Chromosomal inversion polymorphisms are widespread across the species ranges of rough periwinkles (Littorina saxatilis and L. arcana).

Inversions are thought to play a key role in adaptation and speciation, suppressing recombination between diverging populations. Genes influencing adaptive traits cluster in inversions, and changes in inversion frequencies are associated with environmental differences. However, in many organisms, it is unclear if inversions are geographically and taxonomically widespread. The intertidal snail, Littorina saxatilis, is one such example. Strong associations between putative polymorphic inversions and phenotypic differences have been demonstrated between two ecotypes of L. saxatilis in Sweden and inferred elsewhere, but no direct evidence for inversion polymorphism currently exists across the species range. Using whole genome data from 107 snails, most inversion polymorphisms were found to be widespread across the species range. The frequencies of some inversion arrangements were significantly different among ecotypes, suggesting a parallel adaptive role. Many inversions were also polymorphic in the sister species, L. arcana, hinting at an ancient origin.

Differing associations between sex determination and sex-linked inversions in two ecotypes of Littorina saxatilis.

Sexual antagonism is a common hypothesis for driving the evolution of sex chromosomes, whereby recombination suppression is favored between sexually antagonistic loci and the sex-determining locus to maintain beneficial combinations of alleles. This results in the formation of a sex-determining region. Chromosomal inversions may contribute to recombination suppression but their precise role in sex chromosome evolution remains unclear. Because local adaptation is frequently facilitated through the suppression of recombination between adaptive loci by chromosomal inversions, there is potential for inversions that cover sex-determining regions to be involved in local adaptation as well, particularly if habitat variation creates environment-dependent sexual antagonism. With these processes in mind, we investigated sex determination in a well-studied example of local adaptation within a species: the intertidal snail, Littorina saxatilis. Using SNP data from a Swedish hybrid zone, we find novel evidence for a female-heterogametic sex determination system that is restricted to one ecotype. Our results suggest that four putative chromosomal inversions, two previously described and two newly discovered, span the putative sex chromosome pair. We determine their differing associations with sex, which suggest distinct strata of differing ages. The same inversions are found in the second ecotype but do not show any sex association. The striking disparity in inversion-sex associations between ecotypes that are connected by gene flow across a habitat transition that is just a few meters wide indicates a difference in selective regime that has produced a distinct barrier to the spread of the newly discovered sex-determining region between ecotypes. Such sex chromosome-environment interactions have not previously been uncovered in L. saxatilis and are known in few other organisms. A combination of both sex-specific selection and divergent natural selection is required to explain these highly unusual patterns.

Genetic architecture of repeated phenotypic divergence in Littorina saxatilis ecotype evolution.

Chromosomal inversions have been shown to play a major role in a local adaptation by suppressing recombination between alternative arrangements and maintaining beneficial allele combinations. However, so far, their importance relative to the remaining genome remains largely unknown. Understanding the genetic architecture of adaptation requires better estimates of how loci of different effect sizes contribute to phenotypic variation. Here, we used three Swedish islands where the marine snail Littorina saxatilis has repeatedly evolved into two distinct ecotypes along a habitat transition. We estimated the contribution of inversion polymorphisms to phenotypic divergence while controlling for polygenic effects in the remaining genome using a quantitative genetics framework. We confirmed the importance of inversions but showed that contributions of loci outside inversions are of similar magnitude, with variable proportions dependent on the trait and the population. Some inversions showed consistent effects across all sites, whereas others exhibited site-specific effects, indicating that the genomic basis for replicated phenotypic divergence is only partly shared. The contributions of sexual dimorphism as well as environmental factors to phenotypic variation were significant but minor compared to inversions and polygenic background. Overall, this integrated approach provides insight into the multiple mechanisms contributing to parallel phenotypic divergence.

Gene expression studies of plastic and evolutionary responses to global warming.

Phenotypic plasticity can be a rapid response for coping with global warming, yet may be insufficient to protect species from extinction. Evolutionary adaptation may reinforce adaptive or oppose maladaptive plastic responses. With advances in technology whole transcriptomes can provide us with an unprecedented overview of genes and functional processes underlying the interplay between plasticity and evolution. We advocate that insects provide ideal opportunities to study plasticity in non-adapted and thermally adapted populations to infer reaction norms across the whole transcriptome ('reactionomes'). This can advance our understanding of how the interplay between plasticity and evolution shapes responses to warming. So far, a limited number of studies suggest predominantly maladaptive plastic responses to novel environments that are reduced with time, but much more research is needed to infer general patterns.

Genetic variation for adaptive traits is associated with polymorphic inversions in Littorina saxatilis.

Chromosomal inversions have long been recognized for their role in local adaptation. By suppressing recombination in heterozygous individuals, they can maintain coadapted gene complexes and protect them from homogenizing effects of gene flow. However, to fully understand their importance for local adaptation we need to know their influence on phenotypes under divergent selection. For this, the marine snail Littorina saxatilis provides an ideal study system. Divergent ecotypes adapted to wave action and crab predation occur in close proximity on intertidal shores with gene flow between them. Here, we used F2 individuals obtained from crosses between the ecotypes to test for associations between genomic regions and traits distinguishing the Crab-/Wave-adapted ecotypes including size, shape, shell thickness, and behavior. We show that most of these traits are influenced by two previously detected inversion regions that are divergent between ecotypes. We thus gain a better understanding of one important underlying mechanism responsible for the rapid and repeated formation of ecotypes: divergent selection acting on inversions. We also found that some inversions contributed to more than one trait suggesting that they may contain several loci involved in adaptation, consistent with the hypothesis that suppression of recombination within inversions facilitates differentiation in the presence of gene flow.

Genetic variance in fitness and its cross-sex covariance predict adaptation during experimental evolution.

The additive genetic variation (VA ) of fitness in a population is of particular importance to quantify its adaptive potential and predict its response to rapid environmental change. Recent statistical advances in quantitative genetics and the use of new molecular tools have fostered great interest in estimating fitness VA in wild populations. However, the value of VA for fitness in predicting evolutionary changes over several generations remains mostly unknown. In our study, we addressed this question by combining classical quantitative genetics with experimental evolution in the model organism Tribolium castaneum (red flour beetle) in three new environmental conditions (Dry, Hot, Hot-Dry). We tested for potential constraints that might limit adaptation, including environmental and sex genetic antagonisms captured by negative genetic covariance between environments and female and male fitness, respectively. Observed fitness changes after 20 generations mainly matched our predictions. Given that body size is commonly used as a proxy for fitness, we also tested how this trait and its genetic variance (including nonadditive genetic variance) were impacted by environmental stress. In both traits, genetic variances were sex and condition dependent, but they differed in their variance composition, cross-sex and cross-environment genetic covariances, as well as in the environmental impact on VA .

Restoring ancestral phenotypes is a general pattern in gene expression evolution during adaptation to new environments in Tribolium castaneum.

Plasticity and evolution are two processes allowing populations to respond to environmental changes, but how both are related and impact each other remains controversial. We studied plastic and evolutionary responses in gene expression of Tribolium castaneum after exposure of the beetles to new environments that differed from ancestral conditions in temperature, humidity or both. Using experimental evolution with 10 replicated lines per condition, we were able to demonstrate adaptation after 20 generations. We measured whole-transcriptome gene expression with RNA-sequencing to infer evolutionary and plastic changes. We found more evidence for changes in mean expression (shift in the intercept of reaction norms) in adapted lines than for changes in plasticity (shifts in slopes). Plasticity was mainly preserved in selected lines and was responsible for a large part of the phenotypic divergence in expression between ancestral and new conditions. However, we found that genes with the largest evolutionary changes in expression also evolved reduced plasticity and often showed expression levels closer to the ancestral stage. Results obtained in the three different conditions were similar, suggesting that restoration of ancestral expression levels during adaptation is a general evolutionary pattern. With a larger sample in the most stressful condition, we were able to detect a positive correlation between the proportion of genes with reversion of the ancestral plastic response and mean fitness per selection line.

Additive and mostly adaptive plastic responses of gene expression to multiple stress in Tribolium castaneum.

Gene expression is known to be highly responsive to the environment and important for adjustment of metabolism but there is also growing evidence that differences in gene regulation contribute to species divergence and differences among locally adapted populations. However, most studies so far investigated populations when divergence had already occurred. Selection acting on expression levels at the onset of adaptation to an environmental change has not been characterized. Understanding the mechanisms is further complicated by the fact that environmental change is often multivariate, meaning that organisms are exposed to multiple stressors simultaneously with potentially interactive effects. Here we use a novel approach by combining fitness and whole-transcriptome data in a large-scale experiment to investigate responses to drought, heat and their combination in Tribolium castaneum. We found that fitness was reduced by both stressors and their combined effect was almost additive. Expression data showed that stressor responses were acting independently and did not interfere physiologically. Since we measured expression and fitness within the same individuals, we were able to estimate selection on gene expression levels. We found that variation in fitness can be attributed to gene expression variation and that selection pressures were environment dependent and opposite between control and stress conditions. We could further show that plastic responses of expression were largely adaptive, i.e. in the direction that should increase fitness.

High gene flow despite opposite chirality in hybrid zones between enantiomorphic door snails.

We studied differentiation and geneflow patterns between enantiomorphic door-snail species in two hybrid zones in the Bucegi Mountains (Romania) to investigate the effects of intrinsic barriers (complications in copulation) and extrinsic selection by environmental factors. A mitochondrial gene tree confirmed the historical separation of the examined populations into the dextral Alopia livida and the sinistral Alopia straminicollis in accordance with the morphological classification, but also indicated gene flow between the species. By contrast, a network based on amplified fragment length polymorphisms (AFLP) markers revealed local groups of populations as units independent of their species affiliation. Admixture analyses based on AFLP data showed that the genomes of most individuals in the hybrid zones are composed of parts of the genomes of both parental taxa. The introgression patterns of a notable fraction of the examined markers deviated from neutral introgression. However, the patterns of most non-neutral markers were not concordant between the two hybrid zones. There was also no concordance between non-neutral markers in the two genomic clines and markers that were correlated with environmental variables or markers that were correlated with the proportion of dextral individuals in the populations. Neither extrinsic selection by environmental factors nor intrinsic barriers resulting from positive frequency-dependent selection of the prevailing coiling direction were sufficient to maintain the distinctness of A. straminicollis and A. livida. Despite being historically separated units, we conclude that these taxa now merge where they come into contact.

Presumable incipient hybrid speciation of door snails in previously glaciated areas in the Caucasus.

Homoploid hybrid speciation, speciation by hybridization without a change in chromosome number, may be the result of an encounter of closely related species in a habitat that is different from that usually occupied by these species. In the northwestern Caucasus the land snail species Micropontica caucasica and M. circassica form two distinct entities with little admixture at low and intermediate altitudes. However, at higher altitudes in the Lagonaki plateau, which were repeatedly glaciated, Micropontica populations with intermediate characters occur. Admixture analyses based on AFLP data demonstrated that the populations from the Lagonaki plateau are homoploid hybrids that now form a cluster separate from the parental species. The Lagonaki populations are characterized by a mtDNA haplotype clade that has been found in the parental species only once. The fixation of this haplotype clade in most hybrid populations suggests that these haplotypes are better adapted to the cooler conditions in high altitude habitats and have replaced the haplotypes of the parental species in a selective sweep. The fixation of a presumably adaptive mitochondrial haplotype clade in the Lagonaki populations is an important step towards speciation under the differential fitness species concept.