Micro Germline-Restricted Chromosome in Blue Tits: Evidence for Meiotic Functions.

The germline-restricted chromosome (GRC) is likely present in all songbird species but differs widely in size and gene content. This extra chromosome has been described as either a microchromosome with only limited basic gene content or a macrochromosome with enriched gene functions related to female gonad and embryo development. Here, we assembled, annotated, and characterized the first micro-GRC in the blue tit (Cyanistes caeruleus) using high-fidelity long-read sequencing data. Although some genes on the blue tit GRC show signals of pseudogenization, others potentially have important functions, either currently or in the past. We highlight the GRC gene paralog BMP15, which is among the highest expressed GRC genes both in blue tits and in zebra finches (Taeniopygia guttata) and is known to play a role in oocyte and follicular maturation in other vertebrates. The GRC genes of the blue tit are further enriched for functions related to the synaptonemal complex. We found a similar functional enrichment when analyzing published data on GRC genes from two nightingale species (Luscinia spp.). We hypothesize that these genes play a role in maintaining standard maternal inheritance or in recombining maternal and paternal GRCs during potential episodes of biparental inheritance.

Germline-restricted chromosome shows remarkable variation in size among closely related passerine species.

Passerine birds have a supernumerary chromosome in their germ cells called the germline-restricted chromosome (GRC). The GRC was first discovered more than two decades ago in zebra finch but recent studies have suggested that it is likely present in all passerines, the most species rich avian order, encompassing more than half of all modern bird species. Despite its wide taxonomic distribution, studies on this chromosome are still scarce and limited to a few species. Here, we cytogenetically analyzed the GRC in five closely related estrildid finch species of the genus Lonchura. We show that the GRC varies enormously in size, ranging from a tiny micro-chromosome to one of the largest macro-chromosomes in the cell, not only among recently diverged species but also within species and sometimes even between germ cells of a single individual. In Lonchura atricapilla, we also observed variation in GRC copy number among male germ cells of a single individual. Finally, our analysis of hybrids between two Lonchura species with noticeably different GRC size directly supported maternal inheritance of the GRC. Our results reveal the extraordinarily dynamic nature of the GRC, which might be caused by frequent gains and losses of sequences on this chromosome leading to substantial differences in genetic composition of the GRC between and even within species. Such differences might theoretically contribute to reproductive isolation between species and thus accelerate the speciation rate of passerine birds compared to other bird lineages.

Mendelian nightmares: the germline-restricted chromosome of songbirds.

Germline-restricted chromosomes (GRCs) are accessory chromosomes that occur only in germ cells. They are eliminated from somatic cells through programmed DNA elimination during embryo development. GRCs have been observed in several unrelated animal taxa and show peculiar modes of non-Mendelian inheritance and within-individual elimination. Recent cytogenetic and phylogenomic evidence suggests that a GRC is present across the species-rich songbirds, but absent in non-passerine birds, implying that over half of all 10,500 bird species have extensive germline/soma genome differences. Here, we review recent insights gained from genomic, transcriptomic, and cytogenetic approaches with regard to the genetic content, phylogenetic distribution, and inheritance of the songbird GRC. While many questions remain unsolved in terms of GRC inheritance, elimination, and function, we discuss plausible scenarios and future directions for understanding this widespread form of programmed DNA elimination.

Postcopulatory sexual selection reduces Z-linked genetic variation and might contribute to the large Z effect in passerine birds.

The X and Z sex chromosomes play a disproportionately large role in intrinsic postzygotic isolation. The underlying mechanisms of this large X/Z effect are, however, still poorly understood. Here we tested whether faster rates of molecular evolution caused by more intense positive selection or genetic drift on the Z chromosome could contribute to the large Z effect in two closely related passerine birds, the Common Nightingale (Luscinia megarhynchos) and the Thrush Nightingale (L. luscinia). We found that the two species differ in patterns of molecular evolution on the Z chromosome. The Z chromosome of L. megarhynchos showed lower levels of within-species polymorphism and an excess of non-synonymous polymorphisms relative to non-synonymous substitutions. This is consistent with increased levels of genetic drift on this chromosome and may be attributed to more intense postcopulatory sexual selection acting on L. megarhynchos males as was indicated by significantly longer sperm and higher between-male variation in sperm length in L. megarhynchos compared to L. luscinia. Interestingly, analysis of interspecific gene flow on the Z chromosome revealed relatively lower levels of introgression from L. megarhynchos to L. luscinia than vice versa, indicating that the Z chromosome of L. megarhynchos accumulated more hybrid incompatibilities. Our results are consistent with the view that postcopulatory sexual selection may reduce the effective population size of the Z chromosome and thus lead to stronger genetic drift on this chromosome in birds. This can result in relatively faster accumulation of hybrid incompatibilities on the Z and thus contribute to the large Z effect.

Genomic islands of differentiation in two songbird species reveal candidate genes for hybrid female sterility.

Hybrid sterility is a common first step in the evolution of postzygotic reproductive isolation. According to Haldane's Rule, it affects predominantly the heterogametic sex. While the genetic basis of hybrid male sterility in organisms with heterogametic males has been studied for decades, the genetic basis of hybrid female sterility in organisms with heterogametic females has received much less attention. We investigated the genetic basis of reproductive isolation in two closely related avian species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (L. luscinia), that hybridize in a secondary contact zone and produce viable hybrid progeny. In accordance with Haldane's Rule, hybrid females are sterile, while hybrid males are fertile, allowing gene flow to occur between the species. Using transcriptomic data from multiple individuals of both nightingale species, we identified genomic islands of high differentiation (FST ) and of high divergence (Dxy ), and we analysed gene content and patterns of molecular evolution within these islands. Interestingly, we found that these islands were enriched for genes related to female meiosis and metabolism. The islands of high differentiation and divergence were also characterized by higher levels of linkage disequilibrium than the rest of the genome in both species indicating that they might be situated in genomic regions of low recombination. This study provides one of the first insights into genetic basis of hybrid female sterility in organisms with heterogametic females.

Hybrid asexuality as a primary postzygotic barrier between nascent species: On the interconnection between asexuality, hybridization and speciation.

Although sexual reproduction is ubiquitous throughout nature, the molecular machinery behind it has been repeatedly disrupted during evolution, leading to the emergence of asexual lineages in all eukaryotic phyla. Despite intensive research, little is known about what causes the switch from sexual reproduction to asexuality. Interspecific hybridization is one of the candidate explanations, but the reasons for the apparent association between hybridization and asexuality remain unclear. In this study, we combined cross-breeding experiments with population genetic and phylogenomic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis). Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing females that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes. Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of reproductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or inviability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other "classical" forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process.

Interspecific competition promotes habitat and morphological divergence in a secondary contact zone between two hybridizing songbirds.

Interspecific competition is assumed to play an important role in the ecological differentiation of species and speciation. However, empirical evidence for competition's role in speciation remains surprisingly scarce. Here, we studied the role of interspecific competition in the ecological differentiation and speciation of two closely related songbird species, the Common Nightingale (Luscinia megarhynchos) and the Thrush Nightingale (Luscinia luscinia). Both species are insectivorous and ecologically very similar. They hybridize in a secondary contact zone, which is a mosaic of sites where both species co-occur (syntopy) and sites where only one species is present (allotopy). We analysed fine-scale habitat data for both species in both syntopic and allotopic sites and looked for associations between habitat use and bill morphology, which have been previously shown to be more divergent in sympatry than in allopatry. We found that the two nightingale species differ in habitat use in allotopic sites, where L. megarhynchos occurred in drier habitats and at slightly higher elevations, but not in syntopic sites. Birds from allotopic sites also showed higher interspecific divergence in relative bill size compared to birds from syntopic sites. Finally, we found an association between bill morphology and elevation. Our results are consistent with the view that interspecific competition in nightingales has resulted in partial habitat segregation in sympatry and that the habitat-specific food supply has in turn very likely led to bill size divergence. Such ecological divergence may enhance prezygotic as well as extrinsic postzygotic isolation and thus accelerate the completion of the speciation process.

Competition-driven niche segregation on a landscape scale: Evidence for escaping from syntopy towards allotopy in two coexisting sibling passerine species.

The role of interspecific competition for generating patterns in species' distribution is hotly debated and studies taking into account processes occurring at both large and small spatial scales are almost missing. Theoretically, competition between species with overlapping niches should result in divergence of their niches in sympatry to reduce the costs of competition. Many species show a mosaic distribution within sympatric zones, with the syntopic sites occupied by both species, and allotopic sites where only one species occurs. It is unclear whether such mosaics arise as a consequence of competition-driven niche segregation or due to the decline of their abundances towards range edges driven by environmental gradients. If the interspecific competition matters, we should observe (1) a shift in habitat preferences of one or both species between syntopy and allotopy, and (2) between allopatry and allotopy. Moreover, (3) species should show greater divergence in their habitat preferences in allotopy than in allopatry where (4) no differences in habitat preferences may occur. Finally, (5) shifts should be generally greater in the competitively subordinate species than in the dominant species. We used a unique dataset on abundance of two closely related passerine species, the Common Nightingale (Luscinia megarhynchos) and the Thrush Nightingale (Luscinia luscinia), collected across their syntopy, allotopy and allopatry. The predictions were tested within a generalized mixed-effects modelling framework. After accounting for environmental gradients perpendicular to the species' contact zone, we found a strong support for all but one prediction. Habitat preferences of both species shifted markedly between syntopy and allotopy, as well as between allopatry and allotopy. Whereas the species preferred the same habitats in allopatry, their preferences became strikingly different in allotopy where the abundance of the Common Nightingale increased towards dry and warm sites with low coverage of pastures, while the abundance of the Thrush Nightingale showed exactly opposite trends. Fifth prediction was not supported. Our results indicate that the competition between closely related species can result in considerable changes in habitat use across their geographic ranges accompanied with divergence in their habitat preferences in sympatry. Here, the species "escape" from competition to allotopic sites covered by habitats avoided by the competitor. Therefore, we argue that the interspecific competition is an important driver of species' distribution at both large and small spatial scales.

X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids.

Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2(Mmm) allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.

Patterns of gene flow and selection across multiple species of Acrocephalus warblers: footprints of parallel selection on the Z chromosome.

BACKGROUND: Understanding the mechanisms and selective forces leading to adaptive radiations and origin of biodiversity is a major goal of evolutionary biology. Acrocephalus warblers are small passerines that underwent an adaptive radiation in the last approximately 10 million years that gave rise to 37 extant species, many of which still hybridize in nature. Acrocephalus warblers have served as model organisms for a wide variety of ecological and behavioral studies, yet our knowledge of mechanisms and selective forces driving their radiation is limited. Here we studied patterns of interspecific gene flow and selection across three European Acrocephalus warblers to get a first insight into mechanisms of radiation of this avian group. RESULTS: We analyzed nucleotide variation at eight nuclear loci in three hybridizing Acrocephalus species with overlapping breeding ranges in Europe. Using an isolation-with-migration model for multiple populations, we found evidence for unidirectional gene flow from A. scirpaceus to A. palustris and from A. palustris to A. dumetorum. Gene flow was higher between genetically more closely related A. scirpaceus and A. palustris than between ecologically more similar A. palustris and A. dumetorum, suggesting that gradual accumulation of intrinsic barriers rather than divergent ecological selection are more efficient in restricting interspecific gene flow in Acrocephalus warblers. Although levels of genetic differentiation between different species pairs were in general not correlated, we found signatures of apparently independent instances of positive selection at the same two Z-linked loci in multiple species. CONCLUSIONS: Our study brings the first evidence that gene flow occurred during Acrocephalus radiation and not only between sister species. Interspecific gene flow could thus be an important source of genetic variation in individual Acrocephalus species and could have accelerated adaptive evolution and speciation rate in this avian group by creating novel genetic combinations and new phenotypes. Independent instances of positive selection at the same loci in multiple species indicate an interesting possibility that the same loci might have contributed to reproductive isolation in several speciation events.

Sympatry in a nightingale contact zone has no effect on host-specific blood parasite prevalence and lineage diversity.

Parasites are a key driving force behind many ecological and evolutionary processes. Prevalence and diversity of parasites, as well as their effects on hosts, are not uniform across host species. As such, the potential parasite spillover between species can significantly influence outcomes of interspecific interactions. We screened two species of Luscinia nightingales for haemosporidian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) along an approximately 3000 km transect in Europe, incorporating areas of host distant allopatry, close allopatry and sympatry. We found significant differences in infection rates between the two host species, with common nightingales having much lower parasite prevalence than thrush nightingales (36.7% versus 83.8%). This disparity was mostly driven by Haemoproteus prevalence, which was significantly higher in thrush nightingales while common nightingales had a small, but significantly higher, Plasmodium prevalence. Furthermore, we found no effect of proximity to the contact zone on infection rate in either host species. Despite having lower infection prevalence, common nightingales were infected with a significantly higher diversity of parasite lineages than thrush nightingales, and lineage assemblages differed considerably between the two species, even in sympatry. This pattern was mostly driven by the large diversity of comparatively rare lineages, while the most abundant lineages were shared between the two host species. This suggests that, despite the close evolutionary relationships between the two nightingales, there are significant differences in parasite prevalence and diversity, regardless of the distance from the contact zone. This suggests that spillover of haemosporidian blood parasites is unlikely to contribute towards interspecific interactions in this system.

Synthesis and Scope of the Role of Postmating Prezygotic Isolation in Speciation.

How barriers to gene flow arise and are maintained are key questions in evolutionary biology. Speciation research has mainly focused on barriers that occur either before mating or after zygote formation. In comparison, postmating prezygotic (PMPZ) isolation-a barrier that acts after gamete release but before zygote formation-is less frequently investigated but may hold a unique role in generating biodiversity. Here we discuss the distinctive features of PMPZ isolation, including the primary drivers and molecular mechanisms underpinning PMPZ isolation. We then present the first comprehensive survey of PMPZ isolation research, revealing that it is a widespread form of prezygotic isolation across eukaryotes. The survey also exposes obstacles in studying PMPZ isolation, in part attributable to the challenges involved in directly measuring PMPZ isolation and uncovering its causal mechanisms. Finally, we identify outstanding knowledge gaps and provide recommendations for improving future research on PMPZ isolation. This will allow us to better understand the nature of this often-neglected reproductive barrier and its contribution to speciation.

Songbird germline-restricted chromosome as a potential arena of genetic conflicts.

Genetic conflicts can arise between components of the genome with different inheritance strategies. The germline-restricted chromosome (GRC) of songbirds shows unusual mitotic and meiotic transmission compared with the rest of the genome. It is excluded from somatic cells and maintained only in the germline. It is usually present in one copy in the male germline and eliminated during spermatogenesis, while in the female germline, it usually occurs in two copies and behaves as a regular chromosome. Here, we review what is known about the GRC's evolutionary history, genetic content, and expression and discuss how it may be involved in different types of genetic conflicts. Finally, we interrogate the potential role of the GRC in songbird germline development, highlighting several unsolved mysteries.

Rapid gene content turnover on the germline-restricted chromosome in songbirds.

The germline-restricted chromosome (GRC) of songbirds represents a taxonomically widespread example of programmed DNA elimination. Despite its apparent indispensability, we still know very little about the GRC's genetic composition, function, and evolutionary significance. Here we assemble the GRC in two closely related species, the common and thrush nightingale. In total we identify 192 genes across the two GRCs, with many of them present in multiple copies. Interestingly, the GRC appears to be under little selective pressure, with the genetic content differing dramatically between the two species and many GRC genes appearing to be pseudogenized fragments. Only one gene, cpeb1, has a complete coding region in all examined individuals of the two species and shows no copy number variation. The acquisition of this gene by the GRC corresponds with the earliest estimates of the GRC origin, making it a good candidate for the functional indispensability of the GRC in songbirds.

Mechanisms of Intrinsic Postzygotic Isolation: From Traditional Genic and Chromosomal Views to Genomic and Epigenetic Perspectives.

Intrinsic postzygotic isolation typically appears as reduced viability or fertility of interspecific hybrids caused by genetic incompatibilities between diverged parental genomes. Dobzhansky-Muller interactions among individual genes, and chromosomal rearrangements causing problems with chromosome synapsis and recombination in meiosis, have both long been considered as major mechanisms behind intrinsic postzygotic isolation. Recent research has, however, suggested that the genetic basis of intrinsic postzygotic isolation can be more complex and involves, for example, overall divergence of the DNA sequence or epigenetic changes. Here, we review the mechanisms of intrinsic postzygotic isolation from genic, chromosomal, genomic, and epigenetic perspectives across diverse taxa. We provide empirical evidence for these mechanisms, discuss their importance in the speciation process, and highlight questions that remain unanswered.

Sperm morphology and performance in relation to postmating prezygotic isolation in two recently diverged passerine species.

Divergence in sperm phenotype and female reproductive environment may be a common source of postmating prezygotic (PMPZ) isolation between species. However, compared to other reproductive barriers it has received much less attention. In this study, we examined sperm morphology and velocity in two hybridizing passerine species, the common nightingale (Luscinia megarhynchos) and thrush nightingale (L. luscinia). In addition, we for the first time characterized a passerine female reproductive tract fluid proteome. We demonstrate that spermatozoa of the common nightingale have significantly longer and wider midpiece (proximal part of the flagellum containing mitochondria) and longer tail compared to spermatozoa of thrush nightingale. On the other hand, they have significantly shorter and narrower acrosome. Importantly, these differences did not have any effect on sperm velocity. Furthermore, the fluid from the reproductive tract of common nightingale females did not differentially affect velocity of conspecific and heterospecific sperm. Our results indicate that the observed changes in the flagellum and acrosome size are unlikely to contribute to PMPZ isolation through differential sperm velocity of conspecific and heterospecific sperm in the female reproductive tract. However, they could affect other postcopulatory processes, which might be involved in PMPZ isolation, such as sperm storage, longevity or sperm-egg interaction.

Ecological character displacement in the face of gene flow: evidence from two species of nightingales.

BACKGROUND: Ecological character displacement is a process of phenotypic differentiation of sympatric populations caused by interspecific competition. Such differentiation could facilitate speciation by enhancing reproductive isolation between incipient species, although empirical evidence for it at early stages of divergence when gene flow still occurs between the species is relatively scarce. Here we studied patterns of morphological variation in sympatric and allopatric populations of two hybridizing species of birds, the Common Nightingale (Luscinia megarhynchos) and the Thrush Nightingale (L. luscinia). RESULTS: We conducted principal component (PC) analysis of morphological traits and found that nightingale species converged in overall body size (PC1) and diverged in relative bill size (PC3) in sympatry. Closer analysis of morphological variation along geographical gradients revealed that the convergence in body size can be attributed largely to increasing body size with increasing latitude, a phenomenon known as Bergmann's rule. In contrast, interspecific interactions contributed significantly to the observed divergence in relative bill size, even after controlling for the effects of geographical gradients. We suggest that the divergence in bill size most likely reflects segregation of feeding niches between the species in sympatry. CONCLUSIONS: Our results suggest that interspecific competition for food resources can drive species divergence even in the face of ongoing hybridization. Such divergence may enhance reproductive isolation between the species and thus contribute to speciation.

Evolution of B Chromosomes: From Dispensable Parasitic Chromosomes to Essential Genomic Players.

B chromosomes represent additional chromosomes found in many eukaryotic organisms. Their origin is not completely understood but recent genomic studies suggest that they mostly arise through rearrangements and duplications from standard chromosomes. They can occur in single or multiple copies in a cell and are usually present only in a subset of individuals in the population. Because B chromosomes frequently show unstable inheritance, their maintenance in a population is often associated with meiotic drive or other mechanisms that increase the probability of their transmission to the next generation. For all these reasons, B chromosomes have been commonly considered to be nonessential, selfish, parasitic elements. Although it was originally believed that B chromosomes had little or no effect on an organism's biology and fitness, a growing number of studies have shown that B chromosomes can play a significant role in processes such as sex determination, pathogenicity and resistance to pathogens. In some cases, B chromosomes became an essential part of the genome, turning into new sex chromosomes or germline-restricted chromosomes with important roles in the organism's fertility. Here, we review such cases of "cellular domestication" of B chromosomes and show that B chromosomes can be important genomic players with significant evolutionary impact.

How Important Are Structural Variants for Speciation?

Understanding the genetic basis of reproductive isolation is a central issue in the study of speciation. Structural variants (SVs); that is, structural changes in DNA, including inversions, translocations, insertions, deletions, and duplications, are common in a broad range of organisms and have been hypothesized to play a central role in speciation. Recent advances in molecular and statistical methods have identified structural variants, especially inversions, underlying ecologically important traits; thus, suggesting these mutations contribute to adaptation. However, the contribution of structural variants to reproductive isolation between species-and the underlying mechanism by which structural variants most often contribute to speciation-remain unclear. Here, we review (i) different mechanisms by which structural variants can generate or maintain reproductive isolation; (ii) patterns expected with these different mechanisms; and (iii) relevant empirical examples of each. We also summarize the available sequencing and bioinformatic methods to detect structural variants. Lastly, we suggest empirical approaches and new research directions to help obtain a more complete assessment of the role of structural variants in speciation.

Gut microbiota in two recently diverged passerine species: evaluating the effects of species identity, habitat use and geographic distance.

BACKGROUND: It has been proposed that divergence in the gut microbiota composition between incipient species could contribute to their reproductive isolation. Nevertheless, empirical evidence for the role of gut microbiota in speciation is scarce. Moreover, it is still largely unknown to what extent closely related species in the early stages of speciation differ in their gut microbiota composition, especially in non-mammalian taxa, and which factors drive the divergence. Here we analysed the gut microbiota in two closely related passerine species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia). The ranges of these two species overlap in a secondary contact zone, where both species occasionally hybridize and where interspecific competition has resulted in habitat use differentiation. RESULTS: We analysed the gut microbiota from the proximal, middle and distal part of the small intestine in both sympatric and allopatric populations of the two nightingale species using sequencing of bacterial 16S rRNA. We found small but significant differences in the microbiota composition among the three gut sections. However, the gut microbiota composition in the two nightingale species did not differ significantly between either sympatric or allopatric populations. Most of the observed variation in the gut microbiota composition was explained by inter-individual differences. CONCLUSIONS: To our knowledge, this is the first attempt to assess the potential role of the gut microbiota in bird speciation. Our results suggest that neither habitat use, nor geographical distance, nor species identity have strong influence on the nightingale gut microbiota composition. This suggests that changes in the gut microbiota composition are unlikely to contribute to reproductive isolation in these passerine birds.