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Aim: Greenland is one of the places on Earth where the effects of climate change are most evident. The retreat of sea ice has made East Greenland more accessible for longer periods during the year. East Greenland fjords have been notoriously difficult to study due to their remoteness, dense sea ice conditions and lack of infrastructure. As a result, biological monitoring across latitudinal gradients is scarce in East Greenland and relies on sporadic research cruises and trawl data from commercial vessels. We here aim to investigate the transition in fish and marine mammal communities from South to Northeast Greenland using environmental DNA (eDNA). Location: South to Northeast Greenland. Methods: We investigated the transition in fish and marine mammal communities from South to Northeast Greenland using eDNA metabarcoding of seawater samples. We included both surface and mesopelagic samples, collected over approximately 2400 km waterway distance, by sampling from Cape Farewell to Ella Island in August 2021. Results: We demonstrate a clear transition in biological communities from south to northeast, with detected fish and mammal species matching known distributions. Samples from the southern areas were dominated by capelin (Mallotus villosus) and redfish (Sebastes), whereas northeastern samples were dominated by polar cod (Boreogadus saida), sculpins (Myoxocephalus) and ringed seal (Pusa hispida). We provide newly generated 12S rRNA barcodes from 87 fish species, bringing the public DNA database closer to full taxonomic coverage for Greenlandic fish species for this locus. Main Conclusions: Our results demonstrate that eDNA sampling can detect latitudinal shifts in marine biological communities of the Arctic region, which can supplement traditional fish surveys in understanding species distributions and community compositions of marine vertebrates. Importantly, sampling of eDNA can be a feasible approach for detecting northward range expansions in remote areas as climate change progresses.

The anthropogenic impact on the world’s ecosystems is severe and the need for non-invasive, cost-effective tools for monitoring and understanding those impacts are therefore urgent. Here we combine two such methods in a comprehensive multi-year study; camera trapping (CT) and analysis of environmental DNA (eDNA), in river marginal zones of a temperate, wetland Nature Park in Denmark. CT was performed from 2015 to 2019 for a total of 8,778 camera trap days and yielded 24,376 animal observations. The CT observations covered 87 taxa, of which 78 were identified to species level, and 73 were wild native species. For eDNA metabarcoding, a total of 114 freshwater samples were collected from eight sites in all four seasons from 2017 to 2018. The eDNA results yielded a total detection of 80 taxa, of which 74 were identified to species level, and 65 were wild native species. While the number of taxa detected with the two methods were comparable, the species overlap was only 20 %. In combination, CT and eDNA monitoring thus yielded a total of 115 wild species (20 fishes, four amphibians, one snake, 23 mammals and 67 birds), representing half of the species found via conventional surveys over the last ca. 20 years (83% of fishes, 68 % of mammals, 67 % of amphibians, 41 % of birds and 20 % of reptiles). Our study demonstrates that a holistic approach combining two non-invasive methods, CT and eDNA metabarcoding, has great potential as a cost-effective biomonitoring tool for vertebrates.

Several Arctic marine mammal species are predicted to be negatively impacted by rapid sea ice loss associated with ongoing ocean warming. However, consequences for Arctic whales remain uncertain. To investigate how Arctic whales responded to past climatic fluctuations, we analysed 206 mitochondrial genomes from beluga whales (Delphinapterus leucas) sampled across their circumpolar range, and four nuclear genomes, covering both the Atlantic and the Pacific Arctic region. We found four well-differentiated mitochondrial lineages, which were established before the onset of the last glacial expansion ~110 thousand years ago. Our findings suggested these lineages diverged in allopatry, reflecting isolation of populations during glacial periods when the Arctic sea-shelf was covered by multiyear sea ice. Subsequent population expansion and secondary contact between the Atlantic and Pacific Oceans shaped the current geographic distribution of lineages, and may have facilitated mitochondrial introgression. Our demographic reconstructions based on both mitochondrial and nuclear genomes showed markedly lower population sizes during the Last Glacial Maximum (LGM) compared to the preceding Eemian and current Holocene interglacial periods. Habitat modelling similarly revealed less suitable habitat during the LGM (glacial) than at present (interglacial). Together, our findings suggested the association between climate, population size, and available habitat in belugas. Forecasts for year 2100 showed that beluga habitat will decrease and shift northwards as oceans continue to warm, putatively leading to population declines in some beluga populations. Finally, we identified vulnerable populations which, if extirpated as a consequence of ocean warming, will lead to a substantial decline of species-wide haplotype diversity.

Fisheries enforcement relies on visual catch identification and quantification at sea or when landed. Silage (fish dissolved in acid) and fish blocks (block frozen fish) are promising methods for on-board processing and storage of low-value catches. We examined the use of non-destructive sampling and two DNA-based methods, quantitative PCR (qPCR) and metabarcoding, to assess species composition and relative abundance in industrial grade experimental silage and fish blocks. We demonstrate the ability to identify and quantify DNA from fish species in both products. qPCR analysis of small silage samples collected over 21 days detected all target control species. DNA from one species (Atlantic wolffish) was consistently overrepresented while, for three species of gadoids (Atlantic cod, haddock and whiting), the DNA content matched input tissue proportions with high accuracy. qPCR and metabarcoding of fish blocks, sampled as run-off water and exterior swabs, provided consistent species detection, with the highest variance observed in quantification from swab samples. Our analysis shows that DNA-based methods have significant potential as a tool for species identification and quantification of complex on-board-processed seafood products and are readily applicable to taxonomically and morphologically similar fish. There is, however, a need for establishing DNA/weight calibration factors for primary fisheries species.

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.

Monitoring the distribution of marine nonindigenous species is a challenging task. To support this monitoring, we developed and validated the specificity of 12 primer-probe assays for detection of environmental DNA (eDNA) from marine species, all nonindigenous to Europe. The species include sturgeons, a Pacific red algae, oyster thief, a freshwater hydroid from the Black Sea, Chinese mitten crab, Pacific oyster, warty comb jelly, sand gaper, round goby, pink salmon, rainbow trout and North American mud crab. We tested all assays in the laboratory, on DNA extracted from both the target and non-target species to ensure that they only amplified DNA from the intended species. Subsequently, all assays were used to analyse water samples collected at 16 different harbours across two different seasons during 2017. We also included six previously published assays targeting eDNA from goldfish, European carp, two species of dinoflagellates of the genera Karenia and Prorocentrum, two species of the heterokont flagellate genus Pseudochattonella. Conventional monitoring was carried out alongside eDNA sampling but with only one sampling event over the one year. Because eDNA was relatively fast and easy to collect compared to conventional sampling, we sampled eDNA twice during 2017, which showed seasonal changes in the distribution of nonindigenous species. Comparing eDNA levels with salinity gradients did not show any correlation. A significant correlation was observed between number of species detected with conventional monitoring methods and number of species found using eDNA at each location. This supports the use of eDNA for surveillance of the distribution of marine nonindigenous species, where the speed and relative easy sampling in the field combined with fast molecular analysis may provide advantages compared to conventional monitoring methods. Prior validation of assays increases taxonomic precision, and laboratorial setup facilitates analysis of multiple samples simultaneously. The specific eDNA assays presented here can be implemented directly in monitoring programmes across Europe and potentially worldwide to infer a more precise picture of the dynamics in the distribution of marine nonindigenous species.

Canine distemper virus (CDV) and phocine distemper virus (PDV) are major pathogens to terrestrial and marine mammals. Yet little is known about the timing and geographical origin of distemper viruses and to what extent it was influenced by environmental change and human activities. To address this, we (i) performed the first comprehensive time-calibrated phylogenetic analysis of the two distemper viruses, (ii) mapped distemper antibody and virus detection data from marine mammals collected between 1972 and 2018, and (iii) compiled historical reports on distemper dating back to the eighteenth century. We find that CDV and PDV diverged in the early seventeenth century. Modern CDV strains last shared a common ancestor in the nineteenth century with a marked radiation during the 1930s–1950s. Modern PDV strains are of more recent origin, diverging in the 1970s–1980s. Based on the compiled information on distemper distribution, the diverse host range of CDV and basal phylogenetic placement of terrestrial morbilliviruses, we hypothesize a terrestrial CDV-like ancestor giving rise to PDV in the North Atlantic. Moreover, given the estimated timing of distemper origin and radiation, we hypothesize a prominent role of environmental change such as the Little Ice Age, and human activities like globalization and war in distemper virus evolution.

A new endemic species of triplefin Ruanoho scurra is described from deep water (108–216 m) on the shelf region around coastal New Zealand (Northland to Stewart Island). It is differentiated from its congeners by the combination of fresh colour (bright yellow spots on the head and anterior body, oblique lines on the dorsal and anal fins, and sub-vertical lines on the caudal) as well as some proportional measurements. Comments are made on the relationship with its congeners, and evolutionary history of the family in New Zealand waters, along with observations on the habitat in which this new species is found. This paper formally describes the species first mentioned in Stewart & Clements 2015:1523 as the polkadot triplefin.

Aim: Herbivorous reef fishes are considered to have difficulty digesting plant material at extratropical temperatures and are thus largely restricted to tropical waters where they are thought to have evolved. However, the herbivorous Kyphosidae, with both temperate and tropical species, provides an ideal opportunity to test this view. Previous studies have resolved the taxonomy and distribution patterns of all species. Here, we use a calibrated phylogeny to analyse the age, geographical origin and pattern of diversification of kyphosids to determine their origins in space and time, and thus refine hypotheses on the evolutionary origins of herbivory in reef fishes. Location: Pacific Ocean, Atlantic Ocean, Indian Ocean, Western Australia. Methods: The age and geographic origin of Kyphosidae were determined by incorporating fossil calibrations and species distributions onto a phylogeny of all extant species based on fragments from mitochondrial markers and three nuclear markers, and using Bayesian modelling to reconstruct ancestral distributions. Evolution of both diet and tooth shape was also examined using ancestral reconstruction. Results: Ancestral reconstruction suggested a subtropical, Southern Hemisphere Indo-Pacific origin for the family. The chronogram indicates that Kyphosus originated in the early Miocene and that tropical diversity reflects very recent diversification. Main conclusions: Contrary to the general perception that herbivorous reef fishes invaded high latitudes from the tropics, herbivorous kyphosids evolved at intermediate latitudes in the Southern Hemisphere and subsequently diversified into low latitudes where several species dispersed recently to achieve circumglobal distributions. The southern temperate/subtropical reef environment appears to have generated episodes of diversification in several, well-established, widespread reef fish taxa. Some of these reef fish taxa have remained restricted to southern temperate reef environments despite a long tenure in the Cenozoic (e.g. odacines and aplodactylids), and some such as the kyphosids which diversified very recently in both hemispheres resulting in both regional endemics and species with worldwide distributions.

Introduction. The pathogenesis of atopic dermatitis (AD) is not yet fully understood, but the bacterial composition of AD patients’ skin has been shown to have an increased abundance of Staphylococcus aureus . More recently, coagulase-negative Staphylococcus (CoNS) species were shown to be able to inhibit S. aureus , but further studies are required to determine the effects of Staphylococcus community variation in AD. Aim. Here we investigated whether analysing metabarcoding data with the more recently developed DADA2 approach improves metabarcoding analyses compared to the previously used operational taxonomic unit (OTU) clustering, and can be used to study Staphylococcus community dynamics. Methods. The bacterial 16S rRNA region from tape strip samples of the stratum corneum of AD patients (non-lesional skin) and non-AD controls was metabarcoded. We processed metabarcoding data with two different bioinformatic pipelines (an OTU clustering method and DADA2), which were analysed with and without technical replication (sampling strategy). Results. We found that OTU clustering and DADA2 performed well for community-level studies, as demonstrated by the identification of significant differences in the skin bacterial communities associated with AD. However, the OTU clustering approach inflated bacterial richness, which was worsened by not having technical replication. Data processed with DADA2 likely handled sequencing errors more effectively and thereby did not inflate molecular richness. Conclusion. We believe that DADA2 represents an improvement over an OTU clustering approach, and that biological replication rather than technical replication is a more effective use of resources. However, neither OTU clustering nor DADA2 gave insights into Staphylococcus community dynamics, and caution should remain in not overinterpreting the taxonomic assignments at lower taxonomic ranks.

1. The European noble crayfish Astacus astacus is threatened by crayfish plague caused by the oomycete Aphanomyces astaci, which is spread by the invasive North American crayfish (e.g. signal crayfish Pacifastacus leniusculus). Surveillance of crayfish plague status in Norway has traditionally relied on the monitoring survival of cage‐held noble crayfish, a method of ethical concern. Additionally, trapping is used in crayfish population surveillance. Here, we test whether environmental DNA (eDNA) monitoring could provide a suitable alternative to the cage method, and a supplement to trapping. 2. We took advantage of an emerging crayfish plague outbreak in a Norwegian watercourse following illegal introduction of disease‐carrying signal crayfish, and initiated simultaneous eDNA monitoring and cage‐based surveillance, supplemented with trapping. A total of 304 water samples were filtered from several sampling stations over a 4‐year period. eDNA data (species‐specific quantitative real‐time PCR [qPCR]) for the presence of A. astaci, noble and signal crayfish within the water samples were compared to cage mortality and trapping. 3. This is the first study comparing eDNA monitoring and cage surveillance during a natural crayfish plague outbreak. We show that eDNA monitoring corresponds well with the biological status measured in terms of crayfish mortality and trapping results. eDNA analysis also reveals the presence of A. astaci in the water up to 2.5 weeks in advance of the cage method. Estimates of A. astaci and noble crayfish eDNA concentrations increased markedly during mortality and vanished quickly thereafter. eDNA provides a snapshot of the presence, absence or disappearance of crayfish regardless of season, and constitutes a valuable supplement to the trapping method that relies on season and legislation. 4. Synthesis and applications. Simultaneous eDNA monitoring of Aphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish species is well‐suited for early warning of invasion or infection, risk assessments, habitat evaluation and surveillance regarding pathogen and invasive/native crayfish status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach.

Biomass assesment of fish stocks is a difficult task that often involves costly fisheries trawl surveys. Trawl surveys rarely return replicate samples, as trawling attempts are expensive and difficult to reproduce. Furthermore, traditional benthic trawling is often detrimental to habitats and the organisms associated with the sea bottom. The rapidly developing field of environmental DNA (eDNA) analysis offers a new approach to non-invasive monitoring of fish. In the present study, we develop and test species-specific primers and probes for qPCR detection of eDNA from Atlantic herring (Clupea harengus ), Atlantic cod ( Gadus morhua ), European flounder ( Platichthys flesus ), European plaice ( Pleuronectes platessa), and Atlantic mackerel ( Scomber scombrus ) in the Baltic Sea. A recently published qPCR system was applied for European eel (Anguilla anguilla ). Filtered water samples were collected during a stratified benthic trawl survey, enabling parallel comparisons of eDNA concentrations with biomass caught by trawling. No significant correlation was found between eDNA concentrations and the biomass of fish caught by the trawl, although an association was observed between the measured concentrations of eDNA and the known distributions and main abundances of cod, herring, plaice and flounder. This indicates that while eDNA concentrations may not be directly comparable to results from existing methods – likely because aquatic eDNA concentrations are not controlled by the same combinations of factors as e.g. trawl biomass catch – eDNA analysis could prove a useful supplement for monitoring fish stocks in the future.

Summary Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex-GP polyethersulfone, pore size 0·22 μm, hereafter called SX) with commonly used methods. Our experimental set-up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish-free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy® Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq-values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq-values than polycarbonate track-etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq-values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro-organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.

This article comprise the data related to the research article (Møller et al., 2016) [1], and makes it possible to explore and reproduce the topologies that allowed [1] to infer the relationship between the families Bythitidae and Dinematichthyidae. The supplementary data holds nexus-input files for the Bayesian analysis and the ‘.xml’-input files – with and without nucleotide data – that are used in the fossil-calibrated phylogenetic analysis with a relaxed clock model. The resulting topologies are provided as ‘.new’-files together with a characters matrix file for traits to trace across the inferred phylogenies. Keywords: Bythitinae, Aphyonidae, pedomorphism, Coral reef fishes, Deepsea fishes, Cave fishes

The order Ophidiiformes is a large but not very well known group of fishes, unique among teleosts for showing high diversity in both deep sea and shallow reef habitats. The current classification includes more than 500 species, 115 genera and four families, based primarily on mode of reproduction: viviparous Aphyonidae and Bythitidae vs oviparous Carapidae and Ophidiidae. Since 2004 we revised the bythitid tribe Dinematichthyini, described more than 100 new species and noticed that this group has unique morphological characters, perhaps supporting a higher level of classification than the current status. Here we study the viviparous families phylogenetically with partial mitochondrial (nd4, 16s) and nuclear (Rag1) DNA sequences (2194bp). We use a fossil calibration of otolith-based taxa to calibrate the age of the clade comprising bythitid and dinematicththyid representatives, together with fossil calibrations adopted from previous phylogenetic studies. The separation of the order into two major lineages, the viviparous Bythitoidei and the oviparous Ophidioidei is confirmed. At the familial level, however, a new classification is presented for the viviparous clades, placing Aphyonidae as a derived, pedomorphic member of Bythitidae (new diagnosis provided, 33 genera and 118 species). The current subfamily Brosmophycinae is considered polyphyletic and we propose family status for Dinematichthyidae (25 genera, 114 species), supported by unique, morphological synapomorphic characters in the male copulatory apparatus. Previous use of the caudal fin separation or fusion with vertical fins is ambiguous. Age estimates based on calibrated molecular phylogeny agrees with fossil data, giving an origin within the Cretaceous (between 84 and 104mya) for a common ancestor to Ophidiiformes.

Although the Greenland fish fauna has been studied for more than 200 years, new species continue to be discovered. We here take the opportunity of the International Polar Year 2007–08 (IPY) to present an updated check-list of the fishes of Greenland and discuss whether the growing diversity can be explained by global warming. A total of 269 species from 80 families are known from the Greenland Exclusive Economic Zone (EEZ), based on published literature and specimens in museum collections. Since the latest publication covering all known Greenland fishes [Nielsen & Bertelsen 1992], 57 species have been added. Nineteen of these (Harriotta raleighana, Centroscymnus coelolepis, Bathytroctes microlepis, Einara edentula, Ceratoscopelus maderensis, Argyropelecus gigas, Maurolicus muelleri, Polyipnus asteroides, Nansenia oblita, Melanostomias bartonbeani, Polymetme corythaeola, Coryphaenoides mediterraneus, Merlangius merlangus, Guttigadus latifrons, Entelurus aequoreus, Helicolenus dactylopterus, Epigonus telescopus, Lophius piscatorius, Linophryne bicornis) are reported here for the first time. Twenty-nine of the species were added on the basis of taxonomic revisions and/ or identification of specimens caught before 1992, whereas 28 species have been caught in Greenland waters for the first time since 1992. Ten species were new to science described since 1992. Only five of the added species are Arctic – i.e. mainly caught north of the Davis and Denmark Straits. Of the 28 species caught after 1992, five species (Maurolicus muelleri, Merlangius merlangus, Helicolenus dactylopterus, Lophius piscatorius, Entelurus aequoreus) from the southern regions (Atlantic) are mainly from shallow waters (< 400 m) and their arrival is likely to be a result of increasing temperatures. The explanation of the many new records of deep-water fishes is most likely increasing fishing efforts down to depths of 1500 m. The deep waters off Greenland (> 1500 m), however, remain almost unstudied.

Trophic interactions in complex mesopelagic ecosystems are generally poorly understood, but tracing diet remains of predators provides key insights into these. In many cases, however, the prey remains obtained from predator stomachs are unidentifiable by visual inspection and identification depends on new molecular techniques. Here, we search for predators on larvae of the critically endangered European eel (Anguilla anguilla), a species which has shown a dramatic decline in recruitment to the stock. We sampled for predators among mesopelagic fishes using pelagic trawling in the known area of spawning and larval distribution of A. anguilla in the Sargasso Sea. We aimed at developing a species-specific quantitative PCR (qPCR) system, targeting the nd4-region of mitochondrial DNA (mtDNA), to search for eel remains in the stomachs of 17 mesopelagic fish taxa (62 specimens). Remains of A. anguilla were confirmed in 9.7% of all fish stomachs investigated, representing six species (Lampanyctus cuprarius, L. photonotus, Myctophum selenops, Notoscopelus caudispinosus, Melamphaes typhlops, and Chauliodus danae). Thus, our study documents that mesopelagic fishes in the Sargasso Sea to some extent predate the A. anguilla larvae, motivating further studies on upper-level trophic interactions in this oceanic ecosystem.

Europe has a long tradition of exploiting marine fishes and is promoting marine economic activity through its Blue Growth strategy. This increase in anthropogenic pressure, along with climate change, threatens the biodiversity of fishes and food security. Here, we examine the conservation status of 1,020 species of European marine fishes and identify factors that contribute to their extinction risk. Large fish species (greater than 1.5 m total length) are most at risk; half of these are threatened with extinction, predominantly sharks, rays and sturgeons. This analysis was based on the latest International Union for Conservation of Nature (IUCN) European regional Red List of marine fishes, which was coherent with assessments of the status of fish stocks carried out independently by fisheries management agencies: no species classified by IUCN as threatened were considered sustainable by these agencies. A remarkable geographic divergence in stock status was also evident: in northern Europe, most stocks were not overfished, whereas in the Mediterranean Sea, almost all stocks were overfished. As Europe proceeds with its sustainable Blue Growth agenda, two main issues stand out as needing priority actions in relation to its marine fishes: the conservation of marine fish megafauna and the sustainability of Mediterranean fish stocks. Marine fishes exhibit high biodiversity 1,2 and have been culturally and nutritionally important throughout human history 3 . Europe, in particular, has a well-documented history of exploiting marine fish populations, written records of which commence in the classical works of ancient Greece. Although this historical exploitation has undoubtedly altered populations 4,5 and changed many seascapes 6 , marine defaunation in the region has not been as great as in terrestrial systems 7 . However, the use of ocean space and resources is increasing due to Europe’s Blue Growth strategy 8 , the nutritional requirements of an expanding human population are growing 9,10 and marine ecosystems will experience unusually rapid changes in future due to climate change 11,12 . Consequently there are imminent threats both to European marine biodiversity and fish resources 13 . It is important, therefore, to assess the threats of extinction to fish species and to ensure consistency in the management approach by the various agencies involved. We analysed data on the conservation status of 1,020 species of Europe’s marine fishes from the recent International Union for Conservation of Nature (IUCN) Red List assessments 14 to identify characteristics that make Europe’s fishes most susceptible to extinction risk. We then compared the Red List with 115 fish stock assessments (of 31 species) made by intergovernmental agencies charged with providing advice on the exploitation of commercial fishes. Previous comparisons of this sort applied criteria under various modelling assumptions 15,16, 17 or limited the comparison to biomass reference points 18 .

Nature Ecology & Evolution 1, 0170 (2017); published 26 May 2017; corrected 12 June 2017. In the original version of this Article, the European Commission was mistakenly included as an affiliation for Christos D. Maravelias. His contribution to this work was exclusively completed while at the Hellenic Centre for Marine Research.

Coastal marine fish populations are in decline due to overfishing, habitat destruction, climate change and invasive species. Seasonal monitoring is important for detecting temporal changes in the composition of fish communities, but current monitoring is often non-existent or limited to annual or semi-annual surveys. In the present study, we investigate the potential of using environmental DNA (eDNA) metabarcoding of seawater samples to detect the seasonal changes in a coastal marine fish community. Water sampling and snorkelling visual census were performed over 1 year (from 23rd of August 2013 to 11th of August 2014) at a temperate coastal habitat in Denmark (55°45′39″N, 12°35′59″E) and compared to long-term data collected over a 7-year period. We used Illumina sequencing of PCR products to demonstrate that seawater eDNA showed compositional changes in accordance with seasonal changes in the fish community. The vast majority of fish diversity observed in the study area by snorkelling was recovered from sequencing, although the overlap between methods varied widely among sampling events. In total, 24 taxa were detected by both methods, while five taxa were only detected using eDNA and three taxa were only detected by snorkelling. A limitation of the applied primers was the lack of resolution to species level in a few diverse families, and varying sequencing depth between samples represents a potential bias. However, our study demonstrates the utility of eDNA for recovering seasonal variation in marine fish communities, knowledge of which is essential for standardised long-term monitoring of marine biodiversity.

Two morphologically distinct forms of the nominal species Kyphosus sydneyanus (Günther, 1886) (Kyphosidae) were discerned while collecting off Western Australia near Perth in 2009. A morphological comparison with recognized species of Kyphosus and an analysis of mtDNA (Cytochrome b, control region, 12S and 16S) and three nDNA markers (RAG1, RAG2 and Tmo-4C4) demonstrated that the more elongate of these forms was an undescribed species of Kyphosus. It differs from congeners in the Pacific and Indian Oceans in the combination of the following characters: green bar on the operculum, 11–12 dorsal soft fin rays, depth of caudal peduncle 9.9–11.8 % SL, body depth 33.3–41.6 % SL, 55–63 scales in lateral line, 12–16 interorbital scales, 44–55 pored scales in the lateral line, 3–5 gill rakers on upper limb of first gill arch internally, 11–15 gill rakers on lower limb of first gill arch internally, 15–19 total gill rakers on first gill arch, and by having 10 precaudal vertebrae and 16 caudal vertebrae. Examination of museum specimens and available underwater photographs suggests that the new species is restricted to Western Australia, and to date it has been recorded between the Houtman Abrolhos Islands and Albany. Discrepancies between the type specimen and original description of Segutilum klunzingeri Whitley made it impossible to determine the relationship between this taxon and the new species from Western Australia, and thus we consider S. klunzingeri a nomen dubium.

Population genetics is essential for understanding and managing marine ecosystems, but sampling remains challenging. We demonstrate that high-throughput sequencing of seawater environmental DNA can provide useful estimates of genetic diversity in a whale shark (Rhincodon typus) aggregation. We recover similar mitochondrial haplotype frequencies in seawater compared to tissue samples, reliably placing the studied aggregation in a global genetic context and expanding the applications of environmental DNA to encompass population genetics of aquatic organisms.

Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus) showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic habitats. This relates directly to applied fisheries as well as to monitoring effects of ongoing climate change on marine biodiversity-especially in polar ecosystems.

The Red List of marine bony fishes of the Eastern Central Atlantic (ECA) is a review of the conservation status of all native marine bony fishes in ECA according to the global Categories and Criteria of the IUCN Red List of Threatened Species. It identifies those species that are threatened with extinction at the global level and occur within the ECA region. This comprehensive assessment, which is the first of its kind in the ECA, aims to provide improved knowledge of species presence and extinction risk status for the purposes of guiding conservation actions and improved policies for these species both globally and regionally.

Input data files for inferring the relationship among the family Kyphosidae, as presented in (Knudsen and Clements, 2016) [1], is here provided together with resulting topologies, to allow the reader to explore the topologies in detail. The input data files comprise seven nexus-files with sequence alignments of mtDNA and nDNA markers for performing Bayesian analysis. A matrix of recoded character states inferred from the morphology examined in museum specimens representing Dichistiidae, Girellidae, Kyphosidae, Microcanthidae and Scorpididae, is also provided, and can be used for performing a parsimonious analysis to infer the relationship among these perciform families. The nucleotide input data files comprise both multiple and single representatives of the various species to allow for inference of the relationship among the species in Kyphosidae and between the families closely related to Kyphosidae. The ‘.xml’-files with various constrained relationships among the families potentially closely related to Kyphosidae are also provided to allow the reader to rerun and explore the results from the stepping-stone analysis. The resulting topologies are supplied in newick-file formats together with input data files for Bayesian analysis, together with ‘.xml’-files. Re-running the input data files in the appropriate software, will enable the reader to examine log-files and tree-files themselves. Keywords: Sea chub, Drummer, Kyphosus, Scorpis, Girella

Sea chubs of the family Kyphosidae are major consumers of macroalgae on both temperate and tropical reefs, where they can comprise a significant proportion of fish biomass. However, the relationships and taxonomic status of sea chubs (including the junior synonyms Hermosilla, Kyphosus, Neoscorpis and Sectator) worldwide have long been problematical due to perceived lack of character differentiation, complicating ecological assessment. More recently, the situation has been further complicated by publication of conflicting taxonomic treatments. Here, we resolve the relationships, taxonomy and distribution of all known species of sea chubs through a combined analysis of partial fragments from mitochondrial markers (12s, 16s, cytb, tRNA -Pro, -Phe, -Thr and -Val) and three nuclear markers (rag1, rag2, tmo4c4). These new results provide independent evidence for the presence of several junior synonyms among Atlantic and Indo-Pacific taxa, demonstrating that several sea chub species are more widespread than previously thought. In particular, our results can reject the hypothesis of endemic species in the Atlantic Ocean. At a higher taxonomic level, our results shed light on the relationships between Girellidae, Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae and Scorpididae, with Scorpididae resolved as the sister group to Kyphosidae.

This report is the outcome of MONIS 2 – or in full, “Monitoring of Non-Indigenous Species in Danish Marine Water, phase 2” – and includes three deliverable: (1) a national Target Species List including 50 species, (2) a draft Technical Guidance Report, and (3) in silico designed and tested primers and probes for 48 of the 50 species on the Target Species List. The list is based on discussions at a workshop and subsequent scoring and ranking of relevant species. The draft Technical Guidance report is anchored in existing Standard Operating Procedures (i.e. protocols for sampling, storage and analysis) and adapted to the requirements of the Danish NOVANA programme. In addition, the report includes suggestions for next steps to take to implement and improve monitoring and assessment activities in regard to non-indigenous species in Danish marine waters. Danish Nature Agency

Fishes of the order Alepocephaliformes, slickheads and tubeshoulders, constitute a group of deep-sea fishes poorly known in respect to most areas of their biology and systematics. Morphological studies have found alepocephaliform fishes to display a mosaic of synapomorphic and symplesiomorphic characters, resulting in great difficulties when attempting to resolve intra- and interrelationships. Molecular data recently added to the confusion by removing Alepocephaliformes from the Euteleostei and placed them as incertae sedis within the Otocephala. In the present study we attempt to further clarify relationships of Alepocephaliformes by adding newly determined whole mitogenome sequences from 19 alepocephaliforms in order to address 1) phylogenetic position of Alepocephaliformes within the Otocephala; and 2) intrarelationships of Alepocephaliformes. The present study includes 96 taxa of which 30 are alepocephaliforms and unambiguously aligned sequences were subjected to partitioned maximum likelihood and Bayesian analyses. Results from the present study support Alepocephaliformes as a genetically distinct otocephalan order as sister clade to Ostariophysi (mostly freshwater fishes comprising Gonorynchiformes, Cypriniformes, Characiformes, Siluriformes and Gymnotiformes). The disputed family Bathylaconidae was found to be an artificial assemblage of the two genera Bathylaco and Herwigia, with the former as the sister group of the family Alepocephalidae and the latter nested within Alepocephalidae. Platytroctidae was found to be monophyletic as sister clade to the rest of Alepocephaliformes. Previously unrecognized clades within the family Alepocephalidae are presented and a clade comprising Alepocephalus, Conocara and Leptoderma was recovered as the most derived. As long as the current classification is being followed, the genera Alepocephalus, Bathytroctes, Conocara and Narcetes were all found non-monophyletic. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 923–936.

Careproctus kidoi sp. nov. is described from the Baffin Bay between Greenland and Canada, in the northern most part of the western North Atlantic. During a series of bottom trawl surveys conducted in 1988–2004, 22 specimens of an undescribed species of Careproctus were caught at depths between 952 and 1,487 m. It differs from Arctic and North Atlantic congeners in the combination of the characters: pectoral fin rays 21–26, dorsal fin rays 54–60, anal fin rays 50–54, vertebrae 61–64; sucking disc oval, 4.2–6.6% SL; teeth simple; pyloric coeca 3–12; head pore formula (2-6-7-1); color light to dark brown, stomach dusky to dark brown, peritoneum black. It is generally found in deeper waters than the sympatric C. reinhardti.

Liparidae (snailfishes) is one of the most diverse and abundant fish families in polar and deep-sea habitats. However, the evolution of this family is poorly known because of the rarity of many species and difficulties in scoring morphological characters. We perform phylogenetic analyses of Liparidae using sequences from two mtDNA genes, 16S (585 bp) and cytochrome b (426 bp), and 84 morphological characters from 24 species of Liparidae and 4 species of Cyclopteridae (outgroup). The present study confirms earlier hypotheses that the shallow-water genera, such as Liparis and Crystallichthys, occupy basal positions and that deep-water genera, such as Careproctus, Elassodiscus, Rhinoliparis, Paraliparis, Rhodichthys and Psednos, are increasingly derived. The later two genera form a terminal clade which does not include Paraliparis. The topology shows that the family has undergone a reductive type of evolution, with a gradual loss of characters (e.g. sucking disc/pelvic fins, pseudobranchial filaments, skin spinules). Nectoliparis, which had previously been placed either as the basal most genus or among the most derived genera, are found to occupy the most basal position among the taxa analyzed. This result indicates that the sucking disc has been lost at least twice during the evolution of the Liparidae. The basal position of Nectoliparis is supported by its plesiomorphic otolith morphology, whereas an advanced overgrown otolith ostium, unique among teleosts, is found to be apomorphic for a clade containing the derived genera: Paraliparis, Psednos, Rhinoliparis and Rhodichthys. We also identify the presence of probable nuclear inserts of mitochondrial DNA (Numts) in three species of Careproctus and in Elassodiscus caudatus.

For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.

A molecular phylogenetic analysis with complete species sampling of the family Kyphosidae revealed several discrepancies with the current taxonomy. We thus undertook a complete taxonomic revision of all kyphosid genera, i.e. Kyphosus Lacepède, 1801, and the monotypic Hermosilla Jenkins and Evermann, 1889, Sectator Jordan and Evermann, 1903 and Neoscorpis Smith, 1931. Species delimitation was determined on the basis of congruence between (a) monophyletic groupings in the molecular phylogeny, and (b) clusters of morphological variation in type material. Twelve species are supported and redescribed. Both Hermosilla and Sectator are considered junior synonyms of Kyphosus. Kyphosus azureus (JenkinsEvermann, 1889) and K. ocyurus (JordanGilbert, 1882) are redescribed accordingly. We designate a neotype for Kyphosus cornelii (Whitley, 1944), as the original material is lost, and new material was collected at the type locality for this study to facilitate comparison with other species of Kyphosus. Kyphosus sandwicensis (sensu Sauvage, 1880) was found to be a junior synonym of K. elegans (Peters, 1869). Kyphosus incisor (Cuvier in CuvierValenciennes, 1831) and K. analogus (Gill, 1862) are considered junior synonyms of K. vaigiensis (QuoyGaimard, 1825). Kyphosus gallveii (Cunningham, 1910), K. pacificus Sakai and Nakabo, 2004 and K. lutescens (JordanGilbert, 1882) are all considered junior synonyms of K. sectatrix (Linnaeus, 1758). One of the two syntype specimens of K. sectatrix was identified as the holotype of Pimelepterus bosquii (Lacepède, 1802), and proved to be a specimen of K. bigibbus Lacepède, 1801. This specimen is re-assigned as a non-type of K. bigibbus. Full re-descriptions of the following valid species are presented: K. bigibbus, K. cinerascens (Forsskål, 1775), K. cornelii, K. elegans, K. hawaiiensis Sakai and Nakabo, 2004, K. gladius Knudsen and Clements, 2013, K. sydneyanus (Günther, 1886) and K. vaigiensis, together with a key to the family. The distribution of Kyphosus species is reconsidered based on our taxonomic revision, indicating that four species (K. bigibbus, K. cinerascens, K. sectatrix and K. vaigiensis) occur in both the Atlantic and Indo-Pacific regions.

Two morphologically distinct forms of the nominal species Kyphosus sydneyanus (Günther, 1886) (Kyphosidae) were discerned while collecting off Western Australia near Perth in 2009. A morphological comparison with recognized species of Kyphosus and an analysis of mtDNA (Cytochrome b, control region, 12S and 16S) and three nDNA markers (RAG1, RAG2 and Tmo-4C4) demonstrated that the more elongate of these forms was an undescribed species of Kyphosus. It differs from congeners in the Pacific and Indian Oceans in the combination of the following characters: green bar on the operculum, 11-12 dorsal soft fin rays, depth of caudal peduncle 9.9-11.8 % SL, body depth 33.3-41.6 % SL, 55-63 scales in lateral line, 12-16 interorbital scales, 44-55 pored scales in the lateral line, 3-5 gill rakers on upper limb of first gill arch internally, 11-15 gill rakers on lower limb of first gill arch internally, 15-19 total gill rakers on first gill arch, and by having 10 precaudal vertebrae and 16 caudal vertebrae. Examination of museum specimens and available underwater photographs suggests that the new species is restricted to Western Australia, and to date it has been recorded between the Houtman Abrolhos Islands and Albany. Discrepancies between the type specimen and original description of Segutilum klunzingeri Whitley made it impossible to determine the relationship between this taxon and the new species from Western Australia, and thus we consider S. klunzingeri a nomen dubium.

The role of the Escherichia coli lacY gene product (the lactose permease) in the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible promoters was studied in E. coli and P. fluorescens. This was done by comparing strains containing a lacIPOZYA chromosomal insert with newly constructed strains containing inserts without the lacY gene (lacIPOZ). The lactose operon inserts were introduced as single-copy chromosomal inserts to eliminate differences in expression caused by differences in copy number. Comparison between the two types of inserts showed that the lactose permease was essential to allow growth on lactose by both bacteria and that the lactose permease plays an important role in transporting the inducer IPTG across the membrane of P. fluorescens. The use of a functional lactose permease allows expression of beta-galactosidase to increase more than fivefold from a wild-type lac promoter in P. fluorescens SS1001. We suggest that an increase in the rate of protein synthesis from lac-type promoters could be enhanced if an active lactose permease is present as well.

Environmental changes are prominent in Arctic ecosystems, where the distribution, abundance, life history, and health of marine organisms such as the bowhead whale (Balaena mysticetus) are tightly connected to sea ice and sea temperature. However, due to logistical and other challenges of data collection in the Arctic, appropriate assessments of past, present and future effects of climate change and human activities are lacking for many Arctic species. Environmental DNA (eDNA) is emerging as a noninvasive and cost‐effective way of obtaining genetic material from the environment and has the potential to complement traditional methods for biodiversity and genetic monitoring. In this study, we investigate whether eDNA isolated from seawater samples has the capacity to capture the genetic diversity of bowhead whales in Disko Bay, West Greenland, for the implementation of long‐term genetic monitoring programs of key Arctic marine species. A total of 41 eDNA “footprint” samples were obtained from the water surface after a whale had dived and an additional 54 eDNA samples were collected along transect lines. Samples were screened for bowhead DNA using a species‐specific qPCR primer and probe assay, and a subset of 30 samples were successfully Sanger‐sequenced to generate individual mitochondrial control region haplotypes. Moreover, by shotgun sequencing ten footprint samples on an Illumina NovaSeq platform we show that footprints generally contain less than 1% endogenous DNA, resulting in partial mitochondrial genomes in four samples out of ten samples. Our findings suggest that sampling in the footprint or wake of traveling animals is a promising method for capturing the genetic diversity of bowhead whales and other marine megafauna. With optimization of sampling and target DNA sequencing for higher endogenous DNA yield, seawater eDNA samples have a large potential for implementation in the long‐term population genetic monitoring of marine megafauna in the Arctic and elsewhere.

Project manager Jesper Harbo Andersen This MONIS 5 report focuses on freshwater crayfish and was funded by the Danish Environmental Protection Agency. The aim of the present study was to develop species-specific systems for tracking environmental DNA (eDNA) from nine species of freshwater crayfish in water samples. Among these nine species, only one (Astacus astacus) can be considered indigenous in Scandinavia, and among the other eight non-indigenous species; two (Astacus leptodactylus and Pacifastacus leniusculus) are frequently encountered in Scandinavian freshwater systems. The remaining seven non-indigenous species are rarely encountered in Scandinavia but have been recorded in the past from other places in Europe. It is our aim that the nine species-specific eDNA assays presented here will allow for continuous monitoring of both the one indigenous species, the two non-indigenous species more frequently encountered and help produce early warnings of the seven non-indigenous species that might disperse to Denmark. Danish Environmental Protection Agency