Aim To assess the role of the Balkan Peninsula as a Pleistocene refugium for the smooth newt, Lissotriton vulgaris, and to test whether its genetic differentiation is temporally compatible with the southern refugia model. Location The Balkan Peninsula. Methods Phylogeographical analyses were conducted using mitochondrial DNA (mtDNA) sequences sampled from 49 populations of L. vulgaris. A fossil‐calibrated estimate of divergence times among major mtDNA clades was obtained. Results We detected seven parapatrically distributed mtDNA clades with very little admixture among populations. Whereas most clades diverged in the Pleistocene, the earliest splits between Caucasian, Anatolian and Balkan clades occurred in the Pliocene. Clades C, D, K and M have local distributions within the Balkans and have evolved in isolation from other groups. Clade L originated in the Pannonian Basin and northern margin of the Balkan Peninsula and recently expanded across central and western Europe. Clades H and E have recently arrived in the Balkans from source populations in the Apennine Peninsula and Anatolia, respectively. Main conclusions The history of L. vulgaris involves multiple, independent refugial populations in the Balkans. Only one of them, located at the northern periphery of the peninsula, showed evidence of post‐glacial expansion into western and northern Europe. The Balkans have therefore served as a reservoir of old diversity for L. vulgaris. By contrast, populations at the northern and eastern frontiers of the peninsula have experienced non‐equilibrium dynamics. Our dating revealed that very little, if any, pre‐Pliocene genetic diversity has survived in Europe, despite an extensive fossil record for this species in the Miocene and Pliocene. Differentiation of the European mtDNA clades thus seems to have been primarily moulded by Pleistocene climate change. None of the currently recognized subspecies present in the Balkans are reciprocally monophyletic in their mtDNA. We hypothesize that incomplete lineage sorting and mtDNA introgression account for the observed discrepancies.

The importance of interspecific introgression as a source of adaptive variation is increasingly recognized. Theory predicts that beneficial genetic variants cross species boundaries easily even when interspecific hybridization is rare and gene flow is strongly constrained throughout the genome. However, it remains unclear whether certain classes of genes are particularly prone to adaptive introgression. Genes affected by balancing selection (BS) may constitute such a class, because forms of BS that favour novel, initially rare alleles, should facilitate introgression. We tested this hypothesis in hybridizing newts by comparing 13 genes with signatures of BS, in particular an excess of common non-synonymous polymorphisms, to the genomic background (154 genes). Parapatric hybridizing taxa were less differentiated in BS candidate genes than more closely related allopatric lineages, while the opposite was observed in the control genes. Coalescent and forward simulations that explored neutral and BS scenarios under isolation and migration showed that processes other than differential gene flow are unlikely to account for this pattern. We conclude that BS, probably involving a form of novel allele advantage, promotes introgression. This mechanism may be a source of adaptively relevant variation in hybridizing species over prolonged periods.

Because reproductive isolation often evolves gradually, differentiating lineages may retain the potential for genetic exchange for prolonged periods, providing an opportunity to quantify and to understand the fundamental role of gene flow during speciation. Here we delimit evolutionary lineages, reconstruct the phylogeny and infer gene flow in newts of the Lissotriton vulgaris species complex based on 74 nuclear markers sampled from 127 localities. We demonstrate that distinct lineages along the speciation continuum in newts exchange nontrivial amounts of genes, affecting their evolutionary trajectories. By integrating a wide array of methods, we delimit nine evolutionary lineages and show that two principal factors have driven their genetic differentiation: time since the last common ancestor determining levels of shared ancestral polymorphism, and shifts in geographic distributions determining the extent of secondary contact. Post-divergence gene flow, indicative of evolutionary non-independence, has been most extensive in Central Europe, while four southern European lineages have acquired the population-genetic hallmarks of independent species (L. graecus, L. kosswigi, L. lantzi, L. schmidtleri). We obtained strong statistical support for widespread mtDNA introgression following secondary contact, previously suggested by discordance between mtDNA phylogeny and morphology. Our study reveals long-term evolutionary persistence of evolutionary lineages that may periodically exchange genes with one another: although some of these lineages may become extinct or fuse, others will acquire complete reproductive isolation and will carry signatures of this complex history in their genomes.

Bacterial communities play a crucial role in the biology, ecology, and evolution of multicellular organisms. In this research, the microbiome of 24 selected beetle species representing five families (Carabidae, Staphylinidae, Curculionidae, Chrysomelidae, Scarabaeidae) and three trophic guilds (carnivorous, herbivorous, detrivorous) was examined using 16S rDNA sequencing on the Illumina platform. The aim of the study was to compare diversity within and among species on various levels of organization, including evaluation of the impact of endosymbiotic bacteria. Collected data showed that beetles possess various bacterial communities and that microbiota of individuals of particular species hosts are intermixed. The most diverse microbiota were found in Carabidae and Scarabaeidae; the least diverse, in Staphylinidae. On higher organization levels, the diversity of bacteria was more dissimilar between families, while the most distinct with respect to their microbiomes were trophic guilds. Moreover, eight taxa of endosymbiotic bacteria were detected including common genera such as Wolbachia, Rickettsia, and Spiroplasma, as well as the rarely detected Cardinium, Arsenophonus, Buchnera, Sulcia, Regiella, and Serratia. There were no correlations among the abundance of the most common Wolbachia and Rickettsia; a finding that does not support the hypothesis that these bacteria occur interchangeably. The abundance of endosymbionts only weakly and negatively correlates with diversity of the whole microbiome in beetles. Overall, microbiome diversity was found to be more dependent on host phylogeny than on the abundance of endosymbionts. This is the first study in which bacteria diversity is compared between numerous species of beetles in a standardized manner.Electronic supplementary materialThe online version of this article (10.1007/s00248-019-01358-y) contains supplementary material, which is available to authorized users.

Applications that require resequencing of hundreds or thousands of predefined genomic regions in numerous samples are common in studies of non-model organisms. However few approaches at the scale intermediate between multiplex PCR and sequence capture methods are available. Here we explored the utility of Molecular Inversion Probes (MIPs) for the medium-scale targeted resequencing in a non-model system. Markers targeting 112 bp of exonic sequence were designed from transcriptome of Lissotriton newts. We assessed performance of 248 MIP markers in a sample of 85 individuals. Among the 234 (94.4%) successfully amplified markers 80% had median coverage within one order of magnitude, indicating relatively uniform performance; coverage uniformity across individuals was also high. In the analysis of polymorphism and segregation within family, 77% of 248 tested MIPs were confirmed as single copy Mendelian markers. Genotyping concordance assessed using replicate samples exceeded 99%. MIP markers for targeted resequencing have a number of advantages: high specificity, high multiplexing level, low sample requirement, straightforward laboratory protocol, no need for preparation of genomic libraries and no ascertainment bias. We conclude that MIP markers provide an effective solution for resequencing targets of tens or hundreds of kb in any organism and in a large number of samples.

Genomic heterogeneity of divergence between hybridizing species may reflect heterogeneity of introgression, but also processes unrelated to hybridization. Heterogeneous introgression and its repeatability can be directly tested in natural hybrid zones by examining multiple transects. Here, we studied hybrid zones between the European newts Lissotriton montandoni and two lineages of Lissotriton vulgaris, with replicate transects within each zone. Over 1,000 nuclear genes located on a linkage map and mitochondrial DNA were investigated using geographical and genomic clines. Overall, the five transects were all similar, showing hallmarks of strong reproductive isolation: bimodal distribution of genotypes in central populations and narrow allele frequency clines. However, the extent of introgression differed between the zones, possibly as a consequence of their different ages, as suggested by the analysis of heterozygosity runs in diagnostic markers. In three transects genomic signatures of small‐scale (~2 km) zone movements were detected. We found limited overlap of cline outliers between transects, and only weak evidence of stronger differentiation of introgression between zones than between transects within zones. Introgression was heterogeneous across linkage groups, with patterns of heterogeneity similar between transects and zones. Predefined candidates for increased or reduced introgression exhibited only a subtle tendency in the expected direction, suggesting that interspecific differentiation is not a reliable indicator for the strength of introgression. These hierarchically sampled hybrid zones of apparently different ages show how introgression unfolds with time and offer an excellent opportunity to dissect the dynamics of hybridization and architecture of reproductive isolation at advanced stages of speciation.

Genetic variation within species is commonly structured in a hierarchical manner which may result from superimposition of processes acting at different spatial and temporal scales. In organisms of limited dispersal ability, signatures of past subdivision are detectable for a long time. Studies of contemporary genetic structure in such taxa inform about the history of isolation, range changes and local admixture resulting from geographically restricted hybridization with related species. Here we use a set of 139 transcriptome-derived, unlinked nuclear single nucleotide polymorphisms (SNP) to assess the genetic structure of the Carpathian newt (Lissotriton montandoni, Lm) and introgression from its congener, the smooth newt (L. vulgaris, Lv). Two substantially differentiated groups of Lm populations likely originated from separate refugia, both located in the Eastern Carpathians. The colonization of the present range in north-western and south-western directions was accompanied by a modest loss of variation; admixture between the two groups has occurred in the middle of the Eastern Carpathians. Local, apparently recent introgression of Lv alleles into several Lm populations was detected, demonstrating increased power for admixture detection in comparison to a previous study based on a limited number of microsatellite markers. The level of introgression was higher in Lm populations classified as admixed than in syntopic populations. We discuss the possible causes and propose further tests to distinguish between alternatives. Several outlier loci were identified in tests of interspecific differentiation, suggesting genomic heterogeneity of gene flow between species.

Information about demographic history is essential for the understanding of the processes of divergence and speciation. Patterns of genetic variation within and between closely related species provide insights into the history of their interactions. Here, we investigated historical demography and genetic exchange between the Carpathian (Lissotriton montandoni, Lm) and smooth (L. vulgaris, Lv) newts. We combine an extensive geographical sampling and multilocus nuclear sequence data with the approximate Bayesian computation framework to test alternative scenarios of divergence and reconstruct the temporal and spatial pattern of gene flow between species. A model of recent (last glacial period) interspecific gene flow was favoured over alternative models. Thus, despite the relatively old divergence (4-6 mya) and presumably long periods of isolation, the species have retained the ability to exchange genes. Nevertheless, the low migration rates (ca. 10(-6) per gene copy per generation) are consistent with strong reproductive isolation between the species. Models allowing demographic changes were favoured, suggesting that the effective population sizes of both species at least doubled as divergence reaching the current ca. 0.2 million in Lm and 1 million in Lv. We found asymmetry in rates of interspecific gene flow between Lm and one evolutionary lineage of Lv. We suggest that intraspecific polymorphism for hybrid incompatibilities segregating within Lv could explain this pattern and propose further tests to distinguish between alternative explanations. Our study highlights the importance of incorporating intraspecific genetic structure into the models investigating the history of divergence.