Monitoring of environmental DNA from nonindigenous species of algae, dinoflagellates and animals in the North East Atlantic.

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.

Emergence and radiation of distemper viruses in terrestrial and marine mammals.

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.

Circumpolar phylogeography and demographic history of beluga whales reflect past climatic fluctuations.

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.

Comparing DADA2 and OTU clustering approaches in studying the bacterial communities of atopic dermatitis.

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.

Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples.

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.

Environmental DNA from Seawater Samples Correlate with Trawl Catches of Subarctic, Deepwater Fishes.

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.

Input data for inferring species distributions in Kyphosidae world-wide.

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.

Molecular, morphological and fossil input data for inferring relationship among viviparous brotulas (Bythitidae) - Resulting in a family status change for Dinematichthyidae.

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.

World-wide species distributions in the family Kyphosidae (Teleostei: Perciformes).

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.

A new classification of viviparous brotulas (Bythitidae) - with family status for Dinematichthyidae - based on molecular, morphological and fossil data.

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.

Population characteristics of a large whale shark aggregation inferred from seawater environmental DNA.

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.

Revision of the fish family Kyphosidae (Teleostei: Perciformes).

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 (Jenkins & Evermann, 1889) and K. ocyurus (Jordan & Gilbert, 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 Cuvier & Valenciennes, 1831) and K. analogus (Gill, 1862) are considered junior synonyms of K. vaigiensis (Quoy & Gaimard, 1825). Kyphosus gallveii (Cunningham, 1910), K. pacificus Sakai and Nakabo, 2004 and K. lutescens (Jordan & Gilbert, 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.

Kyphosus gladius, a new species of sea chub from Western Australia (Teleostei: Kyphosidae), with comments on Segutilum klunzingeri Whitley.

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.

Revision of the fish family Kyphosidae (Teleostei: Perciformes).

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 (Jenkins & Evermann, 1889) and K. ocyurus (Jordan & Gilbert, 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 Cuvier & Valenciennes, 1831) and K. analogus (Gill, 1862) are considered junior synonyms of K. vaigiensis (Quoy & Gaimard, 1825). Kyphosus gallveii (Cunningham, 1910), K. pacificus Sakai and Nakabo, 2004 and K. lutescens (Jordan & Gilbert, 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.