Sébastien Renaut

On the origin of species: insights from the ecological genomics of lake whitefish

In contrast to the large amount of ecological information supporting the role of natural selection as a main cause of population divergence and speciation, an understanding of the genomic basis underlying those processes is in its infancy. In this paper, we review the main findings of a long-term research programme that we have been conducting on the ecological genomics of sympatric forms of whitefish (Coregonus spp.) engaged in the process of speciation. We present this system as an example of how applying a combination of approaches under the conceptual framework of the theory of adaptive radiation has yielded substantial insight into evolutionary processes in a non-model species. We also discuss how the joint use of recent biotechnological developments will provide a powerful means to address issues raised by observations made to date. Namely, we present data illustrating the potential offered by combining next generation sequencing technologies with other genomic approaches to reveal the genomic bases of adaptive divergence and reproductive isolation. Given increasing access to these new genomic tools, we argue that non-model species studied in their ecological context such as whitefish will play an increasingly important role in generalizing knowledge of speciation.

Mining transcriptome sequences towards identifying adaptive single nucleotide polymorphisms in lake whitefish species pairs (Coregonus spp. Salmonidae)

Next‐generation sequencing allows the discovery of large numbers of single nucleotide polymorphisms (SNPs) in species where little genomic information was previously available. Here, we assembled, de novo, over 130 mb of non‐normalized cDNA using 454 pyrosequencing data from dwarf and normal lake whitefish and backcross hybrids. Our main goals were to gather a large data set of SNP markers, document their distribution within coding regions, evaluate the effect of species divergence on allele frequencies and combine results with previous genomic studies to identify candidate genes underlying the adaptive divergence of lake whitefish. We identified 6094 putative SNPs in 2674 contigs (mean size: 576 bp, range: 101–6116) and 1540 synonymous and 1734 non‐synonymous mutations for a genome‐wide non‐synonymous to synonymous substitution rate ratio (pN/pS) of 0.37. As expected based on the young age (<15 000 years) of whitefish species pair, the overall level of divergence between them was relatively weak. Yet, 89 SNPs showed pronounced allele frequency differences between sympatric normal and dwarf whitefish. Among these, SNPs in genes annotated to energy metabolic functions were the most abundant and this, in addition to previous experimental data at the gene expression and phenotypic level, brings compelling evidence that genes involved in energy metabolism are prime candidates explaining the adaptive divergence of lake whitefish species pairs. Finally, we unexpectedly identified 44 contigs annotated to transposable elements and these were predominantly composed of backcross hybrids sequences. This indicates an elevated activity of transposable elements, which could potentially contribute to the reduced fitness of hybrids previously documented.

Genomic islands of divergence are not affected by geography of speciation in sunflowers

Genomic studies of speciation often report the presence of highly differentiated genomic regions interspersed within a milieu of weakly diverged loci. The formation of these speciation islands is generally attributed to reduced inter-population gene flow near loci under divergent selection, but few studies have critically evaluated this hypothesis. Here, we report on transcriptome scans among four recently diverged pairs of sunflower (Helianthus) species that vary in the geographical context of speciation. We find that genetic divergence is lower in sympatric and parapatric comparisons, consistent with a role for gene flow in eroding neutral differences. However, genomic islands of divergence are numerous and small in all comparisons, and contrary to expectations, island number and size are not significantly affected by levels of interspecific gene flow. Rather, island formation is strongly associated with reduced recombination rates. Overall, our results indicate that the functional architecture of genomes plays a larger role in shaping genomic divergence than does the geography of speciation.

The availability of research data declines rapidly with article age.

Policies ensuring that research data are available on public archives are increasingly being implemented at the government [1], funding agency [2-4], and journal [5, 6] level. These policies are predicated on the idea that authors are poor stewards of their data, particularly over the long term [7], and indeed many studies have found that authors are often unable or unwilling to share their data [8-11]. However, there are no systematic estimates of how the availability of research data changes with time since publication. We therefore requested data sets from a relatively homogenous set of 516 articles published between 2 and 22 years ago, and found that availability of the data was strongly affected by article age. For papers where the authors gave the status of their data, the odds of a data set being extant fell by 17% per year. In addition, the odds that we could find a working e-mail address for the first, last, or corresponding author fell by 7% per year. Our results reinforce the notion that, in the long term, research data cannot be reliably preserved by individual researchers, and further demonstrate the urgent need for policies mandating data sharing via public archives.

RAD in the realm of next‐generation sequencing technologies

The first North American RAD Sequencing and Genomics Symposium, sponsored by Floragenex (http://www.floragenex.com/radmeeting/), took place in Portland, Oregon (USA) on 19 April 2011. This symposium was convened to promote and discuss the use of restriction‐site‐associated DNA (RAD) sequencing technologies. RAD sequencing is one of several strategies recently developed to increase the power of data generated via short‐read sequencing technologies by reducing their complexity ( Baird et al. 2008 ; Huang et al. 2009 ; Andolfatto et al. 2011 ; Elshire et al. 2011 ). RAD sequencing, as a form of genotyping by sequencing, has been effectively applied in genetic mapping and quantitative trait loci (QTL) analyses in a range of organisms including nonmodel, genetically highly heterogeneous organisms ( Table 1 ; Baird et al. 2008 ; Baxter et al. 2011 ; Chutimanitsakun et al. 2011 ; Pfender et al. 2011 ). RAD sequencing has recently found applications in phylogeography ( Emerson et al. 2010 ) and population genomics ( Hohenlohe et al. 2010 ). Considering the diversity of talks presented during this meeting, more developments are to be expected in the very near future.

Genome-wide patterns of divergence during speciation: the lake whitefish case study

The nature, size and distribution of the genomic regions underlying divergence and promoting reproductive isolation remain largely unknown. Here, we summarize ongoing efforts using young (12 000 yr BP) species pairs of lake whitefish (Coregonus clupeaformis) to expand our understanding of the initial genomic patterns of divergence observed during speciation. Our results confirmed the predictions that: (i) on average, phenotypic quantitative trait loci (pQTL) show higher FST values and are more likely to be outliers (and therefore candidates for being targets of divergent selection) than non-pQTL markers; (ii) large islands of divergence rather than small independent regions under selection characterize the early stages of adaptive divergence of lake whitefish; and (iii) there is a general trend towards an increase in terms of numbers and size of genomic regions of divergence from the least (East L.) to the most differentiated species pair (Cliff L.). This is consistent with previous estimates of reproductive isolation between these species pairs being driven by the same selective forces responsible for environment specialization. Altogether, dwarf and normal whitefish species pairs represent a continuum of both morphological and genomic differentiation contributing to ecological speciation. Admittedly, much progress is still required to more finely map and circumscribe genomic islands of speciation. This will be achieved through the use of next generation sequencing data but also through a better quantification of phenotypic traits moulded by selection as organisms adapt to new environmental conditions.

SNP signatures of selection on standing genetic variation and their association with adaptive phenotypes along gradients of ecological speciation in lake whitefish species pairs (Coregonus spp.).

As populations adapt to novel environments, divergent selection will promote heterogeneous genomic differentiation via reductions in gene flow for loci underlying adaptive traits. Using a data set of over 100 SNP markers, genome scans were performed to investigate the effect of natural selection maintaining differentiation in five lakes harbouring sympatric pairs of normal and dwarf lake whitefish (Coregonus clupeaformis). A variable proportion of SNPs (between 0% and 12%) was identified as outliers, which corroborated the predicted intensity of competitive interactions unique to each lake. Moreover, strong reduction in heterozygosity was typically observed for outlier loci in dwarf but not in normal whitefish, indicating that directional selection has been acting on standing genetic variation more intensively in dwarf whitefish. SNP associations in backcross hybrid progeny identified 16 genes exhibiting genotype–phenotype associations for four adaptive traits (growth, swimming activity, gill rakers and condition factor). However, neither simple relationship between elevated levels of genetic differentiation with adaptive phenotype nor conspicuous genetic signatures for parallelism at outlier loci were detected, which underscores the importance of independent evolution among lakes. The integration of phenotypic, transcriptomic and functional genomic information identified two candidate genes (sodium potassium ATPase and triosephosphate isomerase) involved in the recent ecological divergence of lake whitefish. Finally, the identification of several markers under divergent selection suggests that many genes, in an environment‐specific manner, are recruited by selection and ultimately contributed to the repeated ecological speciation of a dwarf phenotype.

The transcriptomics of sympatric dwarf and normal lake whitefish (Coregonus clupeaformis spp., Salmonidae) divergence as revealed by next-generation sequencing

Gene expression divergence is one of the mechanisms thought to be involved in the emergence of incipient species. Next‐generation sequencing has become an extremely valuable tool for the study of this process by allowing whole transcriptome sequencing, or RNA‐Seq. We have conducted a 454 GS‐FLX pyrosequencing experiment to refine our understanding of adaptive divergence between dwarf and normal lake whitefish species (Coregonus clupeaformis spp.). The objectives were to: (i) investigate transcriptomic divergence as measured by liver RNA‐Seq; (ii) test the correlation between divergence in expression and sequence polymorphism; and (iii) investigate the extent of allelic imbalance. We also compared the results of RNA‐seq with those of a previous microarray study performed on the same fish. Following de novo assembly, results showed that normal whitefish overexpressed more contigs associated with protein synthesis while dwarf fish overexpressed more contigs related to energy metabolism, immunity and DNA replication and repair. Moreover, 63 SNPs showed significant allelic imbalance, and this phenomenon prevailed in the recently diverged dwarf whitefish. Results also showed an absence of correlation between gene expression divergence as measured by RNA‐Seq and either polymorphism rate or sequence divergence between normal and dwarf whitefish. This study reiterates an important role for gene expression divergence, and provides evidence for allele‐specific expression divergence as well as evolutionary decoupling of regulatory and coding sequences in the adaptive divergence of normal and dwarf whitefish. It also demonstrates how next‐generation sequencing can lead to a more comprehensive understanding of transcriptomic divergence in a young species pair.

The phenomics and expression quantitative trait locus mapping of brain transcriptomes regulating adaptive divergence in lake whitefish species pairs (Coregonus sp.)

We used microarrays and a previously established linkage map to localize the genetic determinants of brain gene expression for a backcross family of lake whitefish species pairs (Coregonus sp.). Our goals were to elucidate the genomic distribution and sex specificity of brain expression QTL (eQTL) and to determine the extent to which genes controlling transcriptional variation may underlie adaptive divergence in the recently evolved dwarf (limnetic) and normal (benthic) whitefish. We observed a sex bias in transcriptional genetic architecture, with more eQTL observed in males, as well as divergence in genome location of eQTL between the sexes. Hotspots of nonrandom aggregations of up to 32 eQTL in one location were observed. We identified candidate genes for species pair divergence involved with energetic metabolism, protein synthesis, and neural development on the basis of colocalization of eQTL for these genes with eight previously identified adaptive phenotypic QTL and four previously identified outlier loci from a genome scan in natural populations. Eighty-eight percent of eQTL-phenotypic QTL colocalization involved growth rate and condition factor QTL, two traits central to adaptive divergence between whitefish species pairs. Hotspots colocalized with phenotypic QTL in several cases, revealing possible locations where master regulatory genes, such as a zinc-finger protein in one case, control gene expression directly related to adaptive phenotypic divergence. We observed little evidence of colocalization of brain eQTL with behavioral QTL, which provides insight into the genes identified by behavioral QTL studies. These results extend to the transcriptome level previous work illustrating that selection has shaped recent parallel divergence between dwarf and normal lake whitefish species pairs and that metabolic, more than morphological, differences appear to play a key role in this divergence.

Gene Expression Divergence and Hybrid Misexpression between Lake Whitefish Species Pairs (Coregonus spp. Salmonidae)

Genomewide analyses of the transcriptome have confirmed that gene misexpression may underlie reproductive isolation mechanisms in interspecific hybrids. Here, using a 16,006 features cDNA microarray, we compared and contrasted gene expression divergence at two ontogenetic stages in incipient species of normal and dwarf whitefish (Coregonus clupeaformis) with that of first generation (normal x dwarf) and second-generation hybrid crosses (backcross: [normal x dwarf] x normal]. Our goal was to identify the main mode of action responsible for gene transcription and to discover key genes misexpressed in hybrids. Very few transcripts (five of 4,950 expressed) differed in mean expression level between parentals and hybrids at the embryonic stage, in contrast to 16-week-old juvenile fish for which 617 out of 5,359 transcripts differed significantly. We also found evidence for more misexpression in backcross hybrids whereby nonadditivity explained a larger fraction of hybrid inheritance patterns in backcross (54%) compared with F1-hybrids (9%). Gene expression in hybrids was more variable than in pure crosses and transgressive patterns of expression were ubiquitous in hybrids. In backcross embryos in particular, the expression of three key developmental genes involved in protein folding and mRNA translation was severely disrupted. Accordingly, gene misexpression in hybrids adds to other factors previously identified as contributing to the reproductive isolation of incipient species of lake whitefish.