Large effective population size masks population genetic structure in Hirondellea amphipods within the deepest marine ecosystem, the Mariana Trench.

The examination of genetic structure in the deep-ocean hadal zone has focused on divergence between tectonic trenches to understand how environment and geography may drive species divergence and promote endemism. There has been little attempt to examine localized genetic structure within trenches, partly because of logistical challenges associated with sampling at an appropriate scale, and the large effective population sizes of species that can be sampled adequately may mask underlying genetic structure. Here we examine genetic structure in the superabundant amphipod Hirondellea gigas in the Mariana Trench at depths of 8126-10,545 m. RAD sequencing was used to identify 3182 loci containing 43,408 single nucleotide polymorphisms (SNPs) across individuals after stringent pruning of loci to prevent paralogous multicopy genomic regions being erroneously merged. Principal components analysis of SNP genotypes resolved no genetic structure between sampling locations, consistent with a signature of panmixia. However, discriminant analysis of principal components identified divergence between all sites driven by 301 outlier SNPs in 169 loci and significantly associated with latitude and depth. Functional annotation of loci identified differences between singleton loci used in analysis and paralogous loci pruned from the data set and also between outlier and nonoutlier loci, all consistent with hypotheses explaining the role of transposable elements driving genome dynamics. This study challenges the traditional perspective that highly abundant amphipods within a trench form a single panmictic population. We discuss the findings in relation to eco-evolutionary and ontogenetic processes operating in the deep sea, and highlight key challenges associated with population genetic analysis in nonmodel systems with inherent large effective population sizes and genomes.

Plasticity is a locally adapted trait with consequences for ecological dynamics in novel environments.

Phenotypic plasticity is predicted to evolve in more variable environments, conferring an advantage on individual lifetime fitness. It is less clear what the potential consequences of that plasticity will have on ecological population dynamics. Here, we use an invertebrate model system to examine the effects of environmental variation (resource availability) on the evolution of phenotypic plasticity in two life history traits-age and size at maturation-in long-running, experimental density-dependent environments. Specifically, we then explore the feedback from evolution of life history plasticity to subsequent ecological dynamics in novel conditions. Plasticity in both traits initially declined in all microcosm environments, but then evolved increased plasticity for age-at-maturation, significantly so in more environmentally variable environments. We also demonstrate how plasticity affects ecological dynamics by creating founder populations of different plastic phenotypes into new microcosms that had either familiar or novel environments. Populations originating from periodically variable environments that had evolved greatest plasticity had lowest variability in population size when introduced to novel environments than those from constant or random environments. This suggests that while plasticity may be costly it can confer benefits by reducing the likelihood that offspring will experience low survival through competitive bottlenecks in variable environments. In this study, we demonstrate how plasticity evolves in response to environmental variation and can alter population dynamics-demonstrating an eco-evolutionary feedback loop in a complex animal moderated by plasticity in growth.

Surviving winter on the Qinghai-Tibetan Plateau: Pikas suppress energy demands and exploit yak feces to survive winter.

The Qinghai-Tibetan Plateau, with low precipitation, low oxygen partial pressure, and temperatures routinely dropping below -30 °C in winter, presents several physiological challenges to its fauna. Yet it is home to many endemic mammalian species, including the plateau pika (Ochotona curzoniae). How these small animals that are incapable of hibernation survive the winter is an enigma. Measurements of daily energy expenditure (DEE) using the doubly labeled water method show that pikas suppress their DEE during winter. At the same body weight, pikas in winter expend 29.7% less than in summer, despite ambient temperatures being approximately 25 °C lower. Combined with resting metabolic rates (RMRs), this gives them an exceptionally low metabolic scope in winter (DEE/RMRt = 1.60 ± 0.30; RMRt is resting metabolic rate at thermoneutrality). Using implanted body temperature loggers and filming in the wild, we show that this is achieved by reducing body temperature and physical activity. Thyroid hormone (T3 and T4) measurements indicate this metabolic suppression is probably mediated via the thyroid axis. Winter activity was lower at sites where domestic yak (Bos grunniens) densities were higher. Pikas supplement their food intake at these sites by eating yak feces, demonstrated by direct observation, identification of yak DNA in pika stomach contents, and greater convergence in the yak/pika microbiotas in winter. This interspecific coprophagy allows pikas to thrive where yak are abundant and partially explains why pika densities are higher where domestic yak, their supposed direct competitors for food, are more abundant.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

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.

Bioaccumulation of persistent organic pollutants in the deepest ocean fauna.

The legacy and reach of anthropogenic influence is most clearly evidenced by its impact on the most remote and inaccessible habitats on Earth. Here we identify extraordinary levels of persistent organic pollutants in the endemic amphipod fauna from two of the deepest ocean trenches (>10,000 metres). Contaminant levels were considerably higher than documented for nearby regions of heavy industrialization, indicating bioaccumulation of anthropogenic contamination and inferring that these pollutants are pervasive across the world's oceans and to full ocean depth.

Estimating demographic contributions to effective population size in an age-structured wild population experiencing environmental and demographic stochasticity.

A population's effective size (Ne ) is a key parameter that shapes rates of inbreeding and loss of genetic diversity, thereby influencing evolutionary processes and population viability. However, estimating Ne , and identifying key demographic mechanisms that underlie the Ne to census population size (N) ratio, remains challenging, especially for small populations with overlapping generations and substantial environmental and demographic stochasticity and hence dynamic age-structure. A sophisticated demographic method of estimating Ne /N, which uses Fisher's reproductive value to account for dynamic age-structure, has been formulated. However, this method requires detailed individual- and population-level data on sex- and age-specific reproduction and survival, and has rarely been implemented. Here, we use the reproductive value method and detailed demographic data to estimate Ne /N for a small and apparently isolated red-billed chough (Pyrrhocorax pyrrhocorax) population of high conservation concern. We additionally calculated two single-sample molecular genetic estimates of Ne to corroborate the demographic estimate and examine evidence for unobserved immigration and gene flow. The demographic estimate of Ne /N was 0.21, reflecting a high total demographic variance (σ2dg) of 0.71. Females and males made similar overall contributions to σ2dg. However, contributions varied among sex-age classes, with greater contributions from 3 year-old females than males, but greater contributions from ≥5 year-old males than females. The demographic estimate of Ne was ~30, suggesting that rates of increase of inbreeding and loss of genetic variation per generation will be relatively high. Molecular genetic estimates of Ne computed from linkage disequilibrium and approximate Bayesian computation were approximately 50 and 30, respectively, providing no evidence of substantial unobserved immigration which could bias demographic estimates of Ne . Our analyses identify key sex-age classes contributing to demographic variance and thus decreasing Ne /N in a small age-structured population inhabiting a variable environment. They thereby demonstrate how assessments of Ne can incorporate stochastic sex- and age-specific demography and elucidate key demographic processes affecting a population's evolutionary trajectory and viability. Furthermore, our analyses show that Ne for the focal chough population is critically small, implying that management to re-establish genetic connectivity may be required to ensure population viability.

Harvested populations are more variable only in more variable environments.

The interaction between environmental variation and population dynamics is of major importance, particularly for managed and economically important species, and especially given contemporary changes in climate variability. Recent analyses of exploited animal populations contested whether exploitation or environmental variation has the greatest influence on the stability of population dynamics, with consequences for variation in yield and extinction risk. Theoretical studies however have shown that harvesting can increase or decrease population variability depending on environmental variation, and requested controlled empirical studies to test predictions. Here, we use an invertebrate model species in experimental microcosms to explore the interaction between selective harvesting and environmental variation in food availability in affecting the variability of stage-structured animal populations over 20 generations. In a constant food environment, harvesting adults had negligible impact on population variability or population size, but in the variable food environments, harvesting adults increased population variability and reduced its size. The impact of harvesting on population variability differed between proportional and threshold harvesting, between randomly and periodically varying environments, and at different points of the time series. Our study suggests that predicting the responses to selective harvesting is sensitive to the demographic structures and processes that emerge in environments with different patterns of environmental variation.

Isolation and Characterization of Microsatellite DNA Markers in the Deep-Sea Amphipod Paralicella tenuipes by Illumina MiSeq Sequencing.

Here, we describe the development of 16 polymorphic microsatellite markers using an Illumina MiSeq sequencing approach in the deep-sea amphipod Paralicella tenuipes A total of 25 577 844 DNA sequences were filtered for microsatellite motifs of which 197 873 sequences were identified. From these sequences, 64 had sufficient flanking regions for primer design and 16 of these loci were polymorphic. Between 5 and 30 alleles were detected per locus, with an average of 13.63 alleles per locus, across a total of 120 individuals from 5 separate deep sea trenches from the Pacific Ocean. For the 16 loci, observed and expected heterozygosity values ranged from 0.116 to 0.414 and 0.422 to 0.820, respectively, with one locus displaying significant deviation from Hardy-Weinberg equilibrium. The microsatellite loci that have been isolated and described here are the first molecular markers developed for deep sea amphipods and will be invaluable for elucidating the genetic population structure and the extent of connectivity between deep ocean trenches.

Evidence of the phenotypic expression of a lethal recessive allele under inbreeding in a wild population of conservation concern.

Deleterious recessive alleles that are masked in outbred populations are predicted to be expressed in small, inbred populations, reducing both individual fitness and population viability. However, there are few definitive examples of phenotypic expression of lethal recessive alleles under inbreeding conditions in wild populations. Studies that demonstrate the action of such alleles, and infer their distribution and dynamics, are required to understand their potential impact on population viability and inform management responses. The Scottish population of red-billed choughs (Pyrrhocorax pyrrhocorax), which currently totals <60 breeding pairs and is of major conservation concern, has recently been affected by lethal blindness in nestlings. We used family data to show that the pattern of occurrence of blindness within and across affected families that produced blind nestlings was exactly 0·25, matching that expected given a single-locus autosomal lethal recessive allele. Furthermore, the observed distribution of blind nestlings within affected families did not differ from that expected given Mendelian inheritance of such an allele. Relatedness estimates showed that individuals from affected families were not more closely related to each other than they were to individuals from unaffected families that did not produce blind nestlings. Blind individuals tended to be less heterozygous than non-blind individuals, as expected if blindness was caused by the expression of a recessive allele under inbreeding. However, there was no difference in the variance in heterozygosity estimates, suggesting that some blind individuals were relatively outbred. These results suggest carriers of the blindness allele may be widely distributed across contemporary families rather than restricted to a single family lineage, implying that the allele has persisted across multiple generations. Blindness occurred at low frequency (affecting 1·6% of observed nestlings since 1981). However, affected families had larger initial brood sizes than unaffected families. Such high fecundity of carriers of a lethal recessive allele might reflect overdominance, potentially reducing purging and increasing allele persistence probability. We thereby demonstrate the phenotypic expression of a lethal recessive allele in a wild population of conservation concern, and provide a general framework for inferring allele distribution and persistence and informing management responses.

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 F(ST) 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.

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 host's 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).

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 (F(SC) = 0.0227; P < 0.001) and genetically (F(SC) = 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 (F(ST) 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.

Eco-evolutionary dynamics in response to selection on life-history.

Understanding the consequences of environmental change on ecological and evolutionary dynamics is inherently problematic because of the complex interplay between them. Using invertebrates in microcosms, we characterise phenotypic, population and evolutionary dynamics before, during and after exposure to a novel environment and harvesting over 20 generations. We demonstrate an evolved change in life-history traits (the age- and size-at-maturity, and survival to maturity) in response to selection caused by environmental change (wild to laboratory) and to harvesting (juvenile or adult). Life-history evolution, which drives changes in population growth rate and thus population dynamics, includes an increase in age-to-maturity of 76% (from 12.5 to 22 days) in the unharvested populations as they adapt to the new environment. Evolutionary responses to harvesting are outweighed by the response to environmental change (~ 1.4 vs. 4% change in age-at-maturity per generation). The adaptive response to environmental change converts a negative population growth trajectory into a positive one: an example of evolutionary rescue.

Disease transmission in an extreme environment: nematode parasites infect reindeer during the Arctic winter.

Parasitic nematodes are found in almost all wild vertebrate populations but few studies have investigated these host-parasite relationships in the wild. For parasites with free-living stages, the external environment has a major influence on life-history traits, and development and survival is generally low at sub-zero temperatures. For reindeer that inhabit the high Arctic archipelago of Svalbard, parasite transmission is expected to occur in the summer, due to the extreme environmental conditions and the reduced food intake by the host in winter. Here we show experimentally that, contrary to most parasitic nematodes, Marshallagia marshalli of Svalbard reindeer is transmitted during the Arctic winter. Winter transmission was demonstrated by removing parasites in the autumn, using a novel delayed-release anthelmintic bolus, and estimating re-infection rates in reindeer sampled in October, February and April. Larval stages of nematodes were identified using molecular tools, whereas adult stages were identified using microscopy. The abundance of M. marshalli adult worms and L4s increased significantly from October to April, indicating that reindeer were being infected with L3s from the pasture throughout the winter. To our knowledge, this study is the first to experimentally demonstrate over-winter transmission of a gastro-intestinal nematode parasite in a wild animal. Potential mechanisms associated with this unusual transmission strategy are discussed in light of our knowledge of the life-history traits of this parasite.

Selection maintains MHC diversity through a natural population bottleneck.

A perceived consequence of a population bottleneck is the erosion of genetic diversity and concomitant reduction in individual fitness and evolutionary potential. Although reduced genetic variation associated with demographic perturbation has been amply demonstrated for neutral molecular markers, the effective management of genetic resources in natural populations is hindered by a lack of understanding of how adaptive genetic variation will respond to population fluctuations, given these are affected by selection as well as drift. Here, we demonstrate that selection counters drift to maintain polymorphism at a major histocompatibility complex (MHC) locus through a population bottleneck in an inbred island population of water voles. Before and after the bottleneck, MHC allele frequencies were close to balancing selection equilibrium but became skewed by drift when the population size was critically low. MHC heterozygosity generally conformed to Hardy-Weinberg expectations except in one generation during the population recovery where there was a significant excess of heterozygous genotypes, which simulations ascribed to strong differential MHC-dependent survival. Low allelic diversity and highly skewed frequency distributions at microsatellite loci indicated potent genetic drift due to a strong founder affect and/or previous population bottlenecks. This study is a real-time examination of the predictions of fundamental evolutionary theory in low genetic diversity situations. The findings highlight that conservation efforts to maintain the genetic health and evolutionary potential of natural populations should consider the genetic basis for fitness-related traits, and how such adaptive genetic diversity will vary in response to both the demographic fluctuations and the effects of selection.

Genetic differentiation among North Atlantic killer whale populations.

Population genetic structure of North Atlantic killer whale samples was resolved from differences in allele frequencies of 17 microsatellite loci, mtDNA control region haplotype frequencies and for a subset of samples, using complete mitogenome sequences. Three significantly differentiated populations were identified. Differentiation based on microsatellite allele frequencies was greater between the two allopatric populations than between the two pairs of partially sympatric populations. Spatial clustering of individuals within each of these populations overlaps with the distribution of particular prey resources: herring, mackerel and tuna, which each population has been seen predating. Phylogenetic analyses using complete mitogenomes suggested two populations could have resulted from single founding events and subsequent matrilineal expansion. The third population, which was sampled at lower latitudes and lower density, consisted of maternal lineages from three highly divergent clades. Pairwise population differentiation was greater for estimates based on mtDNA control region haplotype frequencies than for estimates based on microsatellite allele frequencies, and there were no mitogenome haplotypes shared among populations. This suggests low or no female migration and that gene flow was primarily male mediated when populations spatially and temporally overlap. These results demonstrate that genetic differentiation can arise through resource specialization in the absence of physical barriers to gene flow.

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach.

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.