Genomic insights on conservation priorities for North Sea houting and European lake whitefish (Coregonus spp.).

Population genomics analysis holds great potential for informing conservation of endangered populations. We focused on a controversial case of European whitefish (Coregonus spp.) populations. The endangered North Sea houting is the only coregonid fish that tolerates oceanic salinities and was previously considered a species (C. oxyrhinchus) distinct from European lake whitefish (C. lavaretus). However, no firm evidence for genetic-based salinity adaptation has been available. Also, studies based on microsatellite and mitogenome data suggested surprisingly recent divergence (c. 2500 years bp) between houting and lake whitefish. These data types furthermore have provided no evidence for possible inbreeding. Finally, a controversial taxonomic revision recently classified all whitefish in the region as C. maraena, calling conservation priorities of houting into question. We used whole-genome and ddRAD sequencing to analyse six lake whitefish populations and the only extant indigenous houting population. Demographic inference indicated post-glacial expansion and divergence between lake whitefish and houting occurring not long after the Last Glaciation, implying deeper population histories than previous analyses. Runs of homozygosity analysis suggested not only high inbreeding (FROH up to 30.6%) in some freshwater populations but also FROH up to 10.6% in the houting prompting conservation concerns. Finally, outlier scans provided evidence for adaptation to high salinities in the houting. Applying a framework for defining conservation units based on current and historical reproductive isolation and adaptive divergence led us to recommend that the houting be treated as a separate conservation unit regardless of species status. In total, the results underscore the potential of genomics to inform conservation practices, in this case clarifying conservation units and highlighting populations of concern.

Genome-wide methylation in the panmictic European eel (Anguilla anguilla).

The role of methylation in adaptive, developmental and speciation processes has attracted considerable interest, but interpretation of results is complicated by diffuse boundaries between genetic and non-genetic variation. We studied whole genome genetic and methylation variation in the European eel, distributed from subarctic to subtropical environments, but with panmixia precluding genetically based local adaptation beyond single-generation responses. Overall methylation was 70.9%, with hypomethylation predominantly found in promoters and first exons. Redundancy analyses involving juvenile glass eels showed 0.06% and 0.03% of the variance at SNPs to be explained by localities and environmental variables, respectively, with GO terms of genes associated with outliers primarily involving neural system functioning. For CpGs 2.98% and 1.36% of variance was explained by localities and environmental variables. Differentially methylated regions particularly included genes involved in developmental processes, with Hox clusters featuring prominently. Life stage (adult versus glass eels) was the most important source of inter-individual variation in methylation, probably reflecting both ageing and developmental processes. Demethylation of transposable elements relative to pure European eel was observed in European X American eel hybrids, possibly representing postzygotic barriers in this system characterized by prolonged speciation and ongoing gene flow. Whereas the genetic data are consistent with a role of single-generation selective responses, the methylation results underpin the importance of epigenetics in the life cycle of eels and suggest interactions between local environments, development and phenotypic variation mediated by methylation variation. Eels are remarkable by having retained eight Hox clusters, and the results suggest important roles of methylation at Hox genes for adaptive processes.

Phylogeography using mitogenomes: A rare Dipodidae, Sicista betulina, in North-western Europe.

Repeated climatic and vegetation changes during the Pleistocene have shaped biodiversity in Northern Europe including Denmark. The Northern Birch Mouse (Sicista betulina) was one of the first small rodent species to colonize Denmark after the Late Glacial Maximum. This study analyses complete mitochondrial genomes and two nuclear genes of the Northern Birch Mouse to investigate the phylogeographical pattern in North-western Europe and test whether the species colonized Denmark through several colonization events. The latter was prompt by (i) the present-day distinct northern and southern Danish distribution and (ii) the subfossil record of Northern Birch Mouse, supporting early Weichselian colonization. Samples from Denmark, Norway, Sweden, Russia, Latvia, Estonia, and Slovakia were included. Mitogenomes were obtained from 54 individuals, all representing unique mitogenomes supporting high genetic variation. Bayesian phylogenetic analysis identified two distinct evolutionary linages in Northern Europe diverging within the Elster glaciation period. The results of the two nuclear genomes showed lower genetic differentiation but supported the same evolutionary history. This suggests an allopatric origin of the clades followed by secondary contact. Individuals from southern Denmark were only found in one clade, while individuals from other areas, including northern Denmark, were represented in both clades. Nevertheless, we found no evidence for repeated colonization's explaining the observed fragmented distribution of the species today. The results indicated that the mitogenome pattern of the Northern Birch Mouse population in southern Denmark was either (i) due to the population being founded from northern Denmark, (ii) a result of climatic and anthropogenic effects reducing population size increasing genetic drift or (iii) caused by sampling bias.

Remote, autonomous real-time monitoring of environmental DNA from commercial fish.

Environmental DNA (eDNA) is increasingly used for monitoring marine organisms; however, offshore sampling and time lag from sampling to results remain problematic. In order to overcome these challenges a robotic sampler, a 2nd generation Environmental Sample Processor (ESP), was tested for autonomous analysis of eDNA from four commercial fish species in a 4.5 million liter mesocosm. The ESP enabled in situ analysis, consisting of water collection, filtration, DNA extraction and qPCR analysis, which allowed for real-time remote reporting and archival sample collection, consisting of water collection, filtration and chemical preservation followed by post-deployment laboratory analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify target molecules from the most abundant species (Atlantic mackerel) both in real-time and from the archived samples. In contrast, detection of low abundant species was challenged by both biological and technical aspects coupled to the ecology of eDNA and the 2G ESP instrumentation. Comparison of the in situ analysis and archival samples demonstrated variance, which potentially was linked to diel migration patterns of the Atlantic mackerel. The study demonstrates strong potential for remote autonomous in situ monitoring which open new possibilities for the field of eDNA and marine monitoring.

Speciation history of European (Anguilla anguilla) and American eel (A. rostrata), analysed using genomic data.

Speciation in the ocean could differ from terrestrial environments due to fewer barriers to gene flow. Hence, sympatric speciation might be common, with American and European eel being candidates for exemplifying this. They show disjunct continental distributions on both sides of the Atlantic, but spawn in overlapping regions of the Sargasso Sea from where juveniles are advected to North American, European and North African coasts. Hybridization and introgression are known to occur, with hybrids almost exclusively observed in Iceland. Different speciation scenarios have been suggested, involving either vicariance or sympatric ecological speciation. Using RAD sequencing and whole-genome sequencing data from parental species and F1 hybrids, we analysed speciation history based on the joint allele frequency spectrum (JAFS) and pairwise sequentially Markovian coalescent (PSMC) plots. JAFS supported a model involving a split without gene flow 150,000-160,000 generations ago, followed by secondary contact 87,000-92,000 generations ago, with 64% of the genome experiencing restricted gene flow. This supports vicariance rather than sympatric speciation, likely associated with Pleistocene glaciation cycles and ocean current changes. Whole-genome PSMC analysis of F1 hybrids from Iceland suggested divergence 200,000 generations ago and indicated subsequent gene flow rather than strict isolation. Finally, simulations showed that results from both approaches (JAFS and PSMC) were congruent. Hence, there is strong evidence against sympatric speciation in North Atlantic eels. These results reiterate the need for careful consideration of cases of possible sympatric speciation, as even in seemingly barrier-free oceanic environments palaeoceanographic factors may have promoted vicariance and allopatric speciation.

Spawning by the European eel across 2000 km of the Sargasso Sea.

It has been known for about a century that European eels have a unique life history that includes offshore spawning in the Sargasso Sea about 5000-7000 km away from their juvenile and adult habitats in Europe and northern Africa. Recently hatched eel larvae were historically collected during Danish, German and American surveys in specific areas in the southern Sargasso Sea. During a 31 day period of March and April 2014, Danish and German research ships sampled for European eel larvae along 15 alternating transects of stations across the Sargasso Sea. The collection of recently hatched eel larvae (≤12 mm) from 70° W and eastward to 50° W showed that the European eel had been spawning across a 2000 km wide region of the North Atlantic Ocean. Historical collections made from 1921 to 2007 showed that small larvae had also previously been collected in this wide longitudinal zone, showing that the spatial extent of spawning has not diminished in recent decades, irrespective of the dramatic decline in recruitment. The use of such a wide spawning area may be related to variations in the onset of the silver eel spawning migration, individual differences in their long-term swimming ability, or aspects of larval drift.

Region-wide and ecotype-specific differences in demographic histories of threespine stickleback populations, estimated from whole genome sequences.

We analysed 81 whole genome sequences of threespine sticklebacks from Pacific North America, Greenland and Northern Europe, representing 16 populations. Principal component analysis of nuclear SNPs grouped populations according to geographical location, with Pacific populations being more divergent from each other relative to European and Greenlandic populations. Analysis of mitogenome sequences showed Northern European populations to represent a single phylogeographical lineage, whereas Greenlandic and particularly Pacific populations showed admixture between lineages. We estimated demographic history using a genomewide coalescence with recombination approach. The Pacific populations showed gradual population expansion starting >100 Kya, possibly reflecting persistence in cryptic refuges near the present distributional range, although we do not rule out possible influence of ancient admixture. Sharp population declines ca. 14-15 Kya were suggested to reflect founding of freshwater populations by marine ancestors. In Greenland and Northern Europe, demographic expansion started ca. 20-25 Kya coinciding with the end of the Last Glacial Maximum. In both regions, marine and freshwater populations started to show different demographic trajectories ca. 8-9 Kya, suggesting that this was the time of recolonization. In Northern Europe, this estimate was surprisingly late, but found support in subfossil evidence for presence of several freshwater fish species but not sticklebacks 12 Kya. The results demonstrate distinctly different demographic histories across geographical regions with potential consequences for adaptive processes. They also provide empirical support for previous assumptions about freshwater populations being founded independently from large, coherent marine populations, a key element in the Transporter Hypothesis invoked to explain the widespread occurrence of parallel evolution across freshwater stickleback populations.

Comparative analysis of complete mitochondrial genomes suggests that relaxed purifying selection is driving high nonsynonymous evolutionary rate of the NADH2 gene in whitefish (Coregonus ssp.).

Several studies have recently reported evidence for positive selection acting on the mitochondrial genome (mitogenome), emphasizing its potential role in adaptive divergence and speciation. In this study we searched 107 full mitogenomes of recently diverged species and lineages of whitefish (Coregonus ssp.) for signals of positive selection. These salmonids show several distinct morphological and ecological differences that may be associated with energetics and therefore potentially positive selection at the mitogenome level. We found that purifying selection and genetic drift were the predominant evolutionary forces acting on the analyzed mitogenomes. However, the NADH dehydrogenase 2 gene (ND2) showed a highly elevated dN/dS ratio compared to the other mitochondrial genes, which was significantly higher in whitefish compared to other salmonids. We therefore further examined nonsynonymous evolution in ND2 by (i) mapping amino acid changes to a protein model structure which showed that they were located away from key functional residues of the protein, (ii) locating them in the sequences of other species of fish (Salmonidae, Anguillidae, Scombridae and Percidae) only to find pronounced overlap of nonsynonymous regions. We thus conclude that relaxed purifying selection is driving the evolution of ND2 by affecting mostly regions that have lower functional relevance.

Relationship between amino acid changes in mitochondrial ATP6 and life-history variation in anguillid eels.

Mitochondrial genes are part of the oxidative phosphorylation pathway and important for energy production. Although evidence for positive selection at the mitochondrial level exists, few studies have investigated the link between amino acid changes and phenotype. Here we test the hypothesis that differences in two life-history related traits, migratory distance between spawning and foraging areas and larval phase duration, are associated with divergent selection within the mitochondrial ATP6 gene in anguillid eels. We compare amino acid changes among 18 species with the sequence of the putative ancestral species, believed to have shown short migratory distance and larval phase duration. We find positive correlations between both life-history related traits and (i) the number of amino acid changes and (ii) the strength of the combined physico-chemical and structural changes at positions previously identified as candidates for positive selection. This supports a link between genotype and phenotype driven by positive selection at ATP6.

Genomic footprints of speciation in Atlantic eels (Anguilla anguilla and A. rostrata).

The importance of speciation-with-geneflow scenarios is increasingly appreciated. However, the specific processes and the resulting genomic footprints of selection are subject to much discussion. We studied the genomics of speciation between the two panmictic, sympatrically spawning sister species; European (Anguilla anguilla) and American eel (A. rostrata). Divergence is assumed to have initiated more than 3 Ma, and although low gene flow still occurs, strong postzygotic barriers are present. Restriction-site-associated DNA (RAD) sequencing identified 328 300 SNPs for subsequent analysis. However, despite the presence of 3757 strongly differentiated SNPs (FST > 0.8), sliding window analyses of FST showed no larger genomic regions (i.e. hundreds of thousands to millions of bases) of elevated differentiation. Overall FST was 0.041, and linkage disequilibrium was virtually absent for SNPs separated by more than 1000 bp. We suggest this to reflect a case of genomic hitchhiking, where multiple regions are under directional selection between the species. However, low but biologically significant gene flow and high effective population sizes leading to very low genetic drift preclude accumulation of strong background differentiation. Genes containing candidate SNPs for positive selection showed significant enrichment for gene ontology (GO) terms relating to developmental processes and phosphorylation, which seems consistent with assumptions that differences in larval phase duration and migratory distances underlie speciation. Most SNPs under putative selection were found outside coding regions, lending support to emerging views that noncoding regions may be more functionally important than previously assumed. In total, the results demonstrate the necessity of interpreting genomic footprints of selection in the context of demographic parameters and life-history features of the studied species.

Do North Atlantic eels show parallel patterns of spatially varying selection?

BACKGROUND: The two North Atlantic eel species, the European and the American eel, represent an ideal system in which to study parallel selection patterns due to their sister species status and the presence of ongoing gene flow. A panel of 80 coding-gene SNPs previously analyzed in American eel was used to genotype European eel individuals (glass eels) from 8 sampling locations across the species distribution. We tested for single-generation signatures of spatially varying selection in European eel by searching for elevated genetic differentiation using FST-based outlier tests and by testing for significant associations between allele frequencies and environmental variables. RESULTS: We found signatures of possible selection at a total of 11 coding-gene SNPs. Candidate genes for local selection constituted mainly genes with a major role in metabolism as well as defense genes. Contrary to what has been found for American eel, only 2 SNPs in our study correlated with differences in temperature, which suggests that other explanatory variables may play a role. None of the genes found to be associated with explanatory variables in European eel showed any correlations with environmental factors in the previous study in American eel. CONCLUSIONS: The different signatures of selection between species could be due to distinct selective pressures associated with the much longer larval migration for European eel relative to American eel. The lack of parallel selection in North Atlantic eels could also be due to most phenotypic traits being polygenic, thus reducing the likelihood of selection acting on the same genes in both species.

Mitogenome sequencing reveals shallow evolutionary histories and recent divergence time between morphologically and ecologically distinct European whitefish (Coregonus spp.).

The advent of second-generation sequencing has made it possible to quickly and economically generate whole mitochondrial genome (mitogenome) sequences. To date, mitogenome studies of nonmodel organisms have demonstrated increased power for resolving interspecies relationships. We explored an alternate use of such data to recover relationships and population history of closely related lineages with a shallow evolutionary history. Using a GS-FLX platform, we sequenced 106 mitogenomes from the Coregonus lavaretus (Europe) and Coregonus clupeaformis (North America) species complexes to investigate the evolutionary history of the endangered Danish North Sea houting (NSH) and other closely related Danish and Baltic European lake whitefish (ELW). Two well-supported clades were found within both ELW and NSH, probably reflecting historical introgression via Baltic migrants. Although ELW and NSH are not reciprocally monophyletic, they share no haplotypes, suggesting recent, but strong, reproductive isolation. The divergence time between NSH and the geographically closest ELW population was estimated using IMa, assuming isolation with migration and a new mutation rate estimate chosen to avoid time-dependency effects. The estimate of c. 2700 bp was remarkably similar to results obtained using microsatellite markers. Within North American C. clupeaformis, the divergence time between the two lineages (Atlantic and Acadian) was estimated as between 20,000 and 60,000 bp. Under the assumption that NSH and ELW colonized Denmark following the last glacial maximum, Bayesian Serial SimCoal analysis showed consistency with a scenario of long-term stability, resulting from a rapid initial sixfold population expansion. The findings illustrate the utility of mitogenome data for resolving recent intraspecific divergence events and provide evidence for recent reproductive isolation of the phenotypically divergent NSH.

Satiety and eating patterns in two species of constricting snakes.

Satiety has been studied extensively in mammals, birds and fish but very little information exists on reptiles. Here we investigate time-dependent satiation in two species of constricting snakes, ball pythons (Python regius) and yellow anacondas (Eunectes notaeus). Satiation was shown to depend on both fasting time and prey size. In the ball pythons fed with mice of a relative prey mass RPM (mass of the prey/mass of the snake×100) of 15%, we observed a satiety response that developed between 6 and 12h after feeding, but after 24h pythons regained their appetite. With an RPM of 10% the pythons kept eating throughout the experiment. The anacondas showed a non-significant tendency for satiety to develop between 6 and 12h after ingesting a prey of 20% RPM. Unlike pythons, anacondas remained satiated after 24h. Handling time (from strike until prey swallowed) increased with RPM. We also found a significant decrease in handling time between the first and the second prey and a positive correlation between handling time and the mass of the snake.