Scott A. Pavey

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION DURING SPECIATION‐WITH‐GENE‐FLOW IN LAKE WHITEFISH SPECIES PAIRS ASSESSED BY RAD SEQUENCING

During speciation‐with‐gene‐flow, effective migration varies across the genome as a function of several factors, including proximity of selected loci, recombination rate, strength of selection, and number of selected loci. Genome scans may provide better empirical understanding of the genome‐wide patterns of genetic differentiation, especially if the variance due to the previously mentioned factors is partitioned. In North American lake whitefish (Coregonus clupeaformis), glacial lineages that diverged in allopatry about 60,000 years ago and came into contact 12,000 years ago have independently evolved in several lakes into two sympatric species pairs (a normal benthic and a dwarf limnetic). Variable degrees of reproductive isolation between species pairs across lakes offer a continuum of genetic and phenotypic divergence associated with adaptation to distinct ecological niches. To disentangle the complex array of genetically based barriers that locally reduce the effective migration rate between whitefish species pairs, we compared genome‐wide patterns of divergence across five lakes distributed along this divergence continuum. Using restriction site associated DNA (RAD) sequencing, we combined genetic mapping and population genetics approaches to identify genomic regions resistant to introgression and derive empirical measures of the barrier strength as a function of recombination distance. We found that the size of the genomic islands of differentiation was influenced by the joint effects of linkage disequilibrium maintained by selection on many loci, the strength of ecological niche divergence, as well as demographic characteristics unique to each lake. Partial parallelism in divergent genomic regions likely reflected the combined effects of polygenic adaptation from standing variation and independent changes in the genetic architecture of postzygotic isolation. This study illustrates how integrating genetic mapping and population genomics of multiple sympatric species pairs provide a window on the speciation‐with‐gene‐flow mechanism.

Mapping phenotypic, expression and transmission ratio distortion QTL using RAD markers in the Lake Whitefish (Coregonus clupeaformis)

The evolution of reproductive isolation in an ecological context may involve multiple facets of species divergence on which divergent selection may operate. These include variation in quantitative phenotypic traits, regulation of gene expression, and differential transmission of particular allelic combinations. Thus, an integrative approach to the speciation process involves identifying the genetic basis of these traits, in order to understand how they are affected by divergent selection in nature and how they ultimately contribute to reproductive isolation. In the Lake Whitefish (Coregonus clupeaformis), dwarf and normal species pairs sympatrically occur in several North American postglacial lakes. The limnetic dwarf whitefish distinguishes from its normal benthic relative by numerous life history, behavioural, morphological and gene expression traits, in relation with the exploitation of distinct ecological niches. Here, we have applied the RAD‐Sequencing method to a hybrid backcross family to reconstruct a high‐density genetic linkage map and perform QTL mapping in the Lake Whitefish. The 3061 cM map encompassed 3438 segregating RAD markers distributed over 40 linkage groups, for an average resolution of 0.89 cM. We mapped phenotypic and expression QTL underlying ecologically important traits as well as transmission ratio distortion QTL, and identified genomic regions harbouring clusters of such QTL. A narrow genomic region strongly associated with sex determination was also evidenced. Positional and functional information revealed in this study will be useful in ongoing population genomic studies to illuminate our understanding of the genomic architecture of reproductive isolation between whitefish species pairs.

Gene Coexpression Networks Reveal Key Drivers of Phenotypic Divergence in Lake Whitefish

A functional understanding of processes involved in adaptive divergence is one of the awaiting opportunities afforded by high-throughput transcriptomic technologies. Functional analysis of coexpressed genes has succeeded in the biomedical field in identifying key drivers of disease pathways. However, in ecology and evolutionary biology, functional interpretation of transcriptomic data is still limited. Here, we used Weighted Gene Co-Expression Network Analysis (WGCNA) to identify modules of coexpressed genes in muscle and brain tissue of a lake whitefish backcross progeny. Modules were connected to gradients of known adaptive traits involved in the ecological speciation process between benthic and limnetic ecotypes. Key drivers, that is, hub genes of functional modules related to reproduction, growth, and behavior were identified, and module preservation was assessed in natural populations. Using this approach, we identified modules of coexpressed genes involved in phenotypic divergence and their key drivers, and further identified a module part specifically rewired in the backcross progeny. Functional analysis of transcriptomic data can significantly contribute to the understanding of the mechanisms underlying ecological speciation. Our findings point to bone morphogenetic protein and calcium signaling as common pathways involved in coordinated evolution of trophic behavior, trophic morphology (gill rakers), and reproduction. Results also point to pathways implicating hemoglobins and constitutive stress response (HSP70) governing growth in lake whitefish.

RAD sequencing reveals within‐generation polygenic selection in response to anthropogenic organic and metal contamination in North Atlantic Eels

Measuring the effects of selection on the genome imposed by human‐altered environment is currently a major goal in ecological genomics. Given the polygenic basis of most phenotypic traits, quantitative genetic theory predicts that selection is expected to cause subtle allelic changes among covarying loci rather than pronounced changes at few loci of large effects. The goal of this study was to test for the occurrence of polygenic selection in both North Atlantic eels (European Eel, Anguilla anguilla and American Eel, A. rostrata), using a method that searches for covariation among loci that would discriminate eels from ‘control’ vs. ‘polluted’ environments and be associated with specific contaminants acting as putative selective agents. RAD‐seq libraries resulted in 23 659 and 14 755 filtered loci for the European and American Eels, respectively. A total of 142 and 141 covarying markers discriminating European and American Eels from ‘control’ vs. ‘polluted’ sampling localities were obtained using the Random Forest algorithm. Distance‐based redundancy analyses (db‐RDAs) were used to assess the relationships between these covarying markers and concentration of 34 contaminants measured for each individual eel. PCB153, 4′4′DDE and selenium were associated with covarying markers for both species, thus pointing to these contaminants as major selective agents in contaminated sites. Gene enrichment analyses suggested that sterol regulation plays an important role in the differential survival of eels in ‘polluted’ environment. This study illustrates the power of combining methods for detecting signals of polygenic selection and for associating variation of markers with putative selective agents in studies aiming at documenting the dynamics of selection at the genomic level and particularly so in human‐altered environments.

What is needed for next-generation ecological and evolutionary genomics?

Ecological and evolutionary genomics (EEG) aims to link gene functions and genomic features to phenotypes and ecological factors. Although the rapid development of technologies allows central questions to be addressed at an unprecedented level of molecular detail, they do not alleviate one of the major challenges of EEG, which is that a large fraction of genes remains without any annotation. Here, we propose two solutions to this challenge. The first solution is in the form of a database that regroups associations between genes, organismal attributes and abiotic and biotic conditions. This database would result in an ecological annotation of genes by allowing cross-referencing across studies and taxa. Our second solution is to use new functional techniques to characterize genes implicated in the response to ecological challenges.

Nonparallelism in MHCIIβ diversity accompanies nonparallelism in pathogen infection of lake whitefish (Coregonus clupeaformis) species pairs as revealed by next-generation sequencing.

Major histocompatibility (MHC) immune system genes may evolve in response to pathogens in the environment. Because they also may affect mate choice, they are candidates for having great importance in ecological speciation. Here, we use next‐generation sequencing to test the general hypothesis of parallelism in patterns of MHCIIβ diversity and bacterial infections among five dwarf and normal whitefish sympatric pairs. A second objective was to assess the functional relationships between specific MHCIIβ alleles and pathogens in natural conditions. Each individual had between one and four alleles, indicating two paralogous loci. In Cliff Lake, the dwarf ecotype was monomorphic for the most common allele. In Webster Lake, the skew in the allelic distribution was towards the same allele but in the normal ecotype, underscoring the nonparallel divergence among lakes. Our signal of balancing selection matched putative peptide binding region residues in some cases, but not in others, supporting other recent findings of substantial functional differences in fish MHCIIβ compared with mammals. Individuals with fewer alleles were less likely to be infected; thus, we found no evidence for the heterozygote advantage hypothesis. MHCIIβ alleles and pathogenic bacteria formed distinct clusters in multivariate analyses, and clusters of certain alleles were associated with clusters of pathogens, or sometimes the absence of pathogens, indicating functional relationships at the individual level. Given that patterns of MHCIIβ and bacteria were nonparallel among dwarf and normal whitefish pairs, we conclude that pathogens driving MHCIIβ evolution did not play a direct role in their parallel phenotypic evolution.

Neutral and selective processes shape MHC gene diversity and expression in stocked brook charr populations (Salvelinus fontinalis)

The capacity of an individual to battle infection is an important fitness determinant in wild vertebrate populations. The major histocompatibility complex (MHC) genes are crucial for a hosts adaptive immune system to detect pathogens. However, anthropogenic activities may disrupt natural cycles of co‐evolution between hosts and pathogens. In this study, we investigated the dynamic sequence and expression variation of host parasite interactions in brook charr (Salvelinus fontinalis) in a context of past human disturbance via population supplementation from domestic individuals. To do so, we developed a new method to examine selection shaping MHC diversity within and between populations and found a complex interplay between neutral and selective processes that varied between lakes that were investigated. We provided evidence for a lower introgression rate of domestic alleles and found that parasite infection increased with domestic genomic background of individuals. We also documented an association between individual MHC alleles and parasite taxa. Finally, longer cis‐regulatory minisatellites were positively correlated with MHC II down‐regulation and domestic admixture, suggesting that inadvertent selection during domestication resulted in a lower immune response capacity, through a trade‐off between growth and immunity, which explained the negative selection of domestic alleles at least under certain circumstances.

How does salinity influence habitat selection and growth in juvenile American eels Anguilla rostrata

The influence of salinity on habitat selection and growth in juvenile American eels Anguilla rostrata captured in four rivers across eastern Canada was assessed in controlled experiments in 2011 and 2012. Glass eels were first categorized according to their salinity preferences towards fresh (FW), salt (SW) or brackish water (BW) and the growth rate of each group of elvers was subsequently monitored in controlled FW and BW environments for 7 months. Most glass eels (78-89%) did not make a choice, i.e. they remained in BW. Salinity preferences were not influenced by body condition, although a possible role of pigmentation could not be ruled out. Glass eels that did make a choice displayed a similar preference for FW (60-75%) regardless of their geographic origin but glass eels from the St Lawrence Estuary displayed a significantly higher locomotor activity than those from other regions. Neither the salinity preferences showed by glass eels in the first experiment nor the rearing salinities appeared to have much influence on growth during the experiments. Elvers from Nova Scotia, however, reached a significantly higher mass than those from the St Lawrence Estuary thus supporting the hypothesis of genetically (or epigenetically) based differences for growth between A. rostrata from different origins. These results provide important ecological knowledge for the sustained exploitation and conservation of this threatened species.

Microbiome investigation in the ecological speciation context of lake whitefish (Coregonus clupeaformis) using next‐generation sequencing

Few studies have applied NGS methods to investigate the microbiome of vertebrates in their natural environment and in freshwater fishes in particularly. Here, we used pyrosequencing of the 16S gene rRNA to (i) test for differences in kidney bacterial communities (i.e. microbiota) of dwarf and normal whitefish found as sympatric pairs, (ii) test the hypothesis of higher bacterial diversity in normal compared with dwarf whitefish and (iii) test for the occurrence of parallelism with the presence and composition of bacterial communities across species pairs inhabiting different lakes. The kidney microbiota of 253 dwarf and normal whitefish from five lakes was analysed combining a double‐nested PCR approach with 454 pyrosequencing. Bacteria were detected in 52.6% of the analysed whitefish. There was no overall significant difference among lakes and forms, although the lake × form interaction was found significant. We identified 579 bacterial genera, which is substantially more than previous descriptions using less sensitive techniques of fish bacterial diversity in kidney, pathogenic or not. Ten of these genera contained eighteen pathogenic species. Differences in bacteria composition between whitefish forms were not parallel among lakes. In accordance with the higher diversity of prey types, normal whitefish kidney tissue consistently had a more diverse bacterial community and this pattern was parallel among lakes. These results add to building evidence from previous studies on this system that the adaptive divergence of dwarf, and normal whitefish has been driven by both parallel and nonparallel ecological conditions across lakes.

A fast, highly sensitive double-nested PCR-based method to screen fish immunobiomes

Efficient methods for constructing 16S tag amplicon libraries for pyrosequencing are needed for the rapid and thorough screening of infectious bacterial diversity from host tissue samples. Here we have developed a double‐nested PCR methodology that generates 16S tag amplicon libraries from very small amounts of bacteria/host samples. This methodology was tested for 133 kidney samples from the lake whitefish Coregonus clupeaformis (Salmonidae) sampled in five different lake populations. The double‐nested PCR efficiency was compared with two other PCR strategies: single primer pair amplification and simple nested PCR. The double‐nested PCR was the only amplification strategy to provide highly specific amplification of bacterial DNA. The resulting 16S amplicon libraries were synthesized and pyrosequenced using 454 FLX technology to analyse the variation of pathogenic bacteria abundance. The proportion of the community sequenced was very high (Good’s coverage estimator; mean = 95.4%). Furthermore, there were no significant differences of sequence coverage among samples. Finally, the occurrence of chimeric amplicons was very low. Therefore, the double‐nested PCR approach provides a rapid, informative and cost‐effective method for screening fish immunobiomes and most likely applicable to other low‐density microbiomes as well.