Diving into broad-scale and high-resolution population genomics to decipher drivers of structure and climatic vulnerability in a marine invertebrate.

Species with widespread distributions play a crucial role in our understanding of climate change impacts on population structure. In marine species, population structure is often governed by both high connectivity potential and selection across strong environmental gradients. Despite the complexity of factors influencing marine populations, studying species with broad distribution can provide valuable insights into the relative importance of these factors and the consequences of climate-induced alterations across environmental gradients. We used the northern shrimp Pandalus borealis and its wide latitudinal distribution to identify current drivers of population structure and predict the species' vulnerability to climate change. A total of 1514 individuals sampled across 24° latitude were genotyped at high geographic (54 stations) and genetic (14,331 SNPs) resolutions to assess genetic variation and environmental correlations. Four populations were identified in addition to finer substructure associated with local adaptation. Geographic patterns of neutral population structure reflected predominant oceanographic currents, while a significant proportion of the genetic variation was associated with gradients in salinity and temperature. Adaptive landscapes generated using climate projections suggest a larger genomic offset in the southern extent of the P. borealis range, where shrimp had the largest adaptive standing genetic variation. Our genomic results combined with recent observations point to further deterioration in southern regions and an impending vulnerable status in the regions at higher latitudes for P. borealis. They also provide rare insights into the drivers of population structure and climatic vulnerability of a widespread meroplanktonic species, which is crucial to understanding future challenges associated with invertebrates essential to ecosystem functioning.

Acceptable Loss: Fitness Consequences of Salinity-Induced Cell Death in a Halotolerant Microalga.

AbstractEnvironmentally induced reductions in fitness components (survival, fecundity) are generally considered as passive, maladaptive responses to stress. However, there is also mounting evidence for active, programmed forms of environmentally induced cell death in unicellular organisms. While conceptual work has questioned how such programmed cell death (PCD) might be maintained by natural selection, few experimental studies have investigated how PCD influences genetic differences in longer-term fitness across environments. Here, we tracked the population dynamics of two closely related strains of the halotolerant microalga Dunaliella salina following transfers across salinities. We showed that after a salinity increase, only one of these strains displayed a massive population decline (-69% in 1 h), largely attenuated by exposure to a PCD inhibitor. However, this decline was followed by a rapid demographic rebound, characterized by faster growth than the nondeclining strain, such that sharper decline was correlated with faster subsequent growth across experiments and conditions. Strikingly, the decline was more pronounced in conditions more favorable to growth (more light, more nutrients, less competition), further suggesting that it was not simply passive. We explored several hypotheses that could explain this decline-rebound pattern, which suggests that successive stresses could select for higher environmentally induced death in this system.

Phenotypic plasticity evolves at multiple biological levels in response to environmental predictability in a long-term experiment with a halotolerant microalga.

Phenotypic plasticity, the change in the phenotype of a given genotype in response to its environment of development, is a ubiquitous feature of life, enabling organisms to cope with variation in their environment. Theoretical studies predict that, under stationary environmental variation, the level of plasticity should evolve to match the predictability of selection at the timing of development. However, the extent to which patterns of evolution of plasticity for more integrated traits are mirrored by their underlying molecular mechanisms remains unclear, especially in response to well-characterized selective pressures exerted by environmental predictability. Here, we used experimental evolution with the microalgae Dunaliella salina under controlled environmental fluctuations, to test whether the evolution of phenotypic plasticity in responses to environmental predictability (as measured by the squared autocorrelation ρ2) occurred across biological levels, going from DNA methylation to gene expression to cell morphology. Transcriptomic analysis indicates clear effects of salinity and ρ2 × salinity interaction on gene expression, thus identifying sets of genes involved in plasticity and its evolution. These transcriptomic effects were independent of DNA methylation changes in cis. However, we did find ρ2-specific responses of DNA methylation to salinity change, albeit weaker than for gene expression. Overall, we found consistent evolution of reduced plasticity in less predictable environments for DNA methylation, gene expression, and cell morphology. Our results provide the first clear empirical signature of plasticity evolution at multiple levels in response to environmental predictability, and highlight the importance of experimental evolution to address predictions from evolutionary theory, as well as investigate the molecular basis of plasticity evolution.

Plasticity across levels: Relating epigenomic, transcriptomic, and phenotypic responses to osmotic stress in a halotolerant microalga.

Phenotypic plasticity, the ability of a given genotype to produce alternative phenotypes in response to its environment of development, is an important mechanism for coping with variable environments. While the mechanisms underlying phenotypic plasticity are diverse, their relative contributions need to be investigated quantitatively to better understand the evolvability of plasticity across biological levels. This requires relating plastic responses of the epigenome, transcriptome, and organismal phenotype, and investigating how they vary with the genotype. Here we carried out this approach for responses to osmotic stress in Dunaliella salina, a green microalga that is a model organism for salinity tolerance. We compared two strains that show markedly different demographic responses to osmotic stress, and showed that these phenotypic responses involve strain- and environment-specific variation in gene expression levels, but a relative low-albeit significant-effect of strain × environment interaction. We also found an important genotype effect on the genome-wide methylation pattern, but little contribution from environmental conditions to the latter. However, we did detect a significant marginal effect of epigenetic variation on gene expression, beyond the influence of genetic differences on epigenetic state, and we showed that hypomethylated regions are correlated with higher gene expression. Our results indicate that epigenetic mechanisms are either not involved in the rapid plastic response to environmental change in this species, or involve only few changes in trans that are sufficient to trigger concerted changes in the expression of many genes, and phenotypic responses by multiple traits.

Using phenotypic plasticity to understand the structure and evolution of the genotype-phenotype map.

Deciphering the genotype-phenotype map necessitates relating variation at the genetic level to variation at the phenotypic level. This endeavour is inherently limited by the availability of standing genetic variation, the rate of spontaneous mutation to novo genetic variants, and possible biases associated with induced mutagenesis. An interesting alternative is to instead rely on the environment as a source of variation. Many phenotypic traits change plastically in response to the environment, and these changes are generally underlain by changes in gene expression. Relating gene expression plasticity to the phenotypic plasticity of more integrated organismal traits thus provides useful information about which genes influence the development and expression of which traits, even in the absence of genetic variation. We here appraise the prospects and limits of such an environment-for-gene substitution for investigating the genotype-phenotype map. We review models of gene regulatory networks, and discuss the different ways in which they can incorporate the environment to mechanistically model phenotypic plasticity and its evolution. We suggest that substantial progress can be made in deciphering this genotype-environment-phenotype map, by connecting theory on gene regulatory network to empirical patterns of gene co-expression, and by more explicitly relating gene expression to the expression and development of phenotypes, both theoretically and empirically.

Reduced phenotypic plasticity evolves in less predictable environments.

Phenotypic plasticity is a prominent mechanism for coping with variable environments, and a key determinant of extinction risk. Evolutionary theory predicts that phenotypic plasticity should evolve to lower levels in environments that fluctuate less predictably, because they induce mismatches between plastic responses and selective pressures. However, this prediction is difficult to test in nature, where environmental predictability is not controlled. Here, we exposed 32 lines of the halotolerant microalga Dunaliella salina to ecologically realistic, randomly fluctuating salinity, with varying levels of predictability, for 500 generations. We found that morphological plasticity evolved to lower degrees in lines that experienced less predictable environments. Evolution of plasticity mostly concerned phases with slow population growth, rather than the exponential phase where microbes are typically phenotyped. This study underlines that long-term experiments with complex patterns of environmental change are needed to test theories about population responses to altered environmental predictability, as currently observed under climate change.

Sources of epigenetic variation and their applications in natural populations.

Epigenetic processes manage gene expression and products in a real-time manner, allowing a single genome to display different phenotypes. In this paper, we discussed the relevance of assessing the different sources of epigenetic variation in natural populations. For a given genotype, the epigenetic variation could be environmentally induced or occur randomly. Strategies developed by organisms to face environmental fluctuations such as phenotypic plasticity and diversified bet-hedging rely, respectively, on these different sources. Random variation can also represent a proxy of developmental stability and can be used to assess how organisms deal with stressful environmental conditions. We then proposed the microbiome as an extension of the epigenotype of the host to assess the factors determining the establishment of the community of microorganisms. Finally, we discussed these perspectives in the applied context of conservation.

Epigenetic anticipation for food and reproduction.

Physiological changes in anticipation of cyclic environmental events are common for the persistence of populations in fluctuating environments (e.g. seasons). However, dealing with sporadic resources such as the intermittent production of seed masting trees may be challenging unless reliable cues also make them predictable. To be adaptive, the anticipation of such episodic events would have to trigger the corresponding physiological response. Epigenetic modifications could result in such physiological anticipatory responses to future changes. The eastern chipmunk (Tamias striatus) is known to adjust its reproductive activity to match juvenile weaning with peak seed availability of masting trees, which are essential for their survival. We therefore expected that epigenetic changes would be linked to spring reproductive initiation in anticipation for beech seed availability in fall. We correlated the variation of DNA methylation profiles of 114 adult chipmunks captured in May with beech seeds abundance in September, over 4 years, for three distinct populations, as well as individuals sampled twice during reproductive and non-reproductive years. The significant correlation between spring epigenetic variation and the amount of food in the fall confirmed the phenotypic flexibility of individuals according to environmental fluctuations. Altogether, these results underlined the key role of epigenetic processes in anticipatory responses enabling organisms to persist in fluctuating environments.

Inferring responses to climate warming from latitudinal pattern of clonal hybridization.

Climate warming may affect reproductive isolation between sympatric sister species by modifying reproductive phenology or mate choice. This is expected to result in a latitudinal progression of hybridization in response to the shifting of environmental conditions. The fish species northern redbelly dace (Chrosomus eos) and finescale dace (C. neogaeus) display a wide sympatric distribution in North America. The asexual reproduction of their hybrids allows determining where and when hybridization occurred. The aim of this study was twofold: first, to assess whether temperature affected reproductive isolation, and second, whether the effects of climate warming resulted in a latitudinal progression of hybridization. We performed a 500 km latitudinal survey (51 sites) in southeastern Quebec (Canada) and determined the distribution of clonal hybrid lineages. Results revealed a total of 78 hybrid lineages, including 70 which originated locally. We detected a significant difference between the southern and northern range of the survey in terms of the proportion of sites harboring local hybrids (20/23 vs. 8/28 sites, respectively) and hybrid diversity (57 vs. 13 lineages, respectively). This confirmed that there was more frequent interspecific mating in the warmest sites. In the southern range, diversity of lineages and simulations suggest that hybridization first took place (>7,000 years) in sites characterized by a longer growing season, followed by northerly adjacent sites (ca. 3,500-5,000 years). Moreover, evidence of hybridization occurring in present-day time was detected. This suggests that the current warming episode is going beyond the limits of the previous warmest period of the Holocene.

Unexpected Oogenic Pathways for the Triploid Fish Chrosomus eos-neogaeus.

Triploid vertebrates from unisexual complexes often perpetuate themselves asexually. In the fish Chrosomus eos×eos-neogaeus, triploids are continuously produced by diploid hybrids. However, they are not expected to perpetuate since C. eos are their only known progeny. This study aims to investigate the oogenesis of these triploid hybrids through experimental crosses. A total of 337 larvae from 12 female triploids and 3 2n/3n mosaics fertilized with C. eos sperm were genetically characterized. The detection of C. eos as progeny of triploid hybrids confirmed the occurrence of a pathway similar to meiotic hybridogenesis but only for half of the tripoids. The presence of tetraploid offspring for all these females revealed the formation of unreduced triploid eggs as a probable failure of meiotic hybridogenesis. The remaining female triploids and all mosaics produced diploid and triploid hybrids. Triploids excluded the haplome from paternal leakage and produced eggs with the diploid hybrid genome through an ameiotic hybridogenesis. Both types of hybridogenesis occurred in a mutually exclusive manner. This leads us to consider 2 hypothetical scenarios: First, any female triploids can perform either type of hybridogenesis, allowing the long-term persistence of triploid hybrids by a fraction of the population. Alternatively, ameiotic hybridogenesis occurs in triploids of the first generation (from diploid mothers), while meiotic hybridogenesis occurs in triploids of the second generation (from triploid mothers); triploid hybrids then are not perpetuating lineages. The population dynamics of the C. eos-neogaeus complex appears a step more complicated than previously expected.

A trait-based ecology to assess the acclimation of a sperm-dependent clonal fish compared to its sexual host.

BACKGROUND: Survival in temporally or spatially changing environments is a prerequisite for the perpetuation of a given species. In addition to genetic variation, the role of epigenetic processes is crucial in the persistence of organisms. For instance, mechanisms such as developmental flexibility enable the adjustment of the phenotype of a given individual to changing conditions throughout its development. However, the extent of factors other than genetic variability, like epigenetic processes, in the production of alternative phenotype and the consequences in realized ecological niches is still unclear. METHODS: In this study, we compared the extent of realized niches between asexual and sexual individuals from different environments. We used a trait-based ecology approach exploiting trophic and locomotive structures to infer the environment that each biotype actually used. More specifically, we compared the morphology of the all-female clonal and sperm-dependent fish Chrosomus eos-neogaeus to that of their sexual host species C. eos in common garden and natural conditions. RESULTS: Transfer from natural to controlled conditions resulted in a similar shift in measured morphology for clonal and sexual individuals suggesting comparable level of flexibility in both kinds of organisms. However, clonal, but not sexual, individuals displayed a consistent phenotype when reared in uniform conditions indicating that in absence of genetic variation, one phenotype corresponds to one niche. This contrasted with results from natural conditions where clones were morphologically as variable as sexual individuals within a sampled site. In addition, similar phenotypic changes for both clonal and sexual individuals were observed among the majority of sampled sites, indicating that they responded similarly to the same environments. DISCUSSION: Our results indicated that clones can efficiently use different niches and may evolve in a range of environmental conditions comparable to that of a sexual species, thus underlying the importance of factors other than genetic variability, like epigenetic processes, for coping with environmental heterogeneity.

Gene flow prevents mitonuclear co-adaptation: A comparative portrait of sympatric wild types and cybrids in the fish Chrosomus eos.

Allospecific mtDNA can occasionally be beneficial for the fitness of populations. It is, however, difficult to assess the effect of mtDNA in natural conditions due to genetic and/or environmental interactions. In the fish Chrosomus eos, the transfer of C. neogaeus mitochondria occurs in a single generation and results in natural cybrids. For a few lakes in Quebec, C. eos can harbor either a C. eos mtDNA (wild types) or a C. neogaeus mtDNA (cybrids). Moreover, mtDNA of cybrids originated either from Mississippian or Atlantic glacial refuges. Such diversity provides a useful system for in situ assessment of allospecific mtDNA effects. We determined genetic, epigenetic and transcriptomic variation as well as mitochondrial enzymatic activity (complex IV) changes among wild types and cybrids either in sympatry or allopatry. Wild types and cybrids did not segregate spatially within a lake. Moreover, no significant genetic differentiation was detected among wild types and cybrids indicating sustained gene flow. Mitochondrial complex IV activity was higher for cybrids in both sympatry and allopatry while no difference was detected among cybrid haplotypes. Epigenetic and transcriptomic analyses revealed only subtle differences between sympatric wild types and cybrids compared to differences between sites. Altogether, these results indicate a limited influence of allospecific mtDNA in nuclear gene expression when controlling for genetic and environmental effects. The absence of a reproductive barrier between wild types and cybrids results in random association of either C. eos or C. neogaeus mtDNA with C. eos nDNA at each generation, and prevents mitonuclear co-adaptation in sympatry.

The effects of allospecific mitochondrial genome on the fitness of northern redbelly dace (Chrosomus eos).

Instantaneous mitochondrial introgression events allow the disentangling of the effects of hybridization from those of allospecific mtDNA. Such process frequently occurred in the fish Chrosomus eos, resulting in cybrid individuals composed of a C. eos nuclear genome but with a C. neogaeus mtDNA. This provides a valuable model to address the fundamental question: How well do introgressed individuals perform in their native environment? We infer where de novo production of cybrids occurred to discriminate native environments from those colonized by cybrids in 25 sites from two regions (West-Qc and East-Qc) in Quebec (Canada). We then compared the relative abundance of wild types and cybrids as a measure integrating both fitness and de novo production of cybrids. According to mtDNA variation, 12 introgression events are required to explain the diversity of cybrids. Five cybrid lineages could not be associated with in situ introgression events. This includes one haplotype carried by 93% of the cybrids expected to have colonized West-Qc. These cybrids also displayed a nearly complete allopatric distribution with wild types. We still inferred de novo production of cybrids at seven sites, that accounted for 70% of the cybrids in East-Qc. Wild-type and cybrid individuals coexist in all East-Qc sites while cybrids were less abundant. Allopatry of cybrids restricted to the postglacial expansion suggests the existence of higher fitness for cybrids in specific conditions, allowing for the colonization of different environments and expanding the species' range. However, allospecific mtDNA does not provide a higher fitness to cybrids in their native environment compared to wild types, making the success of an introgressed lineage uncertain.

Imitating the cost of males: A hypothesis for coexistence of all-female sperm-dependent species and their sexual host.

All-female sperm-dependent species are particular asexual organisms that must coexist with a closely related sexual host for reproduction. However, demographic advantages of asexual over sexual species that have to produce male individuals could lead both to extinction. The unresolved question of their coexistence still challenges and fascinates evolutionary biologists. As an alternative hypothesis, we propose those asexual organisms are afflicted by a demographic cost analogous to the production of males to prevent exclusion of the host. Previously proposed hypotheses stated that asexual individuals relied on a lower fecundity than sexual females to cope with demographic advantage. In contrast, we propose that both sexual and asexual species display the same number of offspring, but half of asexual individuals imitate the cost of sex by occupying ecological niches but producing no offspring. Simulations of population growth in closed systems under different demographic scenarios revealed that only the presence of nonreproductive individuals in asexual females can result in long-term coexistence. This hypothesis is supported by the fact that half of the females in some sperm-dependent organisms did not reproduce clonally.

Balancing selection on the number of repeats in the ribosomal intergenic spacer present in naturally occurring yellow perch (Perca flavescens) populations.

The ribosomal intergenic spacer (IGS), responsible for the rate of transcription of rRNA genes, is associated with the growth and fecundity of individuals. A previous study of IGS length variants in a yellow perch (Perca flavescens) population revealed the presence of two predominant alleles differing by 1 kb due to variation in the number of repeat units. This study aims to assess whether length variation of IGS is the result of selection in natural populations. Length variation of IGS and 11 neutral microsatellite loci were assessed in geographically distant yellow perch populations. Most populations displayed the very same IGS alleles; they did not differ in frequencies among populations and the FST was not significantly different from zero. In contrast, diversity at microsatellite loci was high and differed among populations (FST = 0.18). Selection test based on FST identified IGS as a significant outlier from neutral expectations for population differentiation. Heterozygote excess was also detected in one specific cohort, suggesting temporal variation in the selection regime. While the exact mechanism remains to be specified, together the results of this study support the contention that balancing selection is acting to maintain two distinct IGS alleles in natural fish populations.

Asymmetry in dentition and shape of pharyngeal arches in the clonal fish Chrosomus eos-neogaeus: Phenotypic plasticity and developmental instability.

The effect of the environment may result in different developmental outcomes. Extrinsic signals can modify developmental pathways and result in alternative phenotypes (phenotypic plasticity). The environment can also be interpreted as a stressor and increase developmental instability (developmental noise). Directional and fluctuating asymmetry provide a conceptual background to discriminate between these results. This study aims at assessing whether variation in dentition and shape of pharyngeal arches of the clonal fish Chrosomus eos-neogaeus results from developmental instability or environmentally induced changes. A total of 262 specimens of the Chrosomus eos-neogaeus complex from 12 natural sites were analysed. X-ray microcomputed tomography (X-ray micro-CT) was used to visualize the pharyngeal arches in situ with high resolution. Variation in the number of pharyngeal teeth is high in hybrids in contrast to the relative stability observed in both parental species. The basal dental formula is symmetric while the most frequent alternative dental formula is asymmetric. Within one lineage, large variation in the proportion of individuals bearing basal or alternative dental formulae was observed among sites in the absence of genetic difference. Both dentition and arch shape of this hybrid lineage were explained significantly by environmental differences. Only individuals bearing asymmetric dental formula displayed fluctuating asymmetry as well as directional left-right asymmetry for the arches. The hybrids appeared sensitive to environmental signals and intraspecific variation on pharyngeal teeth was not random but reflects phenotypic plasticity. Altogether, these results support the influence of the environment as a trigger for an alternative developmental pathway resulting in left-right asymmetry in dentition and shape of pharyngeal arches.

Inconsistent phylogeographic pattern between a sperm dependent fish and its host: in situ hybridization vs dispersal.

BACKGROUND: Co-dispersal of sperm-dependent hybrids and their sexual relatives is expected to result in consistent spatial patterns between assemblages of hybrids and genetic structure of parental species. However, local hybridization events may blur this signal as assemblages could be organized under different connectivity constraints. This study aims at testing the hypothesis of local hybridization events by comparing the assemblage of hybrid fish Chrosomus eos-neogaeus to the genetic diversity of one of its parental species, Chrosomus eos. RESULTS: An extensive survey performed on a total of 132 sites located in two regions of Southern Quebec (West-Qc and East-Qc) revealed a distinct organization of hybrid lineages. One of the six hybrid lineages detected in West-Qc is widespread throughout this region resulting in a low α-diversity (1.38) and ß-diversity (4.35). On the other hand, 36 hybrid lineages were detected in East-Qc and displayed narrow geographic distributions leading to a high α-diversity (2.30) and ß-diversity (15.68). In addition, the C. eos multilocus haplotype of several of these hybrids is assigned to their respective sympatric C. eos population. Finally, contrasting with hybrids, the paternal species C. eos displayed a higher ρST in West-Qc (0.2300) than in East-Qc (0.0734). CONCLUSION: The unusually high diversity of hybrid lineages in East-Qc as well as the spatial organization and the close genetic relationship with C. eos sympatric populations support the hypothesis that multiple hybridization events occurred in situ. These findings coupled to the near absence of the maternal species Chrosomus neogeaus suggest that the decline of this species could be the trigger event at the origin of the high rates of spontaneous hybridization in this region.

Facing environmental predictability with different sources of epigenetic variation.

Different sources of epigenetic changes can increase the range of phenotypic options. Environmentally induced epigenetic changes and stochastic epimutations are, respectively, associated with phenotypic plasticity and diversifying bet-hedging. Their relative contribution is thus expected to reflect the capacity of a genotype to face distinct changes since these strategies are differentially selected according to environmental uncertainty. To test this hypothesis, we assessed the sources of epigenetic changes on clonal fish from predictable (lakes) or unpredictable (intermittent streams) environments. DNA methylation of clones from natural conditions revealed contrasting contribution of environmentally induced versus stochastic changes according to their origins. These differences were validated in common garden experiments. Consistent with theoretical models, distinct sources of epigenetic variation prevail according to the environmental uncertainty. However, both sources act conjointly, suggesting that plasticity and random processes are complementary strategies. This represents a rigorous approach for further exploring the capacity of organisms to respond to environmental conditions.