Comparative Population Transcriptomics Provide New Insight into the Evolutionary History and Adaptive Potential of World Ocean Krill.

Genetic variation is instrumental for adaptation to changing environments but it is unclear how it is structured and contributes to adaptation in pelagic species lacking clear barriers to gene flow. Here, we applied comparative genomics to extensive transcriptome datasets from 20 krill species collected across the Atlantic, Indian, Pacific, and Southern Oceans. We compared genetic variation both within and between species to elucidate their evolutionary history and genomic bases of adaptation. We resolved phylogenetic interrelationships and uncovered genomic evidence to elevate the cryptic Euphausia similis var. armata into species. Levels of genetic variation and rates of adaptive protein evolution vary widely. Species endemic to the cold Southern Ocean, such as the Antarctic krill Euphausia superba, showed less genetic variation and lower evolutionary rates than other species. This could suggest a low adaptive potential to rapid climate change. We uncovered hundreds of candidate genes with signatures of adaptive evolution among Antarctic Euphausia but did not observe strong evidence of adaptive convergence with the predominantly Arctic Thysanoessa. We instead identified candidates for cold-adaptation that have also been detected in Antarctic fish, including genes that govern thermal reception such as TrpA1. Our results suggest parallel genetic responses to similar selection pressures across Antarctic taxa and provide new insights into the adaptive potential of important zooplankton already affected by climate change.

Unmasking microsatellite deceptiveness and debunking hybridization with SNPs in four marine copepod species of Calanus.

Interspecific hybridization events are on the rise in natural systems due to climate change disrupting species barriers. Across taxa, microsatellites have long been the molecular markers of choice to identify admixed individuals. However, with the advent of high-throughput sequencing easing the generation of genome-wide datasets, incorrect reports of hybridization resulting from microsatellite technical artefacts have been uncovered in a growing number of taxa. In the marine zooplankton genus Calanus (Copepoda), whose species are used as climate change indicators, microsatellite markers have suggested hybridization between C. finmarchicus and C. glacialis, while other nuclear markers (InDels) never detected any admixed individuals, leaving the scientific community divided. Here, for the first time, we investigated the potential for hybridization among C. finmarchicus, C. glacialis, C. helgolandicus and C. hyperboreus using two large and independent SNP datasets. These were derived firstly from a protocol of target-capture applied to 179 individuals collected from 17 sites across the North Atlantic and Arctic Oceans, including sympatric areas, and second from published RNA sequences. All SNP-based analyses were congruent in showing that Calanus species are distinct and do not appear to hybridize. We then thoroughly re-assessed the microsatellites showing hybrids, with the support of published transcriptomes, and identified technical issues plaguing eight out of 10 microsatellites, including size homoplasy, paralogy, potential for null alleles and even two primer pairs targeting the same locus. Our study illustrates how deceptive microsatellites can be when applied to the investigation of hybridization.

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.

The phylogeography and ecology of Oligobrachia frenulate species suggest a generalist chemosynthesis-based fauna in the arctic.

We used ancient DNA (aDNA) extraction methods to sequence museum voucher samples of Oligobrachia webbi, a frenulate siboglinid polychaete described from a northern Norwegian fjord over fifty years ago. Our sequencing results indicate a genetic match with the cryptic seep species, Oligobrachia haakonmosbiensis (99% pairwise identity for 574 bp mtCOI fragments). Due to its similarity with O. webbi, the identity of O. haakonmosbiensis has been a matter of debate since its description, which we have now resolved. Furthermore, our results demonstrate that chemosynthesis-based siboglinids, that constitute the bulk of the biomass at Arctic seeps are not seep specialists. Our data on sediment geochemistry and carbon and nitrogen content reveal reduced conditions in fjords/sounds, similar to those at seep systems. Accumulation and decomposition of both terrestrial and marine organic matter results in the buildup of methane and sulfide that apparently can sustain chemosymbiotic fauna. The occurrence of fjords and by extension, highly reducing habitats, could have led to Arctic chemosymbiotic species being relatively generalist with their habitat, as opposed to being seep or vent specialists. Our stable isotope analyses indicate the incorporation of photosynthetically derived carbon in some individuals, which aligns with experiments conducted on frenulates before the discovery of chemosynthesis that demonstrated their ability to take up organic molecules from the surrounding sediment. Since reduced gases in non-seep environments are ultimately sourced from photosynthetic processes, we suggest that the extreme seasonality of the Arctic has resulted in Arctic chemosymbiotic animals seasonally changing their degree of reliance on chemosynthetic partners. Overall, the role of chemosynthesis in Arctic benthos and marine ecosystems and links to photosynthesis may be complex, and more extensive than currently known.

Insights into the species evolution of Calanus copepods in the northern seas revealed by de novo transcriptome sequencing.

Copepods of the zooplankton genus Calanus play a key role in marine ecosystems in the northern seas. Although being among the most studied organisms on Earth, due to their ecological importance, genomic resources for Calanus spp. remain scarce, mostly due to their large genome size (from 6 to 12 Gbps). As an alternative to whole-genome sequencing in Calanus spp., we sequenced and de novo assembled transcriptomes of five Calanus species: Calanus glacialis, C. hyperboreus, C. marshallae, C. pacificus, and C. helgolandicus. Functional assignment of protein families based on clusters of orthologous genes (COG) and gene ontology (GO) annotations showed analogous patterns of protein functions across species. Phylogenetic analyses using maximum likelihood (ML) of 191 protein-coding genes mined from RNA-seq data fully resolved evolutionary relationships among seven Calanus species investigated (five species sequenced for this study and two species with published datasets), with gene and site concordance factors showing that 109 out of 191 protein-coding genes support a separation between three groups: the C. finmarchicus group (including C. finmarchicus, C. glacialis, and C. marshallae), the C. helgolandicus group (including C. helgolandicus, C. sinicus, and C. pacificus) and the monophyletic C. hyperboreus group. The tree topology obtained in ML analyses was similar to a previously proposed phylogeny based on morphological criteria and cleared certain ambiguities from past studies on evolutionary relationships among Calanus species.

Customize and get the most out of your reduced-representation sequencing experiment with the new simulation software RADinitio.

Whole-genome sequencing is still often a difficult, costly and time-consuming task. The emergence of various genome reduced-representation sequencing (RRS) protocols such as restriction site-associated DNA sequencing (RADseq) has facilitated the access to genome-wide information, without the need for whole-genome sequencing. Reaching the full potential of RRS protocols though requires adjustments and tailoring to the species under investigation. To that end, simulation software has been developed to guide researchers in the customization of their RADseq experiment, but the extent to which these tools mimic the behaviour of a protocol in generating sequencing data is limited. In this current issue of Molecular Ecology Resources, Rivera-Colón et al. (2020) introduce RADinitio, a new software for simulating RADseq data designed to perform simulations at the highest level of representativeness. By taking into account the effects of library preparation and sequencing parameters on the resulting sequences, RADinitio allows the precise identification of the sources of failure when designing a RADseq experiment. This new software represents a considerable advance in RADseq data simulation and will likely lead to increased success in RADseq experiments.

Lipid metabolism in Calanus finmarchicus is sensitive to variations in predation risk and food availability.

Late developmental stages of the marine copepods in the genus Calanus can spend extended periods in a dormant stage (diapause) that is preceded by the accumulation of large lipid stores. We assessed how lipid metabolism during development from the C4 stage to adult is altered in response to predation risk and varying food availability, to ultimately understand more of the metabolic processes during development in Calanus copepods. We used RNA sequencing to assess if perceived predation risk in combination with varied food availability affects expression of genes associated with lipid metabolism and diapause preparation in C. finmarchicus. The lipid metabolism response to predation risk differed depending on food availability, time and life stage. Predation risk caused upregulation of lipid catabolism with high food, and downregulation with low food. Under low food conditions, predation risk disrupted lipid accumulation. The copepods showed no clear signs of diapause preparation, supporting earlier observations of the importance of multiple environmental cues in inducing diapause in C. finmarchicus. This study demonstrates that lipid metabolism is a sensitive endpoint for the interacting environmental effects of predation pressure and food availability. As diapause may be controlled by lipid accumulation, our findings may contribute towards understanding processes that can ultimately influence diapause timing.

Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance.

BACKGROUND: Pteropods are planktonic gastropods that are considered as bio-indicators to monitor impacts of ocean acidification on marine ecosystems. In order to gain insight into their adaptive potential to future environmental changes, it is critical to use adequate molecular tools to delimit species and population boundaries and to assess their genetic connectivity. We developed a set of target capture probes to investigate genetic variation across their large-sized genome using a population genomics approach. Target capture is less limited by DNA amount and quality than other genome-reduced representation protocols, and has the potential for application on closely related species based on probes designed from one species. RESULTS: We generated the first draft genome of a pteropod, Limacina bulimoides, resulting in a fragmented assembly of 2.9 Gbp. Using this assembly and a transcriptome as a reference, we designed a set of 2899 genome-wide target capture probes for L. bulimoides. The set of probes includes 2812 single copy nuclear targets, the 28S rDNA sequence, ten mitochondrial genes, 35 candidate biomineralisation genes, and 41 non-coding regions. The capture reaction performed with these probes was highly efficient with 97% of the targets recovered on the focal species. A total of 137,938 single nucleotide polymorphism markers were obtained from the captured sequences across a test panel of nine individuals. The probes set was also tested on four related species: L. trochiformis, L. lesueurii, L. helicina, and Heliconoides inflatus, showing an exponential decrease in capture efficiency with increased genetic distance from the focal species. Sixty-two targets were sufficiently conserved to be recovered consistently across all five species. CONCLUSION: The target capture protocol used in this study was effective in capturing genome-wide variation in the focal species L. bulimoides, suitable for population genomic analyses, while providing insights into conserved genomic regions in related species. The present study provides new genomic resources for pteropods and supports the use of target capture-based protocols to efficiently characterise genomic variation in small non-model organisms with large genomes.

A Crude Awakening: Effects of Crude Oil on Lipid Metabolism in Calanoid Copepods Terminating Diapause.

Calanus finmarchicus and Calanus glacialis are keystone zooplankton species in North Atlantic and Arctic marine ecosystems because they form a link in the trophic transfer of nutritious lipids from phytoplankton to predators on higher trophic levels. These calanoid copepods spend several months of the year in deep waters in a dormant state called diapause, after which they emerge in surface waters to feed and reproduce during the spring phytoplankton bloom. Disruption of diapause timing could have dramatic consequences for marine ecosystems. In the present study, Calanus C5 copepodites were collected in a Norwegian fjord during diapause and were subsequently experimentally exposed to the water-soluble fraction of a naphthenic North Sea crude oil during diapause termination. The copepods were sampled repeatedly while progressing toward adulthood and were analyzed for utilization of lipid stores and for differential expression of genes involved in lipid metabolism. Our results indicate that water-soluble fraction exposure led to a temporary pause in lipid catabolism, suggested by (i) slower utilization of lipid stores in water-soluble fraction-exposed C5 copepodites and (ii) more genes in the ß-oxidation pathway being downregulated in water-soluble fraction-exposed C5 copepodites than in the control C5 copepodites. Because lipid content and/or composition may be an important trigger for termination of diapause, our results imply that the timing of diapause termination and subsequent migration to the surface may be delayed if copepods are exposed to oil pollution during diapause or diapause termination. This delay could have detrimental effects on ecosystem dynamics.

The ß-oxidation pathway is downregulated during diapause termination in Calanus copepods.

Calanus copepods are keystone species in marine ecosystems, mainly due to their high lipid content, which is a nutritious food source for e.g. juvenile fish. Accumulated lipids are catabolized to meet energy requirements during dormancy (diapause), which occurs during the last copepodite stage (C5). The current knowledge of lipid degradation pathways during diapause termination is limited. We characterized changes in lipid fullness and generated transcriptional profiles in C5s during termination of diapause and progression towards adulthood. Lipid fullness of C5s declined linearly during developmental progression, but more ß-oxidation genes were upregulated in early C5s compared to late C5s and adults. We identified four possible master regulators of energy metabolism, which all were generally upregulated in early C5s, compared to late C5s and adults. We discovered that one of two enzymes in the carnitine shuttle is absent from the calanoid copepod lineage. Based on the geographical location of the sampling site, the field-samples were initially presumed to consist of C. finmarchicus. However, the identification of C. glacialis in some samples underlines the need for performing molecular analyses to reliably identify Calanus species. Our findings contributes to a better understanding of molecular events occurring during diapause and diapause termination in calanoid copepods.

Towards population genomics in non-model species with large genomes: a case study of the marine zooplankton Calanus finmarchicus.

Advances in next-generation sequencing technologies and the development of genome-reduced representation protocols have opened the way to genome-wide population studies in non-model species. However, species with large genomes remain challenging, hampering the development of genomic resources for a number of taxa including marine arthropods. Here, we developed a genome-reduced representation method for the ecologically important marine copepod Calanus finmarchicus (haploid genome size of 6.34 Gbp). We optimized a capture enrichment-based protocol based on 2656 single-copy genes, yielding a total of 154 087 high-quality SNPs in C. finmarchicus including 62 372 in common among the three locations tested. The set of capture probes was also successfully applied to the congeneric C. glacialis. Preliminary analyses of these markers revealed similar levels of genetic diversity between the two Calanus species, while populations of C. glacialis showed stronger genetic structure compared to C. finmarchicus. Using this powerful set of markers, we did not detect any evidence of hybridization between C. finmarchicus and C. glacialis. Finally, we propose a shortened version of our protocol, offering a promising solution for population genomics studies in non-model species with large genomes.

Remote sensing of zooplankton swarms.

Zooplankton provide the key link between primary production and higher levels of the marine food web and they play an important role in mediating carbon sequestration in the ocean. All commercially harvested fish species depend on zooplankton populations. However, spatio-temporal distributions of zooplankton are notoriously difficult to quantify from ships. We know that zooplankton can form large aggregations that visibly change the color of the sea, but the scale and mechanisms producing these features are poorly known. Here we show that large surface patches (>1000 km2) of the red colored copepod Calanus finmarchicus can be identified from satellite observations of ocean color. Such observations provide the most comprehensive view of the distribution of a zooplankton species to date, and alter our understanding of the behavior of this key zooplankton species. Moreover, our findings suggest that high concentrations of astaxanthin-rich zooplankton can degrade the performance of standard blue-green reflectance ratio algorithms in operational use for retrieving chlorophyll concentrations from ocean color remote sensing.

Genetics redraws pelagic biogeography of Calanus.

Planktonic copepods of the genus Calanus play a central role in North Atlantic/Arctic marine food webs. Here, using molecular markers, we redrew the distributional ranges of Calanus species inhabiting the North Atlantic and Arctic Oceans and revealed much wider and more broadly overlapping distributions than previously described. The Arctic shelf species, C. glacialis, dominated the zooplankton assemblage of many Norwegian fjords, where only C. finmarchicus has been reported previously. In these fjords, high occurrences of the Arctic species C. hyperboreus were also found. Molecular markers revealed that the most common method of species identification, prosome length, cannot reliably discriminate the species in Norwegian fjords. Differences in degree of genetic differentiation among fjord populations of the two species suggested that C. glacialis is a more permanent resident of the fjords than C. finmarchicus We found no evidence of hybridization between the species. Our results indicate a critical need for the wider use of molecular markers to reliably identify and discriminate these morphologically similar copepod species, which serve as important indicators of climate responses.

The complete mitochondrial genome of the copepod Calanus glacialis.

Calanus glacialis, a marine planktonic copepod, is a keystone species in the Arctic Ocean. In this study, we shotgun sequenced the total DNA of one C. glacialis individual, using the NextSeq® Illumina platform, in order to determine its mitochondrial genome sequence. We successfully assembled and annotated this 20,674 bp long sequence, which included 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes. Common gene-coding regions of 19 other species were used to reconstruct a phylogenetic tree, using mitogenomes of the phylogenetically closest copepods available. The new resource described here constitutes a tool of interest for better understanding the structure and dynamics of C. glacialis populations.