I found evidence of rapid evolution of sperm morphology, suggesting that rapid evolution of gametes may be an important factor involved in the early stages of speciation. Finally, I studied the black-and-white Ficedula flycatchers, a group of species suggested to have undergone recent speciation, in order to investigate variation in the rate of evolution between the Z chromosome (i.e. sex chromosome) and the autosomes. In this system, I found contrasting patterns in the evolution of the Z chromosome versus the autosomes. Specifically, my results revealed increased divergence and reduced variation on the Z chromosome compared to the autosomes, a finding that is best explained by the faster-Z hypothesis. As the Z chromosome has been linked to sexually selected traits in the Ficedula flycatchers, I suggest the contrasting pattern of evolution on the Z vs. autosome may have implications for the process of speciation processes in these species. In conclusion, my thesis highlights the utility of combining patterns of genetic and phenotypic divergence to identify at what stage of the speciation process taxa occur and how variation in evolutionary rates between traits can contribute to our understanding of the speciation process

Mulitlocus sequence analyses of the near threatened Semi-collared flycatcher (Ficedula semitorquata) and a comparison with three other Ficedula flycatcher species

The Semi-collared flycatcher (Ficedula semitorquata) is a member of the black-and-white flycatcher species complex. It is poorly studied, rare, and is currently classified as “near threatened”, in the IUCN red lists of threatened species. The Semi-collared flycatcher has a patchy distribution in Europe and part of the Middle East. In this study, I analyzed sequence variation at nuclear loci of Semi-collared flycatchers, and compared the patterns found with those of three other black-and-white flycatcher species, the Pied (F. hypoleuca), Collared (F.albicollis) and Atlas flycatchers (F. speculigera). Genetic variation was found to be relatively high, compared to the other three flycatcher species and there were no signs of inbreeding. All four flycatcher species had less variation at Z-linked loci compared to autosomal loci. A comparison showed that all species combinations had fewer shared polymorphisms and more fixed substitutions at Z-linked than at autosomal loci. Selective sweeps on the Z-chromosome during the evolutionary history of these species is likely to have contributed to this pattern. A mismatch distribution showed signs of a recent population expansion in all four species, and a phylogenetic reconstruction confirmed a relatively deep split and that each species is monophyletic. This study supports the classification of the Semi-collared flycatcher as a separate species. Even though no signs of a small population size are found here, it is important to keep monitoring this bird, since it has gone through several declines across Europe during the last decades. Much of the decline is probably due to habitat destruction, so it is important to retain old forest in the habitat of the species

Data from: Ecological speciation by temporal isolation in a population of the stonefly Leuctra hippopus (Plecoptera, Leuctridae)

Stream dwelling invertebrates are ideal candidates for the study of ecological speciation as they are often adapted to particular environmental conditions within a stream and inhabit only certain reaches of a drainage basin, separated by unsuitable habitat. We studied an atypical population of the stonefly Leuctra hippopus at a site in central Norway, the Isterfoss rapids, in relation to three nearby and two remote conspecific populations. Adults of this population emerge about a month earlier than those of nearby populations, live on large boulders emerging from the rapids, and are short-lived. This population also has distinct morphological features and was studied earlier during the period 1975–1990. We reassessed morphological distinctness with new measurements and added several analyses of genetic distinctness based on mitochondrial and nuclear sequence markers, as well as AFLP fingerprinting and SNPs mined from RAD sequences. The Isterfoss population is shown to be most closely related to its geographical neighbors, yet clearly morphologically and genetically distinct and homogeneous. We conclude that this population is in the process of sympatric speciation, with temporal isolation being the most important direct barrier to gene flow. The shift in reproductive season results from the particular temperature and water level regime in the Isterfoss rapids. The distinct adult body shape and loss of flight are hypothesized to be an adaptation to the unusual habitat. Ecological diversification on small spatial and temporal scales is one of the likely causes of the high diversity of aquatic insects

Deep sympatric mitochondrial divergence without reproductive isolation in the common redstart Phoenicurus phoenicurus

Mitochondrial DNA usually shows low sequence variation within and high sequence divergence among species, which makes it a useful marker for phylogenetic inference and DNA barcoding. A previous study on the common redstart (Phoenicurus phoenicurus) revealed two very different mtDNA haplogroups (5% K2P distance). This divergence is comparable to that among many sister species; however, both haplogroups coexist and interbreed in Europe today. Herein, we describe the phylogeographic pattern of these lineages and test hypotheses for how such high diversity in mtDNA has evolved. We found no evidence for mitochondrial pseudogenes confirming that both haplotypes are of mitochondrial origin. When testing for possible reproductive barriers, we found no evidence for lineage-specific assortative mating and no difference in sperm morphology, indicating that they are not examples of cryptic species, nor likely to reflect the early stages of speciation. A gene tree based on a short fragment of cytochrome c oxidase subunit 1 from the common redstart and 10 other Phoenicurus species, showed no introgression from any of the extant congenerics. However, introgression from an extinct congeneric cannot be excluded. Sequences from two nuclear introns did not show a similar differentiation into two distinct groups. Mismatch distributions indicated that the lineages have undergone similar demographic changes. Taken together, these results confirm that deeply divergent mitochondrial lineages can coexist in biological species. Sympatric mtDNA divergences are relatively rare in birds, but the fact that they occur argues against the use of threshold mtDNA divergences in species delineation. Assortative mating, mtDNA, reproductive isolation, sympatric divergencepublishedVersio

Sperm performance in conspecific and heterospecific female fluid

Divergent sexual selection within allopatric populations may result in divergent sexual phenotypes, which can act as reproductive barriers between populations upon secondary contact. This hypothesis has been most tested on traits involved in precopulatory sexual selection, with less work focusing on traits that act after copulation and before fertilization (i.e., postcopulatory prezygotic traits), particularly in internally fertilizing vertebrates. However, postcopulatory sexual selection within species can also drive trait divergence, resulting in reduced performance of heterospecific sperm within the female reproductive tract. Such incompatibilities, arising as a by-product of divergent postcopulatory sexual selection in allopatry, can represent reproductive barriers, analogous to species-assortative mating preferences. Here, we tested for postcopulatory prezygotic reproductive barriers between three pairs of taxa with diverged sperm phenotypes and moderate-to-high opportunity for postcopulatory sexual selection (barn swallows Hirundo rustica versus sand martins Riparia riparia, two subspecies of bluethroats, Luscinia svecica svecica versus L. s. namnetum, and great tits Parus major versus blue tits Cyanistes caeruleus). We tested sperm swimming performance in fluid from the outer reproductive tract of females, because the greatest reduction in sperm number in birds occurs as sperm swim across the vagina. Contrary to our expectations, sperm swam equally well in fluid from conspecific and heterospecific females, suggesting that postcopulatory prezygotic barriers do not act between these taxon pairs, at this stage between copulation and fertilization. We therefore suggest that divergence in sperm phenotypes in allopatry is insufficient to cause widespread postcopulatory prezygotic barriers in the form of impaired sperm swimming performance in passerine birds

Evolutionary analysis of the female-specific avian W chromosome

The typically repetitive nature of the sex-limited chromosome means that it is often excluded from or poorly covered in genome assemblies, hindering studies of evolutionary and population genomic processes in non-recombining chromosomes. Here, we present a draft assembly of the non-recombining region of the collared flycatcher W chromosome, containing 46 genes without evidence of female-specific functional differentiation. Survival of genes during Wchromosome degeneration has been highly non-random and expression data suggest that this can be attributed to selection for maintaining gene dose and ancestral expression levels of essential genes. Re-sequencing of large population samples revealed dramatically reduced levels of within-species diversity and elevated rates of between-species differentiation (lineage sorting), consistent with low effective population size. Concordance between W chromosome and mitochondrial DNA phylogenetic trees demonstrates evolutionary stable matrilineal inheritance of this nuclear–cytonuclear pair of chromosomes. Our results show both commonalities and differences between W chromosome and Y chromosome evolution

Data from: Sperm performance in conspecific and heterospecific female fluid

Divergent sexual selection within allopatric populations may result in divergent sexual phenotypes, which can act as reproductive barriers between populations upon secondary contact. This hypothesis has been most tested on traits involved in precopulatory sexual selection, with less work focusing on traits that act after copulation and before fertilization (i.e., postcopulatory prezygotic traits), particularly in internally fertilizing vertebrates. However, postcopulatory sexual selection within species can also drive trait divergence, resulting in reduced performance of heterospecific sperm within the female reproductive tract. Such incompatibilities, arising as a by-product of divergent postcopulatory sexual selection in allopatry, can represent reproductive barriers, analogous to species-assortative mating preferences. Here, we tested for postcopulatory prezygotic reproductive barriers between three pairs of taxa with diverged sperm phenotypes and moderate-to-high opportunity for postcopulatory sexual selection (barn swallows Hirundo rustica versus sand martins Riparia riparia, two subspecies of bluethroats, Luscinia svecica svecica versus L. s. namnetum, and great tits Parus major versus blue tits Cyanistes caeruleus). We tested sperm swimming performance in fluid from the outer reproductive tract of females, because the greatest reduction in sperm number in birds occurs as sperm swim across the vagina. Contrary to our expectations, sperm swam equally well in fluid from conspecific and heterospecific females, suggesting that postcopulatory prezygotic barriers do not act between these taxon pairs, at this stage between copulation and fertilization. We therefore suggest that divergence in sperm phenotypes in allopatry is insufficient to cause widespread postcopulatory prezygotic barriers in the form of impaired sperm swimming performance in passerine birds

Linked selection and recombination rate variation drive the evolution of the genomic landscape of differentiation across the speciation continuum of Ficedula flycatchers

Speciation is a continuous process during which genetic changes gradually accumulate in the genomes of diverging species. Recent studies have documented highly heterogeneous differentiation landscapes, with distinct regions of elevated differentiation (“differentiation islands”) widespread across genomes. However, it remains unclear which processes drive the evolution of differentiation islands; how the differentiation landscape evolves as speciation advances; and ultimately, how differentiation islands are related to speciation. Here, we addressed these questions based on population genetic analyses of 200 resequenced genomes from 10 populations of four Ficedula flycatcher sister species. We show that a heterogeneous differentiation landscape starts emerging among populations within species, and differentiation islands evolve recurrently in the very same genomic regions among independent lineages. Contrary to expectations from models that interpret differentiation islands as genomic regions involved in reproductive isolation that are shielded from gene flow, patterns of sequence divergence (dxy and relative node depth) do not support a major role of gene flow in the evolution of the differentiation landscape in these species. Instead, as predicted by models of linked selection, genome-wide variation in diversity and differentiation can be explained by variation in recombination rate and the density of targets for selection. We thus conclude that the heterogeneous landscape of differentiation in Ficedula flycatchers evolves mainly as the result of background selection and selective sweeps in genomic regions of low recombination. Our results emphasize the necessity of incorporating linked selection as a null model to identify genome regions involved in adaptation and speciation.Peer reviewe

All data associated with Cramer et al. Ecology and Evolution

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