Brute force prey metabarcoding to explore the diets of small invertebrates.

Prey metabarcoding has become a popular tool in molecular ecology for resolving trophic interactions at high resolution, from various sample types and animals. To date, most predator-prey studies of small-sized animals (<1 mm) have met the problem of overabundant predator DNA in dietary samples by adding blocking primers/peptide nucleic acids. These primers aim to limit the PCR amplification and detection of the predator DNA but may introduce bias to the prey composition identified by interacting with sequences that are similar to those of the predator. Here we demonstrate the use of an alternative method to explore the prey of small marine copepods using whole-body DNA extracts and deep, brute force metabarcoding of an 18S rDNA fragment. After processing and curating raw data from two sequencing runs of varying depths (0.4 and 5.4 billion raw reads), we isolated 1.3 and 52.2 million prey reads, with average depths of ~15,900 and ~120,000 prey reads per copepod individual, respectively. While data from both sequencing runs were sufficient to distinguish dietary compositions from disparate seasons, locations, and copepod species, greater sequencing depth led to better separation of clusters. As computation and sequencing are becoming ever more powerful and affordable, we expect the brute force approach to become a general standard for prey metabarcoding, as it offers a simple and affordable solution to consumers that is impractical to dissect or unknown to science.

Phylogeography and molecular diversity of two highly abundant Themisto amphipod species in a rapidly changing Arctic Ocean.

Rapid warming in the Arctic is drastically impacting marine ecosystems, affecting species communities and food-web structure. Pelagic Themisto amphipods are a major component of the Arctic zooplankton community and represent a key link between secondary producers and marine vertebrates at higher trophic levels. Two co-existing species dominate in the region: the larger Themisto libellula, considered a true polar species and the smaller Themisto abyssorum, a sub-Arctic, boreal-Atlantic species. Recent changes in abundance and distribution ranges have been detected in both species, likely due to the Atlantification of the Arctic. The ecology and genetic structure of these species are understudied, despite their high biomass and importance in the food web. For both species, we assessed genetic diversity, patterns of spatial genetic structure and demographic history using samples from the Greenland shelf, Fram Strait and Svalbard. This was achieved by analysing variation on the mitochondrial cytochrome c oxidase subunit 1 gene (mtCOI). The results revealed contrasting levels of mtCOI diversity: low levels in T. libellula and high levels in T. abyssorum. A lack of spatial genetic structure and a high degree of genetic connectivity were detected in both species in the study region. These patterns of diversity are potentially linked to the impacts of the Last Glacial Maximum. T. libellula populations may have been isolated in glacial refugia, undergoing gene flow restriction and vicariant effects, followed by a population expansion after deglaciation. Whereas T. abyssorum likely maintained a stable, widely distributed metapopulation further south, explaining the high diversity and connectivity. This study provides new data on the phylogeography of two ecologically important species, which can contribute to predicting how zooplankton communities and food-web structure will manifest in the rapidly changing Arctic.

Molecular tools prove little auks from Svalbard are extremely selective for Calanus glacialis even when exposed to Atlantification.

Two Calanus species, C. glacialis and C. finmarchicus, due to different life strategies and environmental preferences act as an ecological indicators of Arctic Atlantification. Their high lipid content makes them important food source for higher trophic levels of Arctic ecosystems including the most abundant Northern Hemisphere's seabird, the little auk (Alle alle). Recent studies indicate a critical need for the use of molecular methods to reliably identify these two sympatric Calanus species. We performed genetic and morphology-based identification of 2600 Calanus individuals collected in little auks foraging grounds and diet in summer seasons 2019-2021 in regions of Svalbard with varying levels of Atlantification. Genetic identification proved that 40% of Calanus individuals were wrongly classified as C. finmarchicus according to morphology-based identification in both types of samples. The diet of little auks consisted almost entirely of C. glacialis even in more Atlantified regions. Due to the substantial bias in morphology-based identification, we expect that the scale of the northern expansion of boreal C. finmarchicus may have been largely overestimated and that higher costs for birds exposed to Atlantification could be mostly driven by a decrease in the size of C. glacialis rather than by shift from C. glacialis to C. finmarchicus.

Genome-wide DNA methylation predicts environmentally driven life history variation in a marine fish.

Epigenetic modifications are thought to be one of the molecular mechanisms involved in plastic adaptive responses to environmental variation. However, studies reporting associations between genome-wide epigenetic changes and habitat-specific variations in life history traits (e.g., lifespan, reproduction) are still scarce, likely due to the recent application of methylome resequencing methods to non-model species. In this study, we examined associations between whole genome DNA methylation and environmentally driven life history variation in 2 lineages of a marine fish, the capelin (Mallotus villosus), from North America and Europe. In both lineages, capelin harbor 2 contrasting life history tactics (demersal vs. beach-spawning). Performing whole genome and methylome sequencing, we showed that life history tactics are associated with epigenetic changes in both lineages, though the effect was stronger in European capelin. Genetic differentiation between the capelin harboring different life history tactics was negligible, but we found genome-wide methylation changes in both lineages. We identified 9,125 European and 199 North American differentially methylated regions (DMRs) due to life history. Gene ontology (GO) enrichment analysis for both lineages revealed an excess of terms related to neural function. Our results suggest that environmental variation causes important epigenetic changes that are associated with contrasting life history tactics in lineages with divergent genetic backgrounds, with variable importance of genetic variation in driving epigenetic variation. Our study emphasizes the potential role of genome-wide epigenetic variation in adaptation to environmental variation.

Using DNA Metabarcoding to Characterize the Prey Spectrum of Two Co-Occurring Themisto Amphipods in the Rapidly Changing Atlantic-Arctic Gateway Fram Strait.

The two congeneric hyperiids Themisto libellula and T. abyssorum provide an important trophic link between lower and higher trophic levels in the rapidly changing Arctic marine ecosystem. These amphipods are characterized by distinct hydrographic affinities and are hence anticipated to be impacted differently by environmental changes, with major consequences for the Arctic food web. In this study, we applied DNA metabarcoding to the stomach contents of these Themisto species, to comprehensively reveal their prey spectra at an unprecedented-high-taxonomic-resolution and assess the regional variation in their diet across the Fram Strait. Both species feed on a wide variety of prey but their diet strongly differed in the investigated summer season, showing overlap for only a few prey taxa, such as calanoid copepods. The spatially structured prey field of T. libellula clearly differentiated it from T. abyssorum, of which the diet was mainly dominated by chaetognaths. Our approach also allowed the detection of previously overlooked prey in the diet of T. libellula, such as fish species and gelatinous zooplankton. We discuss the reasons for the differences in prey spectra and which consequences these may have in the light of ongoing environmental changes.

Fine-scale differences in eukaryotic communities inside and outside salmon aquaculture cages revealed by eDNA metabarcoding.

Aquaculture impacts on marine benthic ecosystems are widely recognized and monitored. However, little is known about the community changes occurring in the water masses surrounding aquaculture sites. In the present study, we studied the eukaryotic communities inside and outside salmonid aquaculture cages through time to assess the community changes in the neighbouring waters of the farm. Water samples were taken biweekly over five months during the production phase from inside the cages and from nearby points located North and South of the salmon farm. Eukaryotic communities were analyzed by eDNA metabarcoding of the partial COI Leray-XT fragment. The results showed that eukaryotic communities inside the cages were significantly different from those in the outside environment, with communities inside the cages having higher diversity values and more indicator species associated with them. This is likely explained by the appearance of fouling species that colonize the artificial structures, but also by other species that are attracted to the cages by other means. Moreover, these effects were highly localized inside the cages, as the communities identified outside the cages, both North and South, had very similar eukaryotic composition at each point in time. Overall, the eukaryotic communities, both inside and outside the cages, showed similar temporal fluctuations through the summer months, with diversity peaks occurring at the end of July, beginning of September, and in the beginning of November, with the latter showing the highest Shannon diversity and richness values. Hence, our study suggests that seasonality, together with salmonid aquaculture, are the main drivers of eukaryotic community structure in surface waters surrounding the farm.

DNA metabarcoding reveals the importance of gelatinous zooplankton in the diet of Pandalus borealis, a keystone species in the Arctic.

Information about the dietary composition of a species is crucial to understanding their position and role in the food web. Increasingly, molecular approaches such as DNA metabarcoding are used in studying trophic relationships, not least because they may alleviate problems such as low taxonomic resolution or underestimation of digestible taxa in the diet. Here, we used DNA metabarcoding with universal primers for cytochrome c oxidase I (COI) to study the diet composition of the northern shrimp (Pandalus borealis), an Arctic keystone species with large socio-economic importance. Across locations, jellyfish and chaetognaths were the most important components in the diet of P. borealis, jointly accounting for 40%-60% of the total read abundance. This dietary importance of gelatinous zooplankton contrasts sharply with published results based on stomach content analysis. At the same time, diet composition differed between fjord and shelf locations, pointing to different food webs supporting P. borealis in these two systems. Our study underlines the potential of molecular approaches to provide new insights into the diet of marine invertebrates that are difficult to obtain with traditional methods, and calls for a revision of the role of gelatinous zooplankton in the diet of the key Arctic species P. borealis, and in extension, Arctic food webs.

Immunostimulant Bathing Influences the Expression of Immune- and Metabolic-Related Genes in Atlantic Salmon Alevins.

Disease resistance of fish larvae may be improved by bath treatment in water containing immunostimulants. Pattern recognition receptors, such as TLR3, TLR7, and MDA5, work as an "early warning" to induce intracellular signaling and facilitate an antiviral response. A single bath of newly hatched larvae, with Astragalus, upregulated the expression of IFNα, IFNc, ISG15, MDA5, PKR, STAT1, TLR3, and TLR7 immune genes, on day 4 post treatment. Similar patterns were observed for Hyaluronic acid and Poly I:C. Increased expression was observed for ISG15, MDA5, MX, STAT1, TLR3, TLR7, and RSAD2, on day 9 for Imiquimod. Metabolic gene expression was stimulated on day 1 after immunostimulant bath in ULK1, MYC, SLC2A1, HIF1A, MTOR, and SIX1, in Astragalus, Hyaluronic acid, and Imiquimod. Expression of NOS2 in Poly I:C was an average fourfold above that of control at the same timepoint. Throughout the remaining sampling days (2, 4, 9, 16, 32, and 45 days post immunostimulant bath), NOS2 and IL1B were consistently overexpressed. In conclusion, the immunostimulants induced antiviral gene responses, indicating that a single bath at an early life stage could enable a more robust antiviral defense in fish. Additionally, it was demonstrated, based on gene expression data, that cell metabolism was perturbed, where several metabolic genes were co-regulated with innate antiviral genes.

Complex and divergent histories gave rise to genome-wide divergence patterns amongst European whitefish (Coregonus lavaretus).

Pleistocene glaciations dramatically affected species distribution in regions that were impacted by ice cover and subsequent postglacial range expansion impacted contemporary biodiversity in complex ways. The European whitefish, Coregonus lavaretus, is a widely distributed salmonid fish species on mainland Europe, but in Britain it has only seven native populations, all of which are found on the western extremes of the island. The origins and colonization routes of the species into Britain are unknown but likely contributed to contemporary genetic patterns and regional uniqueness. Here, we used up to 25,751 genome-wide polymorphic loci to reconstruct the history and to discern the demographic and evolutionary forces underpinning divergence between British populations. Overall, we found lower genetic diversity in Scottish populations but high differentiation (FST  = 0.433-0.712) from the English/Welsh and other European populations. Differentiation was elevated genome-wide rather than in particular genomic regions. Demographic modelling supported a postglacial colonization into western Scotland from northern refugia and a separate colonization route for the English/Welsh populations from southern refugia, with these two groups having been separated for more than ca. 50 Ky. We found cyto-nuclear discordance at a European scale, with the Scottish populations clustering closely with Baltic population in the mtDNA analysis but not in the nuclear data, and with the Norwegian and Alpine populations displaying the same mtDNA haplotype but being distantly related in the nuclear tree. These findings suggest that neutral processes, primarily drift and regionally distinct pre-glacial evolutionary histories, are important drivers of genomic divergence in British populations of European whitefish. This sheds new light on the establishment of the native British freshwater fauna after the last glacial maximum.

Genome-resolved metagenomics suggests a mutualistic relationship between Mycoplasma and salmonid hosts.

Salmonids are important sources of protein for a large proportion of the human population. Mycoplasma species are a major constituent of the gut microbiota of salmonids, often representing the majority of microbiota. Despite the frequent reported dominance of salmonid-related Mycoplasma species, little is known about the phylogenomic placement, functions and potential evolutionary relationships with their salmonid hosts. In this study, we utilise 2.9 billion metagenomic reads generated from 12 samples from three different salmonid host species to I) characterise and curate the first metagenome-assembled genomes (MAGs) of Mycoplasma dominating the intestines of three different salmonid species, II) establish the phylogeny of these salmonid candidate Mycoplasma species, III) perform a comprehensive pangenomic analysis of Mycoplasma, IV) decipher the putative functionalities of the salmonid MAGs and reveal specific functions expected to benefit the host. Our data provide a basis for future studies examining the composition and function of the salmonid microbiota.

Terrestrial Inputs Shape Coastal Bacterial and Archaeal Communities in a High Arctic Fjord (Isfjorden, Svalbard).

The Arctic is experiencing dramatic changes including increases in precipitation, glacial melt, and permafrost thaw, resulting in increasing freshwater runoff to coastal waters. During the melt season, terrestrial runoff delivers carbon- and nutrient-rich freshwater to Arctic coastal waters, with unknown consequences for the microbial communities that play a key role in determining the cycling and fate of terrestrial matter at the land-ocean interface. To determine the impacts of runoff on coastal microbial (bacteria and archaea) communities, we investigated changes in pelagic microbial community structure between the early (June) and late (August) melt season in 2018 in the Isfjorden system (Svalbard). Amplicon sequences of the 16S rRNA gene were generated from water column, river and sediment samples collected in Isfjorden along fjord transects from shallow river estuaries and glacier fronts to the outer fjord. Community shifts were investigated in relation to environmental gradients, and compared to river and marine sediment microbial communities. We identified strong temporal and spatial reorganizations in the structure and composition of microbial communities during the summer months in relation to environmental conditions. Microbial diversity patterns highlighted a reorganization from rich communities in June toward more even and less rich communities in August. In June, waters enriched in dissolved organic carbon (DOC) provided a niche for copiotrophic taxa including Sulfitobacter and Octadecabacter. In August, lower DOC concentrations and Atlantic water inflow coincided with a shift toward more cosmopolitan taxa usually associated with summer stratified periods (e.g., SAR11 Clade Ia), and prevalent oligotrophic marine clades (OM60, SAR92). Higher riverine inputs of dissolved inorganic nutrients and suspended particulate matter also contributed to spatial reorganizations of communities in August. Sentinel taxa of this late summer fjord environment included taxa from the class Verrucomicrobiae (Roseibacillus, Luteolibacter), potentially indicative of a higher fraction of particle-attached bacteria. This study highlights the ecological relevance of terrestrial runoff for Arctic coastal microbial communities and how its impacts on biogeochemical conditions may make these communities susceptible to climate change.

Space-time dynamics in monitoring neotropical fish communities using eDNA metabarcoding.

The biodiverse Neotropical ecoregion remains insufficiently assessed, poorly managed, and threatened by unregulated human activities. Novel, rapid and cost-effective DNA-based approaches are valuable to improve understanding of the biological communities and for biomonitoring in remote areas. Here, we evaluate the potential of environmental DNA (eDNA) metabarcoding for assessing the structure and distribution of fish communities by analysing water and sediment from 11 locations along the Jequitinhonha River catchment (Brazil). Each site was sampled twice, before and after a major rain event in a five-week period and fish diversity was estimated using high-throughput sequencing of 12S rRNA amplicons. In total, 252 Molecular Operational Taxonomic Units (MOTUs) and 34 fish species were recovered, including endemic, introduced, and previously unrecorded species for this basin. Spatio-temporal variation of eDNA from fish assemblages was observed and species richness was nearly twice as high before the major rain event compared to afterwards. Yet, peaks of diversity were primarily associated with only four of the locations. No correlation between ß-diversity and longitudinal distance or presence of dams was detected, but low species richness observed at sites located near dams might that these anthropogenic barriers may have an impact on local fish diversity. Unexpectedly high α-diversity levels recorded at the river mouth suggest that these sections should be further evaluated as putative "eDNA reservoirs" for rapid monitoring. By uncovering spatio-temporal changes, unrecorded biodiversity components, and putative anthropogenic impacts on fish assemblages, we further strengthen the potential of eDNA metabarcoding as a biomonitoring tool, especially in regions often neglected or difficult to access.

A brain and a head for a different habitat: Size variation in four morphs of Arctic charr (Salvelinus alpinus (L.)) in a deep oligotrophic lake.

Adaptive radiation is the diversification of species to different ecological niches and has repeatedly occurred in different salmonid fish of postglacial lakes. In Lake Tinnsjøen, one of the largest and deepest lakes in Norway, the salmonid fish, Arctic charr (Salvelinus alpinus (L.)), has likely radiated within 9,700 years after deglaciation into ecologically and genetically segregated Piscivore, Planktivore, Dwarf, and Abyssal morphs in the pelagial, littoral, shallow-moderate profundal, and deep-profundal habitats. We compared trait variation in the size of the head, the eye and olfactory organs, as well as the volumes of five brain regions of these four Arctic charr morphs. We hypothesised that specific habitat characteristics have promoted divergent body, head, and brain sizes related to utilized depth differing in environmental constraints (e.g., light, oxygen, pressure, temperature, and food quality). The most important ecomorphological variables differentiating morphs were eye area, habitat, and number of lamellae. The Abyssal morph living in the deepest areas of the lake had the smallest brain region volumes, head, and eye size. Comparing the olfactory bulb with the optic tectum in size, it was larger in the Abyssal morph than in the Piscivore morph. The Piscivore and Planktivore morphs that use more illuminated habitats have the largest optic tectum volume, followed by the Dwarf. The observed differences in body size and sensory capacities in terms of vision and olfaction in shallow and deepwater morphs likely relates to foraging and mating habitats in Lake Tinnsjøen. Further seasonal and experimental studies of brain volume in polymorphic species are needed to test the role of plasticity and adaptive evolution behind the observed differences.

Temperature-dependent egg production and egg hatching rates of small egg-carrying and broadcast-spawning copepods Oithona similis, Microsetella norvegica and Microcalanus pusillus.

Reproductive rates of copepods are temperature-dependent, but poorly known for small copepods at low temperatures, hindering the predictions of population dynamics and secondary production in high-latitude ecosystems. We investigated egg hatching rates, hatching success and egg production of the small copepods Oithona similis and Microsetella norvegica (sac spawners) and Microcalanus pusillus (broadcast spawner) between March and August. Incubations were performed at ecologically relevant temperatures between 1.3 and 13.2°C, and egg production rates were calculated. All egg hatching rates were positively correlated to temperature, although with large species-specific differences. At the lowest temperatures, M. pusillus eggs hatched within 4 days, whereas the eggs from sac spawners took 3-8 weeks to hatch. The egg hatching success was ≤25% for M. pusillus, >75% for O. similis and variable for M. norvegica. The maximum weight-specific egg production rate (µg C µg-1 C d-1) of M. pusillus was higher (0.22) than O. similis (0.12) and M. norvegica (0.06). M. norvegica reproduction peaked at 6-8°C, the prevailing in situ temperatures during its reproductive period. The difference in reproductive rates indicates species-specific thermal plasticity for the three copepods, which could have implications for present and future population dynamics of the species in arctic fjords.

"And if you gaze long into an abyss, the abyss gazes also into thee": four morphs of Arctic charr adapting to a depth gradient in Lake Tinnsjøen.

The origin of species is a central topic in biology. Ecological speciation might be a driver in adaptive radiation, providing a framework for understanding mechanisms, level, and rate of diversification. The Arctic charr Salvelinus alpinus L. is a polymorphic species with huge morphological and life-history diversity in Holarctic water systems. We studied adaptive radiation of Arctic charr in the 460-m-deep Lake Tinnsjøen to (a) document eco-morphology and life-history traits of morphs, (b) estimate reproductive isolation of morphs, and (c) illuminate Holarctic phylogeography and lineages colonizing Lake Tinnsjøen. We compared Lake Tinnsjøen with four Norwegian outgroup populations. Four field-assigned morphs were identified in Lake Tinnsjøen: the planktivore morph in all habitats except deep profundal, the dwarf morph in shallow-moderate profundal, the piscivore morph mainly in shallow-moderate profundal, and a new undescribed abyssal morph in the deep profundal. Morphs displayed extensive life-history variation in age and size. A moderate-to-high concordance was observed among morphs and four genetic clusters from microsatellites. mtDNA suggested two minor endemic clades in Lake Tinnsjøen originating from one widespread colonizing clade in the Holarctic. All morphs were genetically differentiated at microsatellites (F ST: 0.12-0.20), associated with different mtDNA clade frequencies. Analyses of outgroup lakes implied colonization from a river below Lake Tinnsjøen. Our findings suggest postglacial adaptive radiation of one colonizing mtDNA lineage with niche specialization along a depth-temperature-productivity-pressure gradient. Concordance between reproductive isolation and habitats of morphs implies ecological speciation as a mechanism. Particularly novel is the extensive morph diversification with depth into the often unexplored deepwater profundal habitat, suggesting we may have systematically underestimated biodiversity in lakes. In a biological conservation framework, it is imperative to protect endemic below-species-level biodiversity, particularly so since within-species variation comprises an extremely important component of the generally low total biodiversity observed in the northern freshwater systems.

Ecological speciation in European whitefish is driven by a large-gaped predator.

Lake-dwelling fish that form species pairs/flocks characterized by body size divergence are important model systems for speciation research. Although several sources of divergent selection have been identified in these systems, their importance for driving the speciation process remains elusive. A major problem is that in retrospect, we cannot distinguish selection pressures that initiated divergence from those acting later in the process. To address this issue, we studied the initial stages of speciation in European whitefish (Coregonus lavaretus) using data from 358 populations of varying age (26-10,000 years). We find that whitefish speciation is driven by a large-growing predator, the northern pike (Esox lucius). Pike initiates divergence by causing a largely plastic differentiation into benthic giants and pelagic dwarfs: ecotypes that will subsequently develop partial reproductive isolation and heritable differences in gill raker number. Using an eco-evolutionary model, we demonstrate how pike's habitat specificity and large gape size are critical for imposing a between-habitat trade-off, causing prey to mature in a safer place or at a safer size. Thereby, we propose a novel mechanism for how predators may cause dwarf/giant speciation in lake-dwelling fish species.

Shared ancestral polymorphisms and chromosomal rearrangements as potential drivers of local adaptation in a marine fish.

Gene flow has tremendous importance for local adaptation, by influencing the fate of de novo mutations, maintaining standing genetic variation and driving adaptive introgression. Furthermore, structural variation as chromosomal rearrangements may facilitate adaptation despite high gene flow. However, our understanding of the evolutionary mechanisms impending or favouring local adaptation in the presence of gene flow is still limited to a restricted number of study systems. In this study, we examined how demographic history, shared ancestral polymorphism, and gene flow among glacial lineages contribute to local adaptation to sea conditions in a marine fish, the capelin (Mallotus villosus). We first assembled a 490-Mbp draft genome of M. villosus to map our RAD sequence reads. Then, we used a large data set of genome-wide single nucleotide polymorphisms (25,904 filtered SNPs) genotyped in 1,310 individuals collected from 31 spawning sites in the northwest Atlantic. We reconstructed the history of divergence among three glacial lineages and showed that they probably diverged from 3.8 to 1.8 million years ago and experienced secondary contacts. Within each lineage, our analyses provided evidence for large Ne and high gene flow among spawning sites. Within the Northwest Atlantic lineage, we detected a polymorphic chromosomal rearrangement leading to the occurrence of three haplogroups. Genotype-environment associations revealed molecular signatures of local adaptation to environmental conditions prevailing at spawning sites. Our study also suggests that both shared polymorphisms among lineages, resulting from standing genetic variation or introgression, and chromosomal rearrangements may contribute to local adaptation in the presence of high gene flow.

Using mathematical modelling to investigate the adaptive divergence of whitefish in Fennoscandia.

Modern speciation theory has greatly benefited from a variety of simple mathematical models focusing on the conditions and patterns of speciation and diversification in the presence of gene flow. Unfortunately the application of general theoretical concepts and tools to specific ecological systems remains a challenge. Here we apply modeling tools to better understand adaptive divergence of whitefish during the postglacial period in lakes of northern Fennoscandia. These lakes harbor up to three different morphs associated with the three major lake habitats: littoral, pelagic, and profundal. Using large-scale individual-based simulations, we aim to identify factors required for in situ emergence of the pelagic and profundal morphs in lakes initially colonized by the littoral morph. The importance of some of the factors we identify and study - sufficiently large levels of initial genetic variation, size- and habitat-specific mating, sufficiently large carrying capacity of the new niche - is already well recognized. In addition, our model also points to two other factors that have been largely disregarded in theoretical studies: fitness-dependent dispersal and strong predation in the ancestral niche coupled with the lack of it in the new niche(s). We use our theoretical results to speculate about the process of diversification of whitefish in Fennoscandia and to identify potentially profitable directions for future empirical research.

From metabarcoding to metaphylogeography: separating the wheat from the chaff.

Metabarcoding is by now a well-established method for biodiversity assessment in terrestrial, freshwater, and marine environments. Metabarcoding data sets are usually used for α- and ß-diversity estimates, that is, interspecies (or inter-MOTU [molecular operational taxonomic unit]) patterns. However, the use of hypervariable metabarcoding markers may provide an enormous amount of intraspecies (intra-MOTU) information-mostly untapped so far. The use of cytochrome oxidase (COI) amplicons is gaining momentum in metabarcoding studies targeting eukaryote richness. COI has been for a long time the marker of choice in population genetics and phylogeographic studies. Therefore, COI metabarcoding data sets may be used to study intraspecies patterns and phylogeographic features for hundreds of species simultaneously, opening a new field that we suggest to name metaphylogeography. The main challenge for the implementation of this approach is the separation of erroneous sequences from true intra-MOTU variation. Here, we develop a cleaning protocol based on changes in entropy of the different codon positions of the COI sequence, together with co-occurrence patterns of sequences. Using a data set of community DNA from several benthic littoral communities in the Mediterranean and Atlantic seas, we first tested by simulation on a subset of sequences a two-step cleaning approach consisting of a denoising step followed by a minimal abundance filtering. The procedure was then applied to the whole data set. We obtained a total of 563 MOTUs that were usable for phylogeographic inference. We used semiquantitative rank data instead of read abundances to perform AMOVAs and haplotype networks. Genetic variability was mainly concentrated within samples, but with an important between seas component as well. There were intergroup differences in the amount of variability between and within communities in each sea. For two species, the results could be compared with traditional Sanger sequence data available for the same zones, giving similar patterns. Our study shows that metabarcoding data can be used to infer intra- and interpopulation genetic variability of many species at a time, providing a new method with great potential for basic biogeography, connectivity and dispersal studies, and for the more applied fields of conservation genetics, invasion genetics, and design of protected areas.

Polygenic selection drives the evolution of convergent transcriptomic landscapes across continents within a Nearctic sister species complex.

In contrast to the plethora of studies focusing on the genomic basis of adaptive phenotypic divergence, the role of gene expression during speciation has been much less investigated and consequently less understood. Yet, the convergence of differential gene expression patterns between closely related species-pairs might reflect the role of natural selection during the process of ecological speciation. Here, we test for intercontinental convergence in differential transcriptional signatures between limnetic and benthic sympatric species-pairs of Lake Whitefish (Coregonus clupeaformis) and its sister lineage, the European Whitefish (Coregonus lavaretus), using six replicated sympatric species-pairs (two in North America, two in Norway and two in Switzerland). We characterized both sequence variation in transcribed regions and differential gene expression between sympatric limnetic and benthic species across regions and continents. Our first finding was that differentially expressed genes (DEG) between limnetic and benthic whitefish tend to be enriched in shared polymorphism among sister lineages. We then used both genotypes and covariation in expression in order to infer polygenic selection at the gene level. We identified parallel outliers and DEG involving genes primarily overexpressed in limnetic species relative to the benthic species. Our analysis finally revealed the existence of shared genomic bases underlying parallel differential expression across replicated species-pairs from both continents, such as a cis-eQTL affecting the pyruvate kinase expression level involved in glycolysis. Our results are consistent with a long-standing role of natural selection in maintaining trans-continental diversity at phenotypic traits involved in ecological speciation between limnetic and benthic whitefishes.