Isolation and gene flow in a speciation continuum in newts.

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.

Transcriptomics of Intralocus Sexual Conflict: Gene Expression Patterns in Females Change in Response to Selection on a Male Secondary Sexual Trait in the Bulb Mite.

Intralocus sexual conflict (IASC) prevents males and females from reaching their disparate phenotypic optima and is widespread, but little is known about its genetic underpinnings. In Rhizoglyphus robini, a mite species with alternative male morphs, elevated sexual dimorphism of the armored fighter males (compared to more feminized scramblers males) was previously reported to be associated with increased IASC. Because IASC persists if gene expression patterns are correlated between sexes, we compared gene expression patterns of males and females from the replicate lines selected for increased proportion of fighter or scrambler males (F- and S-lines, respectively). Specifically, we tested the prediction that selection for fighter morph caused correlated changes in gene expression patterns in females. We identified 532 differentially expressed genes (FDR < 0.05) between the F-line and S-line males. Consistent with the prediction, expression levels of these genes also differed between females from respective lines. Thus, significant proportion of genes differentially expressed between sexually selected male phenotypes showed correlated expression levels in females, likely contributing to elevated IASC in F-lines reported in a previous study.

Genomic heterogeneity of historical gene flow between two species of newts inferred from transcriptome data.

The role of gene flow in species formation is a major unresolved issue in speciation biology. Progress in this area requires information on the long-term patterns of gene flow between diverging species. Here, we used thousands of single-nucleotide polymorphisms derived from transcriptome resequencing and a method modeling the joint frequency spectrum of these polymorphisms to reconstruct patterns of historical gene flow between two Lissotriton newts: L. vulgaris (Lv) and L. montandoni (Lm). We tested several models of divergence including complete isolation and various scenarios of historical gene flow. The model of secondary contact received the highest support. According to this model, the species split from their common ancestor ca. 5.5 million years (MY) ago, evolved in isolation for ca. 2 MY, and have been exchanging genes for the last 3.5 MY Demographic changes have been inferred in both species, with the current effective population size of ca. 0.7 million in Lv and 0.2 million in Lm. The postdivergence gene flow resulted in two-directional introgression which affected the genomes of both species, but was more pronounced from Lv to Lm. Interestingly, we found evidence for genomic heterogeneity of interspecific gene flow. This study demonstrates the complexity of long-term gene flow between distinct but incompletely reproductively isolated taxa which divergence was initiated millions of years ago.

Constraint and adaptation in newt toll-like receptor genes.

Acute die-offs of amphibian populations worldwide have been linked to the emergence of viral and fungal diseases. Inter and intraspecific immunogenetic differences may influence the outcome of infection. Toll-like receptors (TLRs) are an essential component of innate immunity and also prime acquired defenses. We report the first comprehensive assessment of TLR gene variation for urodele amphibians. The Lissotriton newt TLR repertoire includes representatives of 13 families and is compositionally most similar to that of the anuran Xenopus. Both ancient and recent gene duplications have occurred in urodeles, bringing the total number of TLR genes to at least 21. Purifying selection has predominated the evolution of newt TLRs in both long (∼70 Ma) and medium (∼18 Ma) timescales. However, we find evidence for both purifying and positive selection acting on TLRs in two recently diverged (2-5 Ma) allopatric evolutionary lineages (Lissotriton montandoni and L. vulgaris graecus). Overall, both forms of selection have been stronger in L. v. graecus, while constraint on most TLR genes in L. montandoni appears relaxed. The differences in selection regimes are unlikely to be biased by demographic effects because these were controlled by means of a historical demographic model derived from an independent data set of 62 loci. We infer that TLR genes undergo distinct trajectories of adaptive evolution in closely related amphibian lineages, highlight the potential of TLRs to capture the signatures of different assemblages of pathogenic microorganisms, and suggest differences between lineages in the relative roles of innate and acquired immunity.

Single nucleotide polymorphisms reveal genetic structuring of the carpathian newt and provide evidence of interspecific gene flow in the nuclear genome.

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.

Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing.

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.

Nuclear and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and Bombina variegata supports their independent histories.

Exact location and number of glacial refugia still remain unclear for many European cold-blooded terrestrial vertebrates. We performed a fine-scaled multilocus phylogeographic analysis of two Bombina species combining mitochondrial variation of 950 toads from 385 sites and nuclear genes (Rag-1, Ncx-1) from a subset of samples to reconstruct their colonization and contemporary variation patterns. We identified the lowlands northwest of the Black Sea and the Carpathians to be important refugial areas for B. bombina and B. variegata, respectively. This result emphasizes the importance of Central European refugia for ectothermic terrestrial species, far north of the Mediterranean areas regarded as exclusive glacial refugia for the animals. Additional refugia for B. variegata have been located in the southern Apennines and Balkans. In contrast, no evidence for the importance of other east European plains as refugial regions has been found. The distribution of mtDNA and Ncx-1 variation suggests the presence of local refugia near the Black Sea for B. bombina; however, coalescent simulations did not allow to distinguish whether one or two refugia were present in the region. Strong genetic drift apparently accompanied postglacial expansions reducing diversity in the colonization areas. Extended sampling, coupled with the multilocus isolation with migration analysis, revealed a limited and geographically restricted gene flow from the Balkan to Carpathian populations of B. variegata. However, despite proximity of inferred B. bombina and B. variegata refugia, gene exchange between them was not detected.

jMHC: software assistant for multilocus genotyping of gene families using next-generation amplicon sequencing.

Genotyping of multilocus gene families, such as the major histocompatibility complex (MHC), may be challenging because of problems with assigning alleles to loci and copy number variation among individuals. Simultaneous amplification and genotyping of multiple loci may be necessary, and in such cases, next-generation deep amplicon sequencing offers a great promise as a genotyping method of choice. Here, we describe jMHC, a computer program developed for analysing and assisting in the visualization of deep amplicon sequencing data. Software operates on FASTA files; therefore, output from any sequencing technology may be used. jMHC was designed specifically for MHC studies but it may be useful for analysing amplicons derived from other multigene families or for genotyping other polymorphic systems. The program is written in Java with user-friendly graphical interface (GUI) and can be run on Microsoft Windows, Linux OS and Mac OS.

Heart transcriptome of the bank vole (Myodes glareolus): towards understanding the evolutionary variation in metabolic rate.

BACKGROUND: Understanding the genetic basis of adaptive changes has been a major goal of evolutionary biology. In complex organisms without sequenced genomes, de novo transcriptome assembly using a longer read sequencing technology followed by expression profiling using short reads is likely to provide comprehensive identification of adaptive variation at the expression level and sequence polymorphisms in coding regions. We performed sequencing and de novo assembly of the bank vole heart transcriptome in lines selected for high metabolism and unselected controls. RESULTS: A single 454 Titanium run produced over million reads, which were assembled into 63,581 contigs. Searches against the SwissProt protein database and the ENSEMBL collection of mouse transcripts detected similarity to 11,181 and 14,051 genes, respectively. As judged by the representation of genes from the heart-related Gene Ontology categories and UniGenes detected in the mouse heart, our detection of the genes expressed in the heart was nearly complete (> 95% and almost 90% respectively). On average, 38.7% of the transcript length was covered by our sequences, with notably higher (45.0%) coverage of coding regions than of untranslated regions (24.5% of 5' and 32.7% of 3'UTRs). Lower sequence conservation between mouse and bank vole in untranslated regions was found to be partially responsible for poorer UTR representation. Our data might suggest a widespread transcription from noncoding genomic regions, a finding not reported in previous studies regarding transcriptomes in non-model organisms. We also identified over 19 thousand putative single nucleotide polymorphisms (SNPs). A much higher fraction of the SNPs than expected by chance exhibited variant frequency differences between selection regimes. CONCLUSION: Longer reads and higher sequence yield per run provided by the 454 Titanium technology in comparison to earlier generations of pyrosequencing proved beneficial for the quality of assembly. An almost full representation of genes known to be expressed in the mouse heart was identified. Usage of the extensive genomic resources available for the house mouse, a moderately (20-40 mln years) divergent relative of the voles, enabled a comprehensive assessment of the transcript completeness. Transcript sequences generated in the present study allowed the identification of candidate SNPs associated with divergence of selection lines and constitute a valuable permanent resource forming a foundation for RNAseq experiments aiming at detection of adaptive changes both at the level of gene expression and sequence variants, that would facilitate studies of the genetic basis of evolutionary divergence.

454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher.

BACKGROUND: Because of their functional significance, the Major Histocompatibility Complex (MHC) class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. RESULTS: Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. CONCLUSIONS: 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective, genotyping of MHC systems of virtually any complexity.