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 1.35Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44kb) 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.

Population Genomics Reveals the Underlying Structure of the Small Pelagic European Sardine and Suggests Low Connectivity within Macaronesia.

The European sardine (Sardina pilchardus, Walbaum 1792) is indisputably a commercially important species. Previous studies using uneven sampling or a limited number of makers have presented sometimes conflicting evidence of the genetic structure of S. pilchardus populations. Here, we show that whole genome data from 108 individuals from 16 sampling areas across 5000 km of the species' distribution range (from the Eastern Mediterranean to the archipelago of Azores) support at least three genetic clusters. One includes individuals from Azores and Madeira, with evidence of substructure separating these two archipelagos in the Atlantic. Another cluster broadly corresponds to the center of the distribution, including the sampling sites around Iberia, separated by the Almeria-Oran front from the third cluster that includes all of the Mediterranean samples, except those from the Alboran Sea. Individuals from the Canary Islands appear to belong to the Mediterranean cluster. This suggests at least two important geographical barriers to gene flow, even though these do not seem complete, with many individuals from around Iberia and the Mediterranean showing some patterns compatible with admixture with other genetic clusters. Genomic regions corresponding to the top outliers of genetic differentiation are located in areas of low recombination indicative that genetic architecture also has a role in shaping population structure. These regions include genes related to otolith formation, a calcium carbonate structure in the inner ear previously used to distinguish S. pilchardus populations. Our results provide a baseline for further characterization of physical and genetic barriers that divide European sardine populations, and information for transnational stock management of this highly exploited species towards sustainable fisheries.

Structural Variants and Speciation: Multiple Processes at Play.

Research on the genomic architecture of speciation has increasingly revealed the importance of structural variants (SVs) that affect the presence, abundance, position, and/or direction of a nucleotide sequence. SVs include large chromosomal rearrangements such as fusion/fissions and inversions and translocations, as well as smaller variants such as duplications, insertions, and deletions (CNVs). Although we have ample evidence that SVs play a key role in speciation, the underlying mechanisms differ depending on the type and length of the SV, as well as the ecological, demographic, and historical context. We review predictions and empirical evidence for classic processes such as underdominance due to meiotic aberrations and the coupling effect of recombination suppression before exploring how recent sequencing methodologies illuminate the prevalence and diversity of SVs. We discuss specific properties of SVs and their impact throughout the genome, highlighting that multiple processes are at play, and possibly interacting, in the relationship between SVs and speciation.

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.

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.

The Impact of Chromosomal Rearrangements in Speciation: From Micro- to Macroevolution.

Chromosomal rearrangements (CRs) have been known since almost the beginning of genetics. While an important role for CRs in speciation has been suggested, evidence primarily stems from theoretical and empirical studies focusing on the microevolutionary level (i.e., on taxon pairs where speciation is often incomplete). Although the role of CRs in eukaryotic speciation at a macroevolutionary level has been supported by associations between species diversity and rates of evolution of CRs across phylogenies, these findings are limited to a restricted range of CRs and taxa. Now that more broadly applicable and precise CR detection approaches have become available, we address the challenges in filling some of the conceptual and empirical gaps between micro- and macroevolutionary studies on the role of CRs in speciation. We synthesize what is known about the macroevolutionary impact of CRs and suggest new research avenues to overcome the pitfalls of previous studies to gain a more comprehensive understanding of the evolutionary significance of CRs in speciation across the tree of life.

Integrating Pool-seq uncertainties into demographic inference.

Next-generation sequencing of pooled samples (Pool-seq) is a popular method to assess genome-wide diversity patterns in natural and experimental populations. However, Pool-seq is associated with specific sources of noise, such as unequal individual contributions. Consequently, using Pool-seq for the reconstruction of evolutionary history has remained underexplored. Here we describe a novel Approximate Bayesian Computation (ABC) method to infer demographic history, explicitly modelling Pool-seq sources of error. By jointly modelling Pool-seq data, demographic history and the effects of selection due to barrier loci, we obtain estimates of demographic history parameters accounting for technical errors associated with Pool-seq. Our ABC approach is computationally efficient as it relies on simulating subsets of loci (rather than the whole-genome) and on using relative summary statistics and relative model parameters. Our simulation study results indicate Pool-seq data allows distinction between general scenarios of ecotype formation (single versus parallel origin) and to infer relevant demographic parameters (e.g. effective sizes and split times). We exemplify the application of our method to Pool-seq data from the rocky-shore gastropod Littorina saxatilis, sampled on a narrow geographical scale at two Swedish locations where two ecotypes (Wave and Crab) are found. Our model choice and parameter estimates show that ecotypes formed before colonization of the two locations (i.e. single origin) and are maintained despite gene flow. These results indicate that demographic modelling and inference can be successful based on pool-sequencing using ABC, contributing to the development of suitable null models that allow for a better understanding of the genetic basis of divergent adaptation.

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.

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.

Longest sediment flows yet measured show how major rivers connect efficiently to deep sea.

Here we show how major rivers can efficiently connect to the deep-sea, by analysing the longest runout sediment flows (of any type) yet measured in action on Earth. These seafloor turbidity currents originated from the Congo River-mouth, with one flow travelling >1,130 km whilst accelerating from 5.2 to 8.0 m/s. In one year, these turbidity currents eroded 1,338-2,675 [>535-1,070] Mt of sediment from one submarine canyon, equivalent to 19-37 [>7-15] % of annual suspended sediment flux from present-day rivers. It was known earthquakes trigger canyon-flushing flows. We show river-floods also generate canyon-flushing flows, primed by rapid sediment-accumulation at the river-mouth, and sometimes triggered by spring tides weeks to months post-flood. It is demonstrated that strongly erosional turbidity currents self-accelerate, thereby travelling much further, validating a long-proposed theory. These observations explain highly-efficient organic carbon transfer, and have important implications for hazards to seabed cables, or deep-sea impacts of terrestrial climate change.

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'.

Perfume and Flavor Engineering: A Chemical Engineering Perspective.

In the last two decades, scientific methodologies for the prediction of the design, performance and classification of fragrance mixtures have been developed at the Laboratory of Separation and Reaction Engineering. This review intends to give an overview of such developments. It all started with the question: what do we smell? The Perfumery Ternary Diagram enables us to determine the dominant odor for each perfume composition. Evaporation and 1D diffusion model is analyzed based on vapor-liquid equilibrium and Fick's law for diffusion giving access to perfume performance parameters. The effect of matrix and skin is addressed and the trail of perfumes analyzed. Classification of perfumes with the perfumery radar is discussed. The methodology is extended to flavor and taste engineering. Finally, future research directions are suggested.

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.

Accelerated Evolution of Tissue-Specific Genes Mediates Divergence Amidst Gene Flow in European Green Lizards.

The European green lizards of the Lacerta viridis complex consist of two closely related species, L. viridis and Lacerta bilineata that split less than 7 million years ago in the presence of gene flow. Recently, a third lineage, referred to as the "Adriatic" was described within the L. viridis complex distributed from Slovenia to Greece. However, whether gene flow between the Adriatic lineage and L. viridis or L. bilineata has occurred and the evolutionary processes involved in their diversification are currently unknown. We hypothesized that divergence occurred in the presence of gene flow between multiple lineages and involved tissue-specific gene evolution. In this study, we sequenced the whole genome of an individual of the Adriatic lineage and tested for the presence of gene flow amongst L. viridis, L. bilineata, and Adriatic. Additionally, we sequenced transcriptomes from multiple tissues to understand tissue-specific effects. The species tree supports that the Adriatic lineage is a sister taxon to L. bilineata. We detected gene flow between the Adriatic lineage and L. viridis suggesting that the evolutionary history of the L. viridis complex is likely shaped by gene flow. Interestingly, we observed topological differences between the autosomal and Z-chromosome phylogenies with a few fast evolving genes on the Z-chromosome. Genes highly expressed in the ovaries and strongly co-expressed in the brain experienced accelerated evolution presumably contributing to establishing reproductive isolation in the L. viridis complex.

Inversions and genomic differentiation after secondary contact: When drift contributes to maintenance, not loss, of differentiation.

Due to their effects on reducing recombination, chromosomal inversions may play an important role in speciation by establishing and/or maintaining linked blocks of genes causing reproductive isolation (RI) between populations. This view fits empirical data indicating that inversions typically harbor loci involved in RI. However, previous computer simulations of infinite populations with two to four loci involved in RI implied that, even with gene flux as low as 10-8 per gamete, per generation between alternative arrangements, inversions may not have large, qualitative advantages over collinear regions in maintaining population differentiation after secondary contact. Here, we report that finite population sizes can help counteract the homogenizing consequences of gene flux, especially when several fitness-related loci reside within the inversion. In these cases, the persistence time of differentiation after secondary contact can be similar to when gene flux is absent and notably longer than the persistence time without inversions. Thus, despite gene flux, population differentiation may be maintained for up to 100,000 generations, during which time new incompatibilities and/or local adaptations might accumulate and facilitate progress toward speciation. How often these conditions are met in nature remains to be determined.

Membrane-Supported Layered Coordination Polymer as an Advanced Sustainable Catalyst for Desulfurization.

The application of a catalytic membrane in the oxidative desulfurization of a multicomponent model diesel formed by most refractory sulfur compounds present in fuel is reported here for the first time. The catalytic membrane was prepared by the impregnation of the active lamellar [Gd(H4nmp)(H2O)2]Cl·2H2O (UAV-59) coordination polymer (CP) into a polymethyl methacrylate (PMMA, acrylic glass) supporting membrane. The use of the catalytic membrane in the liquid-liquid system instead of a powder catalyst arises as an enormous advantage associated with the facility of catalyst handling while avoiding catalyst mass loss. The optimization of various parameters allowed to achieve a near complete desulfurization after 3 h under sustainable conditions, i.e., using an aqueous H2O2 as oxidant and an ionic liquid as extraction solvent ([BMIM]PF6, 1:0.5 ratio diesel:[BMIM]PF6). The performance of the catalytic membrane and of the powdered UAV-59 catalyst was comparable, with the advantage that the former could be recycled successfully for a higher number of desulfurization cycles without the need of washing and drying procedures between reaction cycles, turning the catalytic membrane process more cost-efficient and suitable for future industrial application.

Using replicate hybrid zones to understand the genomic basis of adaptive divergence.

Combining hybrid zone analysis with genomic data is a promising approach to understanding the genomic basis of adaptive divergence. It allows for the identification of genomic regions underlying barriers to gene flow. It also provides insights into spatial patterns of allele frequency change, informing about the interplay between environmental factors, dispersal and selection. However, when only a single hybrid zone is analysed, it is difficult to separate patterns generated by selection from those resulting from chance. Therefore, it is beneficial to look for repeatable patterns across replicate hybrid zones in the same system. We applied this approach to the marine snail Littorina saxatilis, which contains two ecotypes, adapted to wave-exposed rocks vs. high-predation boulder fields. The existence of numerous hybrid zones between ecotypes offered the opportunity to test for the repeatability of genomic architectures and spatial patterns of divergence. We sampled and phenotyped snails from seven replicate hybrid zones on the Swedish west coast and genotyped them for thousands of single nucleotide polymorphisms. Shell shape and size showed parallel clines across all zones. Many genomic regions showing steep clines and/or high differentiation were shared among hybrid zones, consistent with a common evolutionary history and extensive gene flow between zones, and supporting the importance of these regions for divergence. In particular, we found that several large putative inversions contribute to divergence in all locations. Additionally, we found evidence for consistent displacement of clines from the boulder-rock transition. Our results demonstrate patterns of spatial variation that would not be accessible without continuous spatial sampling, a large genomic data set and replicate hybrid zones.