Chromosome-scale Genome Assembly of the Rough Periwinkle Littorina saxatilis.

The intertidal gastropod Littorina saxatilis is a model system to study speciation and local adaptation. The repeated occurrence of distinct ecotypes showing different levels of genetic divergence makes L. saxatilis particularly suited to study different stages of the speciation continuum in the same lineage. A major finding is the presence of several large chromosomal inversions associated with the divergence of ecotypes and, specifically, the species offers a system to study the role of inversions in this divergence. The genome of L. saxatilis is 1.35 Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44 kb), and was quite incomplete, with a BUSCO completeness of 80.1% on the Metazoan dataset. A linkage map of one full-sibling family enabled the placement of 587 Mbp of the genome into 17 linkage groups corresponding to the haploid number of chromosomes, but the fragmented nature of this reference genome limited the understanding of the interplay between divergent selection and gene flow during ecotype formation. Here, we present a newly generated reference genome that is highly contiguous, with a N50 of 67 Mb and 90.4% of the total assembly length placed in 17 super-scaffolds. It is also highly complete with a BUSCO completeness of 94.1% of the Metazoa dataset. This new reference will allow for investigations into the genomic regions implicated in ecotype formation as well as better characterization of the inversions and their role in speciation.

Diverse pathways to speciation revealed by marine snails.

Speciation is a key evolutionary process that is not yet fully understood. Combining population genomic and ecological data from multiple diverging pairs of marine snails (Littorina) supports the search for speciation mechanisms. Placing pairs on a one-dimensional speciation continuum, from undifferentiated populations to species, obscured the complexity of speciation. Adding multiple axes helped to describe either speciation routes or reproductive isolation in the snails. Divergent ecological selection repeatedly generated barriers between ecotypes, but appeared less important in completing speciation while genetic incompatibilities played a key role. Chromosomal inversions contributed to genomic barriers, but with variable impact. A multidimensional (hypercube) approach supported framing of questions and identification of knowledge gaps and can be useful to understand speciation in many other systems.

The genetic basis of a recent transition to live-bearing in marine snails.

Key innovations are fundamental to biological diversification, but their genetic basis is poorly understood. A recent transition from egg-laying to live-bearing in marine snails (Littorina spp.) provides the opportunity to study the genetic architecture of an innovation that has evolved repeatedly across animals. Individuals do not cluster by reproductive mode in a genome-wide phylogeny, but local genealogical analysis revealed numerous small genomic regions where all live-bearers carry the same core haplotype. Candidate regions show evidence for live-bearer-specific positive selection and are enriched for genes that are differentially expressed between egg-laying and live-bearing reproductive systems. Ages of selective sweeps suggest that live-bearer-specific alleles accumulated over more than 200,000 generations. Our results suggest that new functions evolve through the recruitment of many alleles rather than in a single evolutionary step.

How chromosomal inversions reorient the evolutionary process.

Inversions are structural mutations that reverse the sequence of a chromosome segment and reduce the effective rate of recombination in the heterozygous state. They play a major role in adaptation, as well as in other evolutionary processes such as speciation. Although inversions have been studied since the 1920s, they remain difficult to investigate because the reduced recombination conferred by them strengthens the effects of drift and hitchhiking, which in turn can obscure signatures of selection. Nonetheless, numerous inversions have been found to be under selection. Given recent advances in population genetic theory and empirical study, here we review how different mechanisms of selection affect the evolution of inversions. A key difference between inversions and other mutations, such as single nucleotide variants, is that the fitness of an inversion may be affected by a larger number of frequently interacting processes. This considerably complicates the analysis of the causes underlying the evolution of inversions. We discuss the extent to which these mechanisms can be disentangled, and by which approach.

Strain-specific metabarcoding reveals rapid evolution of copper tolerance in populations of the coastal diatom Skeletonema marinoi.

Phytoplankton have short generation times, flexible reproduction strategies, large population sizes and high standing genetic diversity, traits that should facilitate rapid evolution under directional selection. We quantified local adaptation of copper tolerance in a population of the diatom Skeletonema marinoi from a mining-exposed inlet in the Baltic Sea and in a non-exposed population 100 km away. We hypothesized that mining pollution has driven evolution of elevated copper tolerance in the impacted population of S. marinoi. Assays of 58 strains originating from sediment resting stages revealed no difference in the average tolerance to copper between the two populations. However, variation within populations was greater at the mining site, with three strains displaying hyper-tolerant phenotypes. In an artificial evolution experiment, we used a novel intraspecific metabarcoding locus to track selection and quantify fitness of all 58 strains during co-cultivation in one control and one toxic copper treatment. As expected, the hyper-tolerant strains enabled rapid evolution of copper tolerance in the mining-exposed population through selection on available strain diversity. Within 42 days, in each experimental replicate a single strain dominated (30%-99% abundance) but different strains dominated the different treatments. The reference population developed tolerance beyond expectations primarily due to slowly developing plastic response in one strain, suggesting that different modes of copper tolerance are present in the two populations. Our findings provide novel empirical evidence that standing genetic diversity of phytoplankton resting stage allows populations to evolve rapidly (20-50 generations) and flexibly on timescales relevant for seasonal bloom progressions.

Psychometric properties of the Patient Health Questionnaire-4 (PHQ-4) in 9230 adults across seven European countries: Findings from the ESTSS ADJUST study.

BACKGROUND: The four-item Patient Health Questionnaire-4 (PHQ-4) is a widely used screening measure for depression and anxiety. OBJECTIVES: This study aimed to test factor structure and measurement invariance in an adult sample of the general population across seven European countries. METHOD: A total sample of 9230 adults, 71.3 % female, Mage = 44.35 (SD = 14.11) from seven countries (Austria, Croatia, Georgia, Germany, Lithuania, Portugal, and Sweden) participated in the study. We applied confirmatory factor analysis (CFA) to examine the factor structure and measurement invariance testing to evaluate measurement equivalence across countries, gender, and age groups. RESULTS: The CFA yielded that a two-factor PHQ-4 model with separate depression and anxiety factors had the best fit. Partial scalar measurement invariance was established across different groups based on gender, age, and country. CONCLUSIONS: The PHQ-4 is a valid and reliable measure that can be applied to screen for depression and anxiety in the general population. LIMITATIONS: The limitation of the study includes the sampling, which resulted in the sample structure with the majority of females, predominantly of high education and from urban communities.

Clones on the run: The genomics of a recently expanded partially clonal species.

Why species that in their core areas mainly reproduce sexually become enriched with clones in marginal populations ("geographic parthenogenesis") remains unclear. Earlier hypotheses have emphasized that selection might promote clonality because it protects locally adapted genotypes. On the other hand, it also hampers recombination and adaptation to changing conditions. The aim of the present study was to investigate the early stages of range expansion in a partially clonal species and what drives an increase in cloning during such expansion. We used genome-wide sequencing to investigate the origin and evolution of large clones formed in a macroalgal species (Fucus vesiculosus) during a recent expansion into the postglacial Baltic Sea. We found low but persistent clonality in core populations, while at range margins, large dominant clonal lineages had evolved repeatedly from different sexual populations. A range expansion model showed that even when asexual recruitment is less favourable than sexual recruitment in core populations, repeated bottlenecks at the expansion front can establish a genetically eroded clonal wave that spreads ahead of a sexual wave into the new area. Genetic variation decreases by drift following repeated bottlenecks at the expansion front. This results in the emerging clones having low expected heterozygosity, which corroborated our empirical observations. We conclude that Baker's Law (clones being favoured by uniparental reproductive assurance in new areas) can play an important role during range expansion in partially clonal species, resulting in a complex spatiotemporal mosaic of clonal and sexual lineages that might persist during thousands of generations.

The molecular background of the aspartate aminotransferase polymorphism in Littorina snails maintained by strong selection on small spatial scales.

Allozymes present several classical examples of divergent selection, including the variation in the cytosolic aspartate aminotransferase (AAT) in the intertidal snails Littorina saxatilis. AAT is a part of the asparate-malate shuttle, in the interidal molluscs involved in the anaerobic respiration during desiccation. Previous allozyme studies reported the sharp gradient in the frequencies of the AAT100and the AAT120 alleles between the low and high shores in the Northern Europe and the differences in their enzymatic activity, supporting the role of AAT in adaptation to desiccation. However, the populations in the Iberian Peninsula showed the opposite allele cline. Using the mRNA sequencing and the genome pool-seq analyses we characterize DNA sequences of the different AAT alleles, report the amino acid replacements behind the allozyme variation and show that same allozyme alleles in Northern and Southern populations have different protein sequences. Gene phylogeny reveals that the AAT100 and the northern AAT120 alleles represent the old polymorphism, shared among the closely related species of Littorina, while the southern AAT120 allele is more recently derived from AAT100. Further, we show that the Aat gene is expressed at constitutive level in different genotypes and conditions, supporting the role of structural variation in regulation of enzyme activity. Finally, we report the location and the structure of the gene in the L. saxatilis genome and the presence of two additional non-functional gene copies. Altogether, we provide a missing link between the classical allozyme studies and the genome scans and bring together the results produced over decades of the genetic research.

A metabarcoding analysis of the wrackbed microbiome indicates a phylogeographic break along the North Sea-Baltic Sea transition zone.

Sandy beaches are biogeochemical hotspots that bridge marine and terrestrial ecosystems via the transfer of organic matter, such as seaweed (termed wrack). A keystone of this unique ecosystem is the microbial community, which helps to degrade wrack and re-mineralize nutrients. However, little is known about this community. Here, we characterize the wrackbed microbiome as well as the microbiome of a primary consumer, the seaweed fly Coelopa frigida, and examine how they change along one of the most studied ecological gradients in the world, the transition from the marine North Sea to the brackish Baltic Sea. We found that polysaccharide degraders dominated both microbiomes, but there were still consistent differences between wrackbed and fly samples. Furthermore, we observed a shift in both microbial communities and functionality between the North and Baltic Sea driven by changes in the frequency of different groups of known polysaccharide degraders. We hypothesize that microbes were selected for their abilities to degrade different polysaccharides corresponding to a shift in polysaccharide content in the different seaweed communities. Our results reveal the complexities of both the wrackbed microbial community, with different groups specialized to different roles, and the cascading trophic consequences of shifts in the near shore algal community.

Ten years of marine evolutionary biology-Challenges and achievements of a multidisciplinary research initiative.

The Centre for Marine Evolutionary Biology (CeMEB) at the University of Gothenburg, Sweden, was established in 2008 through a 10-year research grant of 8.7 m€ to a team of senior researchers. Today, CeMEB members have contributed >500 scientific publications, 30 PhD theses and have organised 75 meetings and courses, including 18 three-day meetings and four conferences. What are the footprints of CeMEB, and how will the centre continue to play a national and international role as an important node of marine evolutionary research? In this perspective article, we first look back over the 10 years of CeMEB activities and briefly survey some of the many achievements of CeMEB. We furthermore compare the initial goals, as formulated in the grant application, with what has been achieved, and discuss challenges and milestones along the way. Finally, we bring forward some general lessons that can be learnt from a research funding of this type, and we also look ahead, discussing how CeMEB's achievements and lessons can be used as a springboard to the future of marine evolutionary biology.

Ten years of demographic modelling of divergence and speciation in the sea.

Understanding population divergence that eventually leads to speciation is essential for evolutionary biology. High species diversity in the sea was regarded as a paradox when strict allopatry was considered necessary for most speciation events because geographical barriers seemed largely absent in the sea, and many marine species have high dispersal capacities. Combining genome-wide data with demographic modelling to infer the demographic history of divergence has introduced new ways to address this classical issue. These models assume an ancestral population that splits into two subpopulations diverging according to different scenarios that allow tests for periods of gene flow. Models can also test for heterogeneities in population sizes and migration rates along the genome to account, respectively, for background selection and selection against introgressed ancestry. To investigate how barriers to gene flow arise in the sea, we compiled studies modelling the demographic history of divergence in marine organisms and extracted preferred demographic scenarios together with estimates of demographic parameters. These studies show that geographical barriers to gene flow do exist in the sea but that divergence can also occur without strict isolation. Heterogeneity of gene flow was detected in most population pairs suggesting the predominance of semipermeable barriers during divergence. We found a weak positive relationship between the fraction of the genome experiencing reduced gene flow and levels of genome-wide differentiation. Furthermore, we found that the upper bound of the 'grey zone of speciation' for our dataset extended beyond that found before, implying that gene flow between diverging taxa is possible at higher levels of divergence than previously thought. Finally, we list recommendations for further strengthening the use of demographic modelling in speciation research. These include a more balanced representation of taxa, more consistent and comprehensive modelling, clear reporting of results and simulation studies to rule out nonbiological explanations for general results.

Local adaptation through countergradient selection in northern populations of Skeletonema marinoi.

Marine microorganisms have the potential to disperse widely with few obvious barriers to gene flow. However, among microalgae, several studies have demonstrated that species can be highly genetically structured with limited gene flow among populations, despite hydrographic connectivity. Ecological differentiation and local adaptation have been suggested as drivers of such population structure. Here we tested whether multiple strains from two genetically distinct Baltic Sea populations of the diatom Skeletonema marinoi showed evidence of local adaptation to their local environments: the estuarine Bothnian Sea and the marine Kattegat Sea. We performed reciprocal transplants of multiple strains between culture media based on water from the respective environments, and we also allowed competition between strains of estuarine and marine origin in both salinities. When grown alone, both marine and estuarine strains performed best in the high-salinity environment, and estuarine strains always grew faster than marine strains. This result suggests local adaptation through countergradient selection, that is, genetic effects counteract environmental effects. However, the higher growth rate of the estuarine strains appears to have a cost in the marine environment and when strains were allowed to compete, marine strains performed better than estuarine strains in the marine environment. Thus, other traits are likely to also affect fitness. We provide evidence that tolerance to pH could be involved and that estuarine strains that are adapted to a more fluctuating pH continue growing at higher pH than marine strains.

An allozyme polymorphism is associated with a large chromosomal inversion in the marine snail Littorina fabalis.

Understanding the genetic targets of natural selection is one of the most challenging goals of population genetics. Some of the earliest candidate genes were identified from associations between allozyme allele frequencies and environmental variation. One such example is the clinal polymorphism in the arginine kinase (Ak) gene in the marine snail Littorina fabalis. While other enzyme loci do not show differences in allozyme frequencies among populations, the Ak alleles are near differential fixation across repeated wave exposure gradients in Europe. Here, we use this case to illustrate how a new sequencing toolbox can be employed to characterize the genomic architecture associated with historical candidate genes. We found that the Ak alleles differ by nine nonsynonymous substitutions, which perfectly explain the different migration patterns of the allozymes during electrophoresis. Moreover, by exploring the genomic context of the Ak gene, we found that the three main Ak alleles are located on different arrangements of a putative chromosomal inversion that reaches near fixation at the opposing ends of two transects covering a wave exposure gradient. This shows Ak is part of a large (3/4 of the chromosome) genomic block of differentiation, in which Ak is unlikely to be the only target of divergent selection. Nevertheless, the nonsynonymous substitutions among Ak alleles and the complete association of one allele with one inversion arrangement suggest that the Ak gene is a strong candidate to contribute to the adaptive significance of the inversion.

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.

Media perception and trust among disaster survivors: Tsunami survivors' interaction with journalists, media exposure, and associations with trust in media and authorities.

A critical part of disaster communication is media coverage in the interface of the afflicted, media, and authorities. One communication key is building trust. Disaster survivors encounter journalists in a high-stress context, but little is known about their perceptions of these interactions and the subsequent media exposure. The aim of this study is to explore how survivors 6 years after a major disaster perceived their encounters with journalists and exposure in the media, as well as their level of trust in the media, compared with government and authorities. Data were used from a longitudinal study of Swedish tourists, repatriated from the 2004 Indian Ocean tsunami, surveyed up to 6 years after the tsunami to assess posttraumatic stress (PTS) and effects on mental health. At 6 years after, the survey included questions about survivors' perceptions of journalist interactions (reported by n = 311), of their own media exposure (n = 177), and survivors' trust in media organizations and public authorities (n = 1,181). Tsunami survivors mainly perceived interactions with journalists as being professional. There were 14% who reported that the interactions were supportive and 17% that the interactions were a strain. Similarly, most participants had a neutral view concerning the subsequent media coverage or exposure, although 12% experienced media exposure as stressful and 12% reported that it had been involuntary. Finally, the survivors indicated higher confidence and trust in Swedish radio and TV as compared to the Swedish authorities, and the participants' level of trust in the media was associated with their perceptions of journalists, r = 0.34, p < 0.001, and media coverage, r = 0.47, p < 0.001. Disaster survivors mainly agreed with emotionally neutral statements about interacting with the media, the performance of journalists on site, and their own media exposure. Nonetheless, a substantial minority found the encounters and exposure to be negative, and the results suggest a link between personal experiences or perceptions and trust in the media.

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.

Inversions and parallel evolution.

Local adaptation leads to differences between populations within a species. In many systems, similar environmental contrasts occur repeatedly, sometimes driving parallel phenotypic evolution. Understanding the genomic basis of local adaptation and parallel evolution is a major goal of evolutionary genomics. It is now known that by preventing the break-up of favourable combinations of alleles across multiple loci, genetic architectures that reduce recombination, like chromosomal inversions, can make an important contribution to local adaptation. However, little is known about whether inversions also contribute disproportionately to parallel evolution. Our aim here is to highlight this knowledge gap, to showcase existing studies, and to illustrate the differences between genomic architectures with and without inversions using simple models. We predict that by generating stronger effective selection, inversions can sometimes speed up the parallel adaptive process or enable parallel adaptation where it would be impossible otherwise, but this is highly dependent on the spatial setting. We highlight that further empirical work is needed, in particular to cover a broader taxonomic range and to understand the relative importance of inversions compared to genomic regions without inversions. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Introduction to the theme issue 'Species' ranges in the face of changing environments'.

Understanding where, when and how species' ranges will be modified is both a fundamental problem and essential to predicting how spatio-temporal environmental changes in abiotic and biotic factors impact biodiversity. Notably, different species may respond disparately to similar environmental changes: some species may overcome an environmental change only with difficulty or not at all, while other species may readily overcome the same change. Ranges may contract, expand or move. The drivers and consequences of this variability in species' responses remain puzzling. Importantly, changes in a species' range creates feedbacks to the environmental conditions, populations and communities in its previous and current range, rendering population genetic, population dynamic and community processes inextricably linked. Understanding these links is critical in guiding biodiversity management and conservation efforts. This theme issue presents current thinking about the factors and mechanisms that limit and/or modify species' ranges. It also outlines different approaches to detect changes in species' distributions, and illustrates cases of range modifications in several taxa. Overall, this theme issue highlights the urgency of understanding species' ranges but shows that we are only just beginning to disentangle the processes involved. One way forward is to unite ecology with evolutionary biology and empirical with modelling approaches. This article is part of the theme issue 'Species' ranges in the face of changing environments (Part II)'.