Lake Whitefish Species Pairs Research Articles

Holobionts and ecological speciation: the intestinal microbiota of lake whitefish species pairs

BackgroundIt is well established that symbionts have considerable impact on their host, yet the investigation of the possible role of the holobiont in the host’s speciation process is still in its infancy. In this study, we compared the intestinal microbiota among five sympatric pairs of dwarf (limnetic) and normal (benthic) lake whitefish Coregonus clupeaformis representing a continuum in the early stage of ecological speciation. We sequenced the 16s rRNA gene V3-V4 regions of the intestinal microbiota present in a total of 108 wild sympatric dwarf and normal whitefish as well as the water bacterial community from five lakes to (i) test for differences between the whitefish intestinal microbiota and the water bacterial community and (ii) test for parallelism in the intestinal microbiota of dwarf and normal whitefish.ResultsThe water bacterial community was distinct from the intestinal microbiota, indicating that intestinal microbiota did not reflect the environment, but rather the intrinsic properties of the host microbiota. Our results revealed a strong influence of the host (dwarf or normal) on the intestinal microbiota with pronounced conservation of the core intestinal microbiota (mean ~ 44% of shared genera). However, no clear evidence for parallelism was observed, whereby non-parallel differences between dwarf and normal whitefish were observed in three of the lakes while similar taxonomic composition was observed for the two other species pairs.ConclusionsThis absence of parallelism across dwarf vs. normal whitefish microbiota highlighted the complexity of the holobiont and suggests that the direction of selection could be different between the host and its microbiota.

Modeling the Multiple Facets of Speciation-with-Gene-Flow toward Inferring the Divergence History of Lake Whitefish Species Pairs (Coregonus clupeaformis).

Parallel divergence across replicated species pairs occurring in similar environmental contrasts may arise through distinct evolutionary scenarios. Deciphering whether such parallelism actually reflects repeated parallel divergence driven by divergent selection or a single divergence event with subsequent gene flow needs to be ascertained. Reconstructing historical gene flow is therefore of fundamental interest to understand how demography and selection jointly shaped genomic divergence during speciation. Here, we use an extended modeling framework to explore the multiple facets of speciation-with-gene-flow with demo-genetic divergence models that capture both temporal and genomic variation in effective population size and migration rate. We investigate the divergence history of replicate sympatric species pairs of Lake Whitefish (normal benthic and dwarf limnetic) characterized by variable degrees of ecological divergence and reproductive isolation. Genome-wide SNPs were used to document the extent of genetic differentiation in each species pair, and 26 divergence models were fitted and compared with the unfolded joint allele frequency spectrum of each pair. We found evidence that a recent (circa 3,000–4,000 generations) asymmetrical secondary contact between expanding postglacial populations has accompanied Whitefish diversification. Our results suggest that heterogeneous genomic differentiation has emerged through the combined effects of linked selection generating variable rates of lineage sorting across the genome during geographical isolation, and heterogeneous introgression eroding divergence at different rates across the genome upon secondary contact. This study thus provides a new retrospective insight into the historical demographic and selective processes that shaped a continuum of divergence associated with ecological speciation.

Collapse of Lake WhitefishCoregonus clupeaformis(Mitchill, 1818) species pair in Como Lake, Ontario

Lake Whitefish (Coregonus clupeaformis) species pairs exist in a small number of northern temperate lakes in North America. Sympatric populations of dwarf and normal-sized whitefish represent a continuum of morphological and genetic divergence among these lakes. In this study, we used morphological and age data to assess the status of the Lake Whitefish species pair in Como Lake, Ontario (Canada). Whitefish were collected with gill-nets from spawning shoals during the fall of 2012 and 2014 and compared with archived (circa 1989) dwarf and normal-sized specimens. Our study indicates that the two previously collected forms have been replaced by a single form with a different morphology and age structure. In comparison to archived specimens, contemporary Lake Whitefish were larger, deeper-bodied and older; with more gill rakers, lateral line scales, smaller eyes and a wider interorbital width. We hypothesize that the recent invasion of Como Lake by Spiny Waterflea (Bythotrephes longimanus) has affected whitefish growth rates and changed the ecosystem conditions that previously maintained the species pair.

Standing chromosomal variation in Lake Whitefish species pairs: the role of historical contingency and relevance for speciation.

The role of chromosome changes in speciation remains a debated topic, although demographic conditions associated with divergence should promote their appearance. We tested a potential relationship between chromosome changes and speciation by studying two Lake Whitefish (Coregonus clupeaformis) lineages that recently colonized postglacial lakes following allopatry. A dwarf limnetic species evolved repeatedly from the normal benthic species, becoming reproductively isolated. Lake Whitefish hybrids experience mitotic and meiotic instability, which may result from structurally divergent chromosomes. Motivated by this observation, we test the hypothesis that chromosome organization differs between Lake Whitefish species pairs using cytogenetics. While chromosome and fundamental numbers are conserved between the species (2n=80, NF=98), we observe extensive polymorphism of subtle karyotype traits. We describe intrachromosomal differences associated with heterochromatin and repetitive DNA, and test for parallelism among three sympatric species pairs. Multivariate analyses support the hypothesis that differentiation at the level of subchromosomal markers mostly appeared during allopatry. Yet we find no evidence for parallelism between species pairs among lakes, consistent with colonization effect or postcolonization differentiation. The reported intrachromosomal polymorphisms do not appear to play a central role in driving adaptive divergence between normal and dwarf Lake Whitefish. We discuss how chromosomal differentiation in the Lake Whitefish system may contribute to the destabilization of mitotic and meiotic chromosome segregation in hybrids, as documented previously. The chromosome structures detected here are still difficult to sequence and assemble, demonstrating the value of cytogenetics as a complementary approach to understand the genomic bases of speciation.

Variable extent of parallelism in respiratory, circulatory, and neurological traits across lake whitefish species pairs

Parallel adaptive radiation events provide a powerful framework for investigations of ecology's contribution to phenotypic diversification. Ecologically driven divergence has been invoked to explain the repeated evolution of sympatric dwarf and normal lake whitefish (Coregonus clupeaformis) species in multiple lakes in eastern North America. Nevertheless, links between most putatively adaptive traits and ecological variation remain poorly defined within and among whitefish species pairs. Here, we examine four species pairs for variation in gill, heart, and brain size; three traits predicted to show strong phenotypic responses to ecological divergence. In each of the species pairs, normals exhibited larger body size standardized gills compared to dwarfs – a pattern that is suggestive of a common ecological driver of gill size divergence. Within lakes, the seasonal hypoxia experienced in the benthic environment is a likely factor leading to the requirement for larger gills in normals. Interestingly, the morphological pathways used to achieve larger gills varied between species pairs from Québec and Maine, which may imply subtle non-parallelism in gill size divergence related to differences in genetic background. There was also a non-significant trend toward larger hearts in dwarfs, the more active species of the two, whereas brain size varied exclusively among the lake populations. Taken together, our results suggest that the diversification of whitefish has been driven by parallel and non-parallel ecological conditions across lakes. Furthermore, the phenotypic response to ecological variation may depend on genetic background of each population.

BAC library construction, screening and clone sequencing of lake whitefish (Coregonus clupeaformis, Salmonidae) towards the elucidation of adaptive species divergence

Genomic DNA sequences and other genomic resources are essential towards the elucidation of the genomic bases of adaptive divergence and reproductive isolation. Here, we describe the construction, characterization and screening of a nonarrayed BAC library for lake whitefish (Coregonus clupeaformis). We then show how the combined use of BAC library screening and next-generation sequencing can lead to efficient full-length assembly of candidate genes. The lake whitefish BAC library consists of 181,050 clones derived from a single heterozygous fish. The mean insert size is 92 Kb, representing 5.2 haploid genome equivalents. Ten BAC clones were isolated following a quantitative real-time PCR screening approach that targeted five previously identified candidate genes. Sequencing of these clones on a 454 GS FLX system yielded 178,000 reads with a mean length of 358 bp, for a total of 63.8 Mb. De novo assembly and annotation then allowed retrieval of contigs corresponding to each candidate gene, which also contained up- and/or downstream noncoding sequences. These results suggest that the lake whitefish BAC library combined with next-generation sequencing technologies will be key resources to achieve a better understanding of both adaptive divergence and reproductive isolation in lake whitefish species pairs as well as salmonid evolution in general.

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.

Transcriptome-wide signature of hybrid breakdown associated with intrinsic reproductive isolation in lake whitefish species pairs (Coregonus spp. Salmonidae)

Genetic analyses of speciation have focused nearly exclusively on retrospective analyses of reproductive isolation between highly divergent species. Yet, a full understanding of the speciation process must encompass analysis of the consequences of genomic divergence in young lineages still capable of exchanging genes under natural conditions. The accumulation of conditionally neutral genetic variation may lead to the evolution of divergent gene networks. In a hybrid background, such mutations may no longer compensate one another, resulting in the appearance of selectively disadvantageous traits, including disruption of gene expression regulation. Here, we documented genome-wide patterns of gene expression divergence between young lineages of normal and dwarf lake whitefish and their backcross hybrids for which strong, yet incomplete post-zygotic isolation barriers exist. A significant proportion (33%) of backcross hybrids showed developmental abnormalities not seen in parental forms and eventually leading to death. Although the transcriptome of parental forms was nearly identical during embryonic development, suggesting a role for stabilizing selection, all hybrids displayed strongly divergent patterns of gene expression. By comparing healthy, surviving hybrids against moribund ones showing abnormal development, we observed that over 2000 genes were misregulated in these abnormal embryos. In particular, misregulation was significantly biased toward essential developmental genes, which were severely underexpressed. Furthermore, genes previously documented to be highly transgressive (exaggerated inter-individual variance) were almost invariably underexpressed in hybrids. Our results thus clearly showed a transcriptome-wide signature of hybrid breakdown in young, incipient species and demonstrated a persuasive link between misexpression of essential developmental genes and post-zygotic isolation.

Role of epibenthic resource opportunities in the parallel evolution of lake whitefish species pairs (Coregonus sp.)

Parallel evolution of a dwarf and normal whitefish has been documented in six post-glacial lakes. Here, we relate the structure and seasonal variations of the epibenthic invertebrate communities to the extent of phenotypic differentiation in these species pairs. The highest phenotypic differentiation occurs in lakes characterized by less overlap in size distribution between limnetic and epibenthic prey which could represent enhanced ecological opportunities for trophic specialization and adaptive divergence. Differences in community assemblages and seasonal variation of biotic and abiotic conditions may also play a role. Accumulating evidence indicates that strong directional selection acting on dwarf whitefish may be more important than divergent selection acting on both sympatric forms in driving whitefish phenotypic divergence and ultimately, ecological speciation. Along with Landry et al. (2007), this study supports the general hypothesis that parallelism in divergence among sympatric dwarf and normal whitefish is associated with parallelism in limnological adaptive landscape.

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 (p(N)/p(S)) 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.

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.

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.

Pervasive Sex-Linked Effects on Transcription Regulation As Revealed by Expression Quantitative Trait Loci Mapping in Lake Whitefish Species Pairs (Coregonus sp., Salmonidae)

Mapping of expression quantitative trait loci (eQTL) is a powerful means for elucidating the genetic architecture of gene regulation. Yet, eQTL mapping has not been applied toward investigating the regulation architecture of genes involved in the process of population divergence, ultimately leading to speciation events. Here, we conducted an eQTL mapping experiment to compare the genetic architecture of transcript regulation in adaptive traits, differentiating the recently evolved limnetic (dwarf) and benthic (normal) species pairs of lake whitefish. The eQTL were mapped in three data sets derived from an F(1) hybrid-dwarf backcrossed family: the entire set of 66 genotyped individuals and the two sexes treated separately. We identified strikingly more eQTL in the female data set (174), compared to both male (54) and combined (33) data sets. The majority of these genes were not differentially expressed between male and female progeny of the backcross family, thus providing evidence for a strong pleiotropic sex-linked effect in transcriptomic regulation. The subtelomeric region of a linkage group segregating in females encompassed >50% of all eQTL, which exhibited the most pronounced additive effects. We also conducted a direct comparison of transcriptomic profiles between pure dwarf and normal progeny reared in controlled conditions. We detected 34 differentially expressed transcripts associated with eQTL segregating only in sex-specific data sets and mostly belonging to functional groups that differentiate dwarf and normal whitefish in natural populations. Therefore, these eQTL are not related to interindividual variation, but instead to the adaptive and historical genetic divergence between dwarf and normal whitefish. This study exemplifies how the integration of genetic and transcriptomic data offers a strong means for dissecting the functional genomic response to selection by separating mapping family-specific effects from genetic factors under selection, potentially involved in the phenotypic divergence of natural populations.

The Genetic Architecture of Ecological Speciation and the Association with Signatures of Selection in Natural Lake Whitefish (Coregonus sp. Salmonidae) Species Pairs

Adaptive evolutionary change is contingent on variation and selection; thus, understanding adaptive divergence and ultimately speciation requires information on both the genetic basis of adaptive traits as well as an understanding of the role of divergent natural selection on those traits. The lake whitefish (Coregonus clupeaformis) consists of several sympatric "dwarf" (limnetic) and normal (benthic) species pairs that co-inhabit northern postglacial lakes. These young species pairs have evolved independently and display parallelism in life history, behavioral, and morphological divergence associated with the use of distinct trophic resources. We identified phenotype-environment associations and determined the genetic architecture and the role of selection modulating population genetic divergence in sympatric dwarf and normal lake whitefish. The genetic architecture of 9 adaptive traits was analyzed in 2 hybrid backcrosses individually phenotyped throughout their life history. Significant quantitative trait loci (QTL) were associated with swimming behavior (habitat selection and predator avoidance), growth rate, morphology (condition factor and gill rakers), and life history (onset of maturity and fecundity). Genome scans among 4 natural sympatric pairs, using loci segregating in the map, revealed a signature of selection for 24 loci. Loci exhibiting a signature of selection were associated with QTL relative to other regions of the genome more often than expected by chance alone. Two parallel QTL outliers for growth and condition factor exhibited segregation distortion in both mapping families, supporting the hypothesis that adaptive divergence contributing to parallel reductions of gene flow among natural populations may cause genetic incompatibilities. Overall, these findings offer evidence that the genetic architecture of ecological speciation is associated with signatures of selection in nature, providing strong support for the hypothesis that divergent natural selection is currently maintaining adaptive differentiation and promoting ecological speciation in lake whitefish species pairs.