Wiesław Babik

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

BackgroundBecause 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.ResultsHere 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 flycatchers MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family.Conclusions454 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.

Mitochondrial phylogeography of the Eurasian beaver Castor fiber L.

Nucleotide variation in an approximately 490 bp fragment of the mitochondrial DNA control region (mtDNA CR) was used to describe the genetic variation and phylogeographical pattern in the Eurasian beaver (Castor fiber) over its entire range. The sampling effort was focused on the relict populations that survived a drastic population bottleneck, caused by overhunting, at the end of the 19th century. A total of 152 individuals grouped into eight populations representing all currently recognized subspecies were studied. Sixteen haplotypes were detected, none of them shared among populations. Intrapopulation sequence variation was very low, most likely a result of the severe bottleneck. Extreme genetic structure could result from human‐mediated extinction of intermediate populations, but it could also be an effect of prior substantial structuring of the beaver populations with watersheds of major Eurasian rivers acting as barriers to gene flow. Phylogenetic analysis revealed the presence of two mtDNA lineages: eastern (Poland, Lithuania, Russia and Mongolia) and western (Germany, Norway and France), the former comprising more divergent haplotypes. The low level of sequence divergence of the entire cytochrome b gene among six individuals representing six subspecies suggests differentiation during the last glacial period and existence of multiple glacial refugia. At least two evolutionary significant units (ESU) can be identified, the western and the eastern haplogroup. The individual relict populations should be regarded as management units, the eastern subspecies possibly also as ESUs. Guidelines for future translocations and reintroductions are proposed.

MHC-DRB3 variation in a free-living population of the European bison, Bison bonasus

MHC genes play a crucial role in pathogen recognition and are the most polymorphic genes in vertebrates. Loss of variation in these genes in bottlenecked species is thought to put their survival at risk. We examined variation at the MHC II DRB3 locus in the European bison, Bison bonasus, a species that has undergone an extreme bottleneck: the current population originated from only 12 founders. We also tested for the association of DRB3 genes with the incidence of posthitis, a disease affecting the reproductive organs of bulls and posing a new threat to the survival of the species. We found very limited MHC diversity, with only four alleles segregating in a sample of 172 individuals from a free‐ranging Białowieża population. The alleles were highly divergent and revealed the hallmark of positive selection acting on them in the past, that is, a significant excess of nonsynonymous substitutions. This excess was concentrated in putative antigen‐binding sites, suggesting that selection was driven by pathogens. However, we did not observe departures from Hardy–Weinberg equilibrium, an indicator of strong ongoing selection. Neither have we found a significant association between DRB3 alleles or genotypes and susceptibility to posthitis. Alleles conferring resistance to males may have been lost during the extreme bottleneck the species had undergone.

Detecting balancing selection in genomes: limits and prospects

In spite of the long‐term interest in the process of balancing selection, its frequency in genomes and evolutionary significance remain unclear due to challenges related to its detection. Current statistical approaches based on patterns of variation observed in molecular data suffer from low power and a high incidence of false positives. This raises the question whether balancing selection is rare or is simply difficult to detect. We discuss genetic signatures produced by this mode of selection and review the current approaches used for their identification in genomes. Advantages and disadvantages of the available methods are presented, and areas where improvement is possible are identified. Increased specificity and reduced rate of false positives may be achieved by using a demographic model, applying combinations of tests, appropriate sampling scheme and taking into account intralocus variation in selection pressures. We emphasize novel solutions, recently developed model‐based approaches and good practices that should be implemented in future studies looking for signals of balancing selection. We also draw attention of the readers to the results of recent theoretical studies, which suggest that balancing selection may be ubiquitous but transient, leaving few signatures detectable by existing methods. Testing this new theory may require the development of novel high‐throughput methods extending beyond genomic scans.

Genetic differentiation among northern and southern populations of the moor frog Rana arvalis Nilsson in central Europe

Starch gel electrophoresis and morphometric characters were used to assess the geographical variation between 14 populations of the moor frog, Rana arvalis, from northern and southern areas in Central Europe. Six of the 13 screened allozyme loci were polymorphic (95% criterion). No fixed differences in allele composition between the two regions were found. Some of the alleles were region specific. Genetic variability as measured by expected heterozygosity (He) and number of alleles per locus was significantly lower in the southern samples than in northern ones (He=0.104 and He=0.156, alleles/locus=1.6 and 1.8 respectively). This is interpreted as a consequence of the different past history of these two groups during the Pleistocene. Population subdivision, as measured by FST, was substantial (0.124 and 0.078 for the southern and northern group, respectively); 59.9% of the between-locality variation is attributed to this division into two geographical groups. Isolation-by-distance was detected by significant negative correlation between the estimate of gene flow (log [Mcirc ]) and log(geographical distance) only for the southern population groups. This indicates that the northern populations have recently recolonized their contemporary distribution area. The mean genetic distance between the northern and southern group of populations was DN=0.062. Despite the relatively low genetic distance between them, the two population groups form two distinct clusters in the maximum likelihood (ML) tree. Discriminant analysis on 11 size adjusted body measurements showed considerable overlap between populations from different geographical areas. An isolated Romanian Reci population which genetically belongs to the southern group of populations was morphologically situated in an intermediate position between northern and other southern populations.

Accuracy of allele frequency estimation using pooled RNA-Seq

For nonmodel organisms, genome‐wide information that describes functionally relevant variation may be obtained by RNA‐Seq following de novo transcriptome assembly. While sequencing has become relatively inexpensive, the preparation of a large number of sequencing libraries remains prohibitively expensive for population genetic analyses of nonmodel species. Pooling samples may be then an attractive alternative. To test whether pooled RNA‐Seq accurately predicts true allele frequencies, we analysed the liver transcriptomes of 10 bank voles. Each sample was sequenced both as an individually barcoded library and as a part of a pool. Equal amounts of total RNA from each vole were pooled prior to mRNA selection and library construction. Reads were mapped onto the de novo assembled reference transcriptome. High‐quality genotypes for individual voles, determined for 23 682 SNPs, provided information on ‘true’ allele frequencies; allele frequencies estimated from the pool were then compared with these values. ‘True’ frequencies and those estimated from the pool were highly correlated. Mean relative estimation error was 21% and did not depend on expression level. However, we also observed a minor effect of interindividual variation in gene expression and allele‐specific gene expression influencing allele frequency estimation accuracy. Moreover, we observed strong negative relationship between minor allele frequency and relative estimation error. Our results indicate that pooled RNA‐Seq exhibits accuracy comparable with pooled genome resequencing, but variation in expression level between individuals should be assessed and accounted for. This should help in taking account the difference in accuracy between conservatively expressed transcripts and these which are variable in expression level.

The genomics of adaptation

The amount and nature of genetic variation available to natural selection affect the rate, course and outcome of evolution. Consequently, the study of the genetic basis of adaptive evolutionary change has occupied biologists for decades, but progress has been hampered by the lack of resolution and the absence of a genome-level perspective. Technological advances in recent years should now allow us to answer many long-standing questions about the nature of adaptation. The data gathered so far are beginning to challenge some widespread views of the way in which natural selection operates at the genomic level. Papers in this Special Feature of Proceedings of the Royal Society B illustrate various aspects of the broad field of adaptation genomics. This introductory article sets up a context and, on the basis of a few selected examples, discusses how genomic data can advance our understanding of the process of adaptation.

Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure

Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre‐eminent system for the study of selective pressures that arise from host–pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population‐genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.

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.

Initial Molecular-Level Response to Artificial Selection for Increased Aerobic Metabolism Occurs Primarily through Changes in Gene Expression

Experimental evolution combined with genome or transcriptome resequencing (Evolve and Resequence) represents a promising approach for advancing our understanding of the genetic basis of adaptation. Here, we applied this strategy to investigate the effect of selection on a complex trait in lines derived from a natural population of a small mammal. We analyzed the liver and heart transcriptomes of bank voles (Myodes [=Clethrionomys] glareolus) that had been selected for increased aerobic metabolism. The organs were sampled from 13th generation voles; at that point, the voles from four replicate selected lines had 48% higher maximum rates of oxygen consumption than those from four control lines. At the molecular level, the response to selection was primarily observed in gene expression: Over 300 genes were found to be differentially expressed between the selected and control lines and the transcriptome-wide pattern of expression distinguished selected lines from controls. No evidence for selection-driven changes of allele frequencies at coding sites was found: No single nucleotide polymorphism (SNP) changed frequency more than expected under drift alone and frequency changes aggregated over all SNPs did not separate selected and control lines. Nevertheless, among genes which showed highest differentiation in allele frequencies between selected and control lines we identified, using information about gene functions and the biology of the selected phenotype, plausible targets of selection; these genes, together with those identified in expression analysis, have been prioritized for further studies. Because our selection lines were derived from a natural population, the amount and the spectrum of variation available for selection probably closely approximated that typically found in populations of small mammals. Therefore, our results are relevant to the understanding of the molecular basis of complex adaptations occurring in natural vertebrate populations.