Marius Alexander Wenzel

Fine‐scale population epigenetic structure in relation to gastrointestinal parasite load in red grouse (Lagopus lagopus scotica)

Epigenetic modification of cytosine methylation states can be elicited by environmental stresses and may be a key process affecting phenotypic plasticity and adaptation. Parasites are potent stressors with profound physiological and ecological effects on their host, but there is little understanding in how parasites may influence host methylation states. Here, we estimate epigenetic diversity and differentiation among 21 populations of red grouse (Lagopus lagopus scotica) in north‐east Scotland and test for association of gastrointestinal parasite load (caecal nematode Trichostrongylus tenuis) with hepatic genome‐wide and locus‐specific methylation states. Following methylation‐sensitive AFLP (MSAP), 129 bands, representing 73 methylation‐susceptible and 56 nonmethylated epiloci, were scored across 234 individuals. The populations differed significantly in genome‐wide methylation levels and were also significantly epigenetically (FSC = 0.0227; P < 0.001) and genetically (FSC = 0.0058; P < 0.001) differentiated. Parasite load was not associated with either genome‐wide methylation levels or epigenetic differentiation. Instead, we found eight disproportionately differentiated epilocus‐specific methylation states (FST outliers) using bayescan software and significant positive and negative association of 35 methylation states with parasite load from bespoke generalized estimating equations (GEE), simple logistic regression (sam) and Bayesian environmental analysis (bayenv2). Following Sanger sequencing, genome mapping and geneontology (go) annotation, some of these epiloci were linked to genes involved in regulation of cell cycle, signalling, metabolism, immune system and notably rRNA methylation, histone acetylation and small RNAs. These findings demonstrate an epigenetic signature of parasite load in populations of a wild bird and suggest intriguing physiological effects of parasite‐associated cytosine methylation.

Genome‐wide association and genome partitioning reveal novel genomic regions underlying variation in gastrointestinal nematode burden in a wild bird

Identifying the genetic architecture underlying complex phenotypes is a notoriously difficult problem that often impedes progress in understanding adaptive eco‐evolutionary processes in natural populations. Host–parasite interactions are fundamentally important drivers of evolutionary processes, but a lack of understanding of the genes involved in the hosts response to chronic parasite insult makes it particularly difficult to understand the mechanisms of host life history trade‐offs and the adaptive dynamics involved. Here, we examine the genetic basis of gastrointestinal nematode (Trichostrongylus tenuis) burden in 695 red grouse (Lagopus lagopus scotica) individuals genotyped at 384 genome‐wide SNPs. We first use genome‐wide association to identify individual SNPs associated with nematode burden. We then partition genome‐wide heritability to identify chromosomes with greater heritability than expected from gene content, due to harbouring a multitude of additive SNPs with individually undetectable effects. We identified five SNPs on five chromosomes that accounted for differences of up to 556 worms per bird, but together explained at best 4.9% of the phenotypic variance. These SNPs were closely linked to genes representing a range of physiological processes including the immune system, protein degradation and energy metabolism. Genome partitioning indicated genome‐wide heritability of up to 29% and three chromosomes with excess heritability of up to 4.3% (total 8.9%). These results implicate SNPs and novel genomic regions underlying nematode burden in this system and suggest that this phenotype is somewhere between being based on few large‐effect genes (oligogenic) and based on a large number of genes with small individual but large combined effects (polygenic).

The role of parasite-driven selection in shaping landscape genomic structure in red grouse (Lagopus lagopus scotica).

Landscape genomics promises to provide novel insights into how neutral and adaptive processes shape genome‐wide variation within and among populations. However, there has been little emphasis on examining whether individual‐based phenotype–genotype relationships derived from approaches such as genome‐wide association (GWAS) manifest themselves as a population‐level signature of selection in a landscape context. The two may prove irreconcilable as individual‐level patterns become diluted by high levels of gene flow and complex phenotypic or environmental heterogeneity. We illustrate this issue with a case study that examines the role of the highly prevalent gastrointestinal nematode Trichostrongylus tenuis in shaping genomic signatures of selection in red grouse (Lagopus lagopus scotica). Individual‐level GWAS involving 384 SNPs has previously identified five SNPs that explain variation in T. tenuis burden. Here, we examine whether these same SNPs display population‐level relationships between T. tenuis burden and genetic structure across a small‐scale landscape of 21 sites with heterogeneous parasite pressure. Moreover, we identify adaptive SNPs showing signatures of directional selection using FST outlier analysis and relate population‐ and individual‐level patterns of multilocus neutral and adaptive genetic structure to T. tenuis burden. The five candidate SNPs for parasite‐driven selection were neither associated with T. tenuis burden on a population level, nor under directional selection. Similarly, there was no evidence of parasite‐driven selection in SNPs identified as candidates for directional selection. We discuss these results in the context of red grouse ecology and highlight the broader consequences for the utility of landscape genomics approaches for identifying signatures of selection.

Digging for gold nuggets: uncovering novel candidate genes for variation in gastrointestinal nematode burden in a wild bird species.

The extent to which genotypic variation at a priori identified candidate genes can explain variation in complex phenotypes is a major debate in evolutionary biology. Whereas some high‐profile genes such as the MHC or MC1R clearly do account for variation in ecologically relevant characters, many complex phenotypes such as response to parasite infection may well be underpinned by a large number of genes, each of small and effectively undetectable effect. Here, we characterize a suite of novel candidate genes for variation in gastrointestinal nematode (Trichostrongylus tenuis) burden among red grouse (Lagopus lagopus scotica) individuals across a network of moors in north‐east Scotland. We test for associations between parasite load and genotypic variation in twelve genes previously identified to be differentially expressed in experimentally infected red grouse or genetically differentiated among red grouse populations with overall different parasite loads. These genes are associated with a broad physiological response including immune system processes. Based on individual‐level generalized linear models, genotypic variants in nine genes were significantly associated with parasite load, with effect sizes accounting for differences of 514–666 worms per bird. All but one of these variants were synonymous or untranslated, suggesting that these may be linked to protein‐coding variants or affect regulatory processes. In contrast, population‐level analyses revealed few and inconsistent associations with parasite load, and little evidence of signatures of natural selection. We discuss the broader significance of these contrasting results in the context of the utility of population genomics and landscape genomics approaches in detecting adaptive genomic signatures.

A transcriptomic investigation of handicap models in sexual selection

Handicap models link the evolution of secondary sexual ornaments to physiological costs and thus provide a mechanistic explanation for signal honesty in sexual selection. Two commonly invoked models, the immunocompetence handicap hypothesis (ICHH) and the oxidative stress handicap hypothesis (OSHH), propose suppression of immunocompetence or increase of oxidative stress by testosterone, but empirical evidence for both models is controversial and based on morphological and physiological assays. Here, we investigated these two models on the gene transcription level using microarrays to quantify the transcriptomic response of red grouse (Lagopus lagopus scoticus) caecal, spleen and liver tissues to experimental manipulation of testosterone levels. We used a geneontology framework to identify genes related to immune function and response to reactive oxygen species and examined how transcription levels changed under experimentally increased testosterone levels in birds with parasites present or absent. Contrary to our expectations, testosterone had virtually no effect on gene transcription in spleen and liver. A small number of genes were significantly differentially regulated in caecum, and while their functions and transcription changes are consistent with the ICHH, we found little support for the OSHH. More genes responded to testosterone in the presence rather than absence of parasites, suggesting that handicap mechanisms may be context dependent and more pronounced in the presence of adverse environmental conditions. These findings illustrate the utility of transcriptomics to investigating handicap models, suggest that classic models may not underlie the handicap mechanism, and indicate that novel emerging models involving different mediators and physiological systems should be examined.

In silico identification and characterisation of 17 polymorphic anonymous non-coding sequence markers (ANMs) for red grouse (Lagopus lagopus scotica)

Anonymous non-coding sequence markers (ANMs) are powerful neutral genetic markers with great utility in phylogeography, population genetics and population genomics. Developing ANMs has previously relied on sequencing random fragments of genomic DNA in the target species and then querying bioinformatics databases to identify unannotated, putatively neutral fragments. Here, we describe an alternative in silico approach that is based on identifying large unannotated genomic regions in model species to provide a priori neutral targets for candidate ANMs that are remote from exonic regions. We illustrate this approach by developing a set of 17 polymorphic ANMs for red grouse (Lagopus lagopus scotica) from c. 1 Mbp non-coding chromosome regions of chicken, turkey and zebrafinch genomes. This pipeline presents a powerful and efficient approach when appropriate model genomes are available for the target species of interest.

Signatures of balancing selection in toll-like receptor (TLRs) genes – novel insights from a free-living rodent

Selective pressure from pathogens is considered a key selective force driving the evolution of components of the immune system. Since single components of the immune system may interact with many pathogens, and single pathogens may be recognized by multiple components of the immune system, gaining a better understanding of the mechanisms of parasite-driven selection requires the study of multiple genes and pathogens. Toll-like receptors (TLRs) are a large gene family that code for antigen-presenting components of the innate immune response. In the present paper we characterize polymorphism and signatures of selection in seven TLRs in free-living bank voles Myodes glareolus. We report the first evidence of balancing selection in several TLR genes, supported by positive values of Fu and Li’s D* in TLR2 and TLR5, and positive values of Tajima’s D in LRR regions within TLR1 and TLR2. We further found significant associations between amino-acid alleles of TLR1 and TLR5 and susceptibility to infection with the blood pathogen Bartonella. Interestingly, selection patterns in TLRs presenting virus-derived motifs (TLR7 and TLR9) differed considerably from those interacting with bacterial PAMPs. In contrast to the highly variable TLRs presenting bacterial motifs, TLR7 and TLR9 had low polymorphism and displayed signatures of directional selection. These findings suggest different functional responses across the TLR gene family and highlight the complexity of parasite-driven selection.

Microbiome composition within a sympatric species complex of intertidal isopods (Jaera albifrons)

The increasingly recognised effects of microbiomes on the eco-evolutionary dynamics of their hosts are promoting a view of the “hologenome” as an integral host-symbiont evolutionary entity. For example, sex-ratio distorting reproductive parasites such as Wolbachia are well-studied pivotal drivers of invertebrate reproductive processes, and more recent work is highlighting novel effects of microbiome assemblages on host mating behaviour and developmental incompatibilities that underpin or reinforce reproductive isolation processes. However, examining the hologenome and its eco-evolutionary effects in natural populations is challenging because microbiome composition is considerably influenced by environmental factors. Here we illustrate these challenges in a sympatric species complex of intertidal isopods (Jaera albifrons spp.) with pervasive sex-ratio distortion and ecological and behavioural reproductive isolation mechanisms. We deep-sequence the bacterial 16S rRNA gene among males and females collected in spring and summer from two coasts in north-east Scotland, and examine microbiome composition with a particular focus on reproductive parasites. Microbiomes of all species were diverse (overall 3,317 unique sequences among 3.8 million reads) and comprised mainly Proteobacteria and Bacteroidetes taxa typical of the marine intertidal zone, in particular Vibrio spp. However, we found little evidence of the reproductive parasites Wolbachia, Rickettsia, Spiroplasma and Cardinium, suggesting alternative causes of sex-ratio distortion. Notwithstanding, a significant proportion of the variance in microbiome composition among samples was explained by sex (14.1 %), nested within geographic (26.9 %) and seasonal (39.6 %) variance components. The functional relevance of this sex signal was difficult to ascertain given the absence of reproductive parasites, the ephemeral nature of the species assemblages and substantial environmental variability. These results establish the Jaera albifrons species complex as an intriguing system for examining the effects of microbiomes on reproductive processes and speciation, and highlight the difficulties associated with snapshot assays of microbiome composition in dynamic and complex environments.

Erratum to: Pronounced genetic structure and low genetic diversity in European red-billed chough (Pyrrhocorax pyrrhocorax) populations

In the original publication, Tables 3 and 6 were published with incorrect estimates of population heterozygosities. All other diversity statistics were correct as originally presented. Updated versions of Tables 3 and 6 with corrected heterozygosity estimates confirmed using Arlequin 3.5 (Excoffier and Lischer 2010) as in Davila et al. (2014) are provided in this erratum. Discrepancies were minor for populations on the British Isles. The correct estimates for Spain are slightly larger than those reported for La Palma by Davila et al. (2014), but this does not necessarily affect their interpretation that choughs on La Palma may have originated from multiple migration events. The original conclusion that chough populations on the British Isles have low genetic diversity compared to continental European populations remains and is now, in fact, strengthened.