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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.

Insects and other terrestrial invertebrates are declining in species richness and abundance. This includes the invertebrates associated with herbivore dung, which have been negatively affected by grazing abandonment and the progressive loss of large herbivores since the Late Pleistocene. Importantly, traditional monitoring of these invertebrates is time‐consuming and requires considerable taxonomic expertise, which is becoming increasingly scarce. In this study, we investigated the potential of environmental DNA (eDNA) metabarcoding of cow dung samples for biomonitoring of dung‐associated invertebrates. From eight cowpats we recovered eDNA from 12 orders, 29 families, and at least 54 species of invertebrates (mostly insects), representing several functional groups. Furthermore, species compositions differed between the three sampled habitats of dry grassland, meadow, and forest. These differences were in accordance with the species’ ecology; for instance, several species known to be associated with humid conditions or lower temperatures were found only in the forest habitat. We discuss potential caveats of the method, as well as directions for future study and perspectives for implementation in research and monitoring.

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied “eDNA haplotyping” to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.

Large carnivores are currently recolonizing Europe following legal protection, but increased mortality in landscapes highly impacted by humans may limit further population expansion. We analyzed mortality and disappearance rates of 35 wolves (of which three emigrated, nine died and 14 disappeared by 1 January 2020) by genetic monitoring in the heavily cultivated and densely populated Jutland peninsula (Denmark and Schleswig‐Holstein, Germany). Annual traffic kill rate estimates ranged from 0.37 (95% CI: 0.11–0.85) to 0.78 (0.51–0.96) in the German part, equivalent to 0.08 (0.02–0.29)–0.25 (0.13–0.46) for the entire region, in the absence of any registered Danish roadkills. In Denmark, annual mortality rate estimates ranged from 0.46 (0.29–0.67) to 0.52 (0.35–0.71), predominantly from cryptic mortality. Despite successful reproductions, we conclude the region is a wolf population sink, primarily driven by cryptic mortality, most likely illegal killing. We hypothesize that frequent encounters between wolves and wolf‐averse persecutors in cultivated landscapes may cause unsustainably high mortality rates despite the majority of hunters respecting protection laws.

Macroinvertebrate communities are crucial for biodiversity monitoring and assessment of ecological status in stream ecosystems. However, traditional monitoring approaches require intensive sampling and rely on invasive morphological identifications that are time‐consuming and dependent on taxonomic expertise. Importantly, sampling is often only carried out once in a year, namely during late winter–spring, where most indicator taxa have larval stages in the streams. Hence, species with divergent phenology might not be detected. Here, we use environmental DNA (eDNA) metabarcoding of filtered water samples collected in both spring and autumn from five streams in Denmark to address seasonal turnover in community composition of stream macroinvertebrates. We find that eDNA read data from the same stream sampling site clearly show different communities in spring and autumn, respectively. For three of the five streams, season even appears to be a more important factor than sampling site for explaining the variation in community composition. Finally, we compare eDNA data with a near‐decadal dataset of taxon occurrences in the same five streams based on kick sampling conducted through a national monitoring program. This comparison reveals an overlap in species composition, but also that the two approaches provide complementary rather than identical insights into community composition. Our study demonstrates that aquatic eDNA metabarcoding is useful for species detection across highly diverse taxa and for identifying seasonal patterns in community composition of freshwater macroinvertebrates. Thus, our results have important implications for both fundamental research in aquatic ecology and for applied biomonitoring.

Environmental DNA (eDNA) extracted from water samples has recently shown potential as a valuable source of population genetic information for aquatic macroorganisms. This approach offers several potential advantages compared with conventional tissue‐based methods, including the fact that eDNA sampling is noninvasive and generally more cost‐efficient. Currently, eDNA approaches have been limited to single‐marker studies of mitochondrial DNA (mtDNA), and the relationship between eDNA haplotype composition and true haplotype composition still needs to be thoroughly verified. This will require testing of bioinformatic and statistical software to correct for erroneous sequences, as well as biases and random variation in relative sequence abundances. However, eDNA‐based population genetic methods have far‐reaching potential for both basic and applied research. In this paper, we present a brief overview of the achievements of eDNA‐based population genetics to date, and outline the prospects for future developments in the field, including the estimation of nuclear DNA (nuDNA) variation and epigenetic information. We discuss the challenges associated with eDNA samples as opposed to those of individual tissue samples and assess whether eDNA might offer additional types of information unobtainable with tissue samples. Lastly, we provide recommendations for determining whether an eDNA approach would be a useful and suitable choice in different research settings. We limit our discussion largely to contemporary aquatic systems, but the advantages, challenges, and perspectives can to a large degree be generalized to eDNA studies with a different spatial and temporal focus.

The relationships of crustaceans and hexapods (Pancrustacea) have been much discussed and partially elucidated following the emergence of phylogenomic data sets. However, major uncertainties still remain regarding the position of iconic taxa such as Branchiopoda, Copepoda, Remipedia, and Cephalocarida, and the sister group relationship of hexapods. We assembled the most taxon-rich phylogenomic pancrustacean data set to date and analyzed it using a variety of methodological approaches. We prioritised low levels of missing data and found that some clades were consistently recovered independently of the analytical approach used. These include, for example, Oligostraca and Altocrustacea. Substantial support was also found for Allotriocarida, with Remipedia as the sister of Hexapoda (i.e., Labiocarida), and Branchiopoda as the sister of Labiocarida, a clade that we name Athalassocarida (=”nonmarine shrimps”). Within Allotriocarida, Cephalocarida was found as the sister of Athalassocarida. Finally, moderate support was found for Hexanauplia (Copepoda as sister to Thecostraca) in alliance with Malacostraca. Mapping key crustacean tagmosis patterns and developmental characters across the revised phylogeny suggests that the ancestral pancrustacean was relatively short-bodied, with extreme body elongation and anamorphic development emerging later in pancrustacean evolution., J.L.-F. was supported by a Marie Skłodowska-Curie Fellowship (655814) and J.O. was supported by a grant from the Danish Agency for Science, Technology and Innovation (0601-12345B). F.P. was supported by the grant CHALLENGEN (CTM2013-48163) from the Spanish Government and a post-doctoral contract funded by the Beatriu de Pinós Programme of the Generalitat de Catalunya (2014-BPB-00038). J.F.F. was supported by a NERC GW4+ PhD studentship and J.V. and D.P. were supported by a NERC BETR grant (NE/P013678/1). M.G. is supported by a European Marie Curie (ITN – IGNITE) PhD studentship

Environmental DNA (eDNA) provides a promising supplement to traditional sampling methods for population genetic inferences, but current studies have almost entirely focused on short mitochondrial markers. Here, we develop one mitochondrial and one nuclear set of target capture probes for the whale shark (Rhincodon typus) and test them on seawater samples collected in Qatar to investigate the potential of target capture for eDNA-based population studies. The mitochondrial target capture successfully retrieved similar to 235x (90x - 352x per base position) coverage of the whale shark mitogenome. Using a minor allele frequency of 5%, we find 29 variable sites throughout the mitogenome, indicative of at least five contributing individuals. We also retrieved numerous mitochondrial reads from an abundant nontarget species, mackerel tuna (Euthynnus affinis), showing a clear relationship between sequence similarity to the capture probes and the number of captured reads. The nuclear target capture probes retrieved only a few reads and polymorphic variants from the whale shark, but we successfully obtained millions of reads and thousands of polymorphic variants with different allele frequencies from E. affinis. We demonstrate that target capture of complete mitochondrial genomes and thousands of nuclear loci is possible from aquatic eDNA samples. Our results highlight that careful probe design, taking into account the range of divergence between target and nontarget sequences as well as presence of nontarget species at the sampling site, is crucial to consider. eDNA sampling coupled with target capture approaches provide an efficient means with which to retrieve population genomic data from aggregating and spawning aquatic species.

The gray wolf Canis lupus range in central Europe is dynamically expanding, reconnecting previously isolated populations. Thus, a recent paper has proposed to merge the current Baltic and Central European (CE) wolf management units, which are no longer isolated by distance. However, recent genetic findings indicate that these two populations are not genetically homogenous. Here we review the most recent data on wolf genetic structure in central Europe and show that even though the CE and Baltic wolves represent the same phylogeographic lineage, their demographic history has resulted in significant genetic structure between these two populations. While the groups are interconnected by moderate gene flow, it is not high enough to reduce the strong founder signal observed in the CE population, suggesting that population dynamics within the CE wolf range are largely independent from those of its source (Baltic) population. Consequently, a management unit combining the CE and Baltic wolves would not form a demographically coherent entity. Thus, we recommend that conservation management units maintain their separate status.

An assessment of vertical distribution, diel migration, taxonomic and functional diversity of fishes was carried out at offshore platforms in The (Arabian–Iranian–Persian) Gulf. Video footage was recorded at the Al Shaheen oil field between 2007 and 2014 using a remotely operated vehicle (ROV). A total of 12 822 individual fishes, from 83 taxonomic groups were recorded around the platforms. All the species identified are considered native to The Gulf, although Cyclichthys orbicularis and Lutjanus indicus were recorded for the first time in Qatari waters. Several trends were uncovered in the vertical distribution of the fish community; most species were observed between 20 and 50 m depth and fish abundance decreased towards the bottom, with the highest abundances recorded in the upper layers, i.e. down to 40 m depth. Vertical variation in fish diversity, however, was generally not accompanied by differences in vertical movements. Carnivores and invertivores were the dominant trophic groups, being found at each depth range from surface to seabed. The functional indices showed no significant differences between water depths or diel cycles. The study demonstrates that oil platforms represent a hotspot of fish diversity and interesting sites for studying fish communities, abundance and behaviour.

Conservation and management of marine biodiversity depends on biomonitoring of marine habitats, but current approaches are resource‐intensive and require different approaches for different organisms. Environmental DNA (eDNA) extracted from water samples is an efficient and versatile approach to detecting aquatic animals. In the ocean, eDNA composition reflects local fauna at fine spatial scales, but little is known about the effectiveness of eDNA‐based monitoring of marine communities at larger scales. We investigated the potential of eDNA to characterize and distinguish marine communities at large spatial scales by comparing vertebrate species composition among marine habitats in Qatar, the Arabian Gulf (also known as the Persian Gulf), based on eDNA metabarcoding of seawater samples. We conducted species accumulation analyses to estimate how much of the vertebrate diversity we detected. We obtained eDNA sequences from a diverse assemblage of marine vertebrates, spanning 191 taxa in 73 families. These included rare and endangered species and covered 36% of the bony fish genera previously recorded in the Gulf. Sites of similar habitat type were also similar in eDNA composition. The species accumulation analyses showed that the number of sample replicates was insufficient for some sampling sites but suggested that a few hundred eDNA samples could potentially capture >90% of the marine vertebrate diversity in the study area. Our results confirm that seawater samples contain habitat‐characteristic molecular signatures and that eDNA monitoring can efficiently cover vertebrate diversity at scales relevant to national and regional conservation and management., Article impact statement: Environmental DNA provides habitat‐characteristic molecular signatures and can be used efficiently to map marine biodiversity at large spatial scales.

International audience

Schistosomiasis is a waterborne, infectious disease with high morbidity and significant economic burdens affecting more than 250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal more sensitive diagnostic tools are needed to monitor progress towards transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA) based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the non-human part of the parasite life cycle occurs. To our knowledge, this is the first report of the successful detection of S. mansoni in freshwater samples using aquatic eDNA. True eDNA was detected in as few as 10 cercariae/L water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising new tool to significantly improve the environmental surveillance of S. mansoni. Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.SignificanceAccurate detection and delineation of schistosomiasis transmission sites will be vital in on-going efforts to control and ultimately eliminate one of the most neglected tropical parasitic diseases affecting more than 250 million people worldwide. Conventional methods to detect parasites in the environment are cumbersome and have low sensitivity. We therefore developed an environmental DNA (eDNA) based method for schistosome detection in aquatic environments. Aquatic eDNA showed higher sensitivity than conventional snail surveys. We conclude that eDNA is a promising non-invasive and sensitive tool for environmental surveillance of schistosomiasis transmission. As the efforts and aims to control the disease are transitioning towards complete transmission interruption, this could be the robust and cost-effective surveillance tool needed in the “end game” of schistosomiasis.

Ocean circulation, geological history, geographic distance, and seascape heterogeneity play an important role in phylogeography of coral‐dependent fishes. Here, we investigate potential genetic population structure within the yellowbar angelfish (Pomacanthus maculosus) across the Northwestern Indian Ocean (NIO). We then discuss our results with respect to the above abiotic features in order to understand the contemporary distribution of genetic diversity of the species. To do so, restriction site‐associated DNA sequencing (RAD‐seq) was utilized to carry out population genetic analyses on P. maculosus sampled throughout the species’ distributional range. First, genetic data were correlated to geographic and environmental distances, and tested for isolation‐by‐distance and isolation‐by‐environment, respectively, by applying the Mantel test. Secondly, we used distance‐based and model‐based methods for clustering genetic data. Our results suggest the presence of two putative barriers to dispersal; one off the southern coast of the Arabian Peninsula and the other off northern Somalia, which together create three genetic subdivisions of P. maculosus within the NIO. Around the Arabian Peninsula, one genetic cluster was associated with the Red Sea and the adjacent Gulf of Aden in the west, and another cluster was associated with the Arabian Gulf and the Sea of Oman in the east. Individuals sampled in Kenya represented a third genetic cluster. The geographic locations of genetic discontinuities observed between genetic subdivisions coincide with the presence of substantial upwelling systems, as well as habitat discontinuity. Our findings shed light on the origin and maintenance of genetic patterns in a common coral reef fish inhabiting the NIO, and reinforce the hypothesis that the evolution of marine fish species in this region has likely been shaped by multiple vicariance events., Metapopulation genetics of coral reef fish. Phylogeography Northwestern Indian Ocean.

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.

Schistosomiasis is a water-based, infectious disease with high morbidity and significant economic burdens affecting >250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal, more sensitive diagnostic tools are needed to monitor progress toward transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA)-based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the nonhuman part of the parasite life cycle occurs. This is a report of the successful detection of S. mansoni in freshwater samples by using aquatic eDNA. True eDNA was detected in as few as 10 cercariae per liter of water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising tool to substantially improve the environmental surveillance of S. mansoni. Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.

The Zebra Shark, in recent years known as Stegostoma fasciatum (Hermann, 1783), is well known for its dramatic ontogenetic change of color pattern, from striped ("zebra'') juveniles to spotted ("leopard'') adults. Nevertheless, many aspects of the species' biology, ecology, and morphology are still unknown or inadequately described, and its nomenclature is contentious. This study introduces a hitherto undescribed color morph of the Zebra Shark and provides an updated diagnosis and redescription of the species. Firstly, we establish that the Zebra Shark remains a single species based on genetic data from mitochondrial COI and ND4 markers. Secondly, through morphological analyses, we conclude that there are two morphs of the species, the known, zebra striped morph and a new, sandy colored morph. Both morphs were studied morphometrically to expose any ontogenetic changes, such as a decrease in the relative length of the tail with increasing total length (TL). The external coloration pattern clearly differentiates the two morphs, and both morphs can be further divided into three stages based on color pattern and size: juveniles (255-562 mm TL), transitionals (562-1395 mm TL), and adults (>1300 mm TL). The transitional sandy morph is dorsally covered by a swirly pattern of thin, dark brown bands edged with freckle-like brown spots. The adults are a uniform sandy beige, partially covered with brown freckles. A mature male of the zebra morph displayed a yet unknown feature of the claspers: a small, triangular spike extruding from the dorsal terminal of the clasper glands. Finally, we reviewed the nomenclature of the species and suggest that the original name Stegostoma tigrinum Forster, 1781 should be used as the senior synonym for the species.

The conservation status of European temperate forests is overall unfavorable, and many associated species are listed in national or European red-lists. A better understanding of factors increasing survival probability of red-listed species is needed for a more efficient conservation effort. Here, we investigated the importance of current forest cover, historical forest cover and a number of soil and climate variables on the incidence and richness of red-listed forest species in Denmark. We considered eight major taxa separately (mammals, saproxylic beetles, butterflies, vascular plants and four groups of fungi), using mainly citizen science data from several national mapping projects. Taxa were selected to represent important forest habitats or properties (soil, dead wood, forest glades and landscape context) and differ in dispersal potential and trophic strategy. For all groups, presence and richness of red-listed species was positively related with current forest cover, but-for most taxa-forest cover 200 years ago was an even better predictor. The intersection of past and current deciduous forest was used to identify the area of continuous, lost and new forest. Continuous and lost deciduous forest cover were strong predictors of red-listed species occurrence in most groups, but surprisingly species richness of butterflies and hydnoid fungi, and presence of mammals, was significantly, positively affected by coniferous forest area. The positive effect of lost deciduous forests on red-listed species, suggest an extinction debt of at least 200 years, with some areas hosting more red-listed species than the current area of old forest can sustain in the long run. Our results suggest that current priorities for forest conservation in Denmark are not efficient in protecting red-listed forest species, and that more focus should be put on conserving deciduous forest with long continuity. Furthermore, a multi-taxa approach including a wide array of organism groups with contrasting habitat affiliations, results in a more comprehensive understanding of the requirements of red-listed forest species and necessitate a more focused approach to conservation planning. (C) 2016 Elsevier B.V. All rights reserved.

Terrestrial arthropods comprise the most species‐rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of arthropod species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity. At the same time, monitoring grassland arthropod diversity is time‐consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non‐invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators, and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant‐associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.

About one third of the oil and gas extracted worldwide comes from offshore sources. There is currently thousands of large-scale oil and gas platforms spread across the seas and coastal oceans, from the North Sea to the seas of South Asia. Although their primary purpose is not related to enrichment of biodiversity, gas and oil platforms can act as large and complex artificial marine habitats for a wide range of marine organisms, including sessile invertebrates and fishes. These structures have shown to enhance ecosystem function, especially secondary fish production, relative to adjacent natural reefs, besides being among the most productive marine fish habitats globally. Indeed oil platforms constitute important fish aggregating devices, especially in areas subjected to a range of environmental perturbations. Hard substratum availability combined with exclusion zones around these structures allows for the development of diverse fish assemblages. We carried out the first assessment of vertical distribution, diel migration, taxonomic and functional diversity of fishes at offshore platforms in the Arabian Gulf. Video footages were recorded at the Al Shaheen Oil Field, between 2007 and 2014 using a Remotely Operated Vehicle (ROV). Indeed diving and snorkeling are strictly forbidden in the exclusion zone around offshore platforms, precluding conventional monitoring techniques such as underwater visual census, collection of sessile organisms, etc. Routine inspection and maintenance surveys, however, are conducted to monitor the state of underwater structures using ROVs. A total of 4,510 video files, containing 120 hours of underwater video recordings, were provided by Maersk Oil Qatar A/S. These videos were recorded over six years (2007, 2009, 2010, 2011, 2012 and 2014), during day and night, at the 9 platform locations (A-I) within the Al Shaheen Oil Field using a ROV of the model SAAB Seaeye Surveyor Plus 229. To assess the local fish community, a total of 242 videos amounting to twenty-one hours of observation were selected randomly, however, if no fish appear in a selected video a new video was randomly selected from the list. This work represents the first assessment of reef fish communities inhabiting oil and gas platforms in the Arabian Gulf. A total of 12,822 fish, belonging to 83 taxonomic groups were recorded around the platforms. Among them, two species are first recorded for Qatari waters: Cyclichthys orbicularis and Lutjanus indicus. In addition two chondrichthyes, one endangered (Stegostoma fasciatum) and a vulnerable species (Taeniurops meyeni), were also observed across the platforms. Several trends were found in the vertical distribution of the fish community, most species were observed between 20 and 50 m depth and higher fish abundance recorded in the upper layers, down to 40 m depth and decreasing with depth. Vertical variation, however, in fish diversity was generally not accompanied by differences in vertical movements. The vertical variation, however, in fish diversity was not accompanied by differences in vertical movements of the fish. These results suggest that the vertical zonation pattern does not change, or only changes slightly, on a daily basis. The mean centers of mass of the most abundant fish species did not overlap, likely due to potential competition or niche differences. The dominant trophic groups were carnivores and invertivores, being well represented at each depth range (each spanning 10 m) from surface to seabed. Diel vertical movement was observed only for Acanthopagrus bifasciatus, which was concentrated at shallow depths during the daytime but migrated to deeper layers at night. The functional indices showed no significant differences between water depths or diel cycles (day / night). Besides the temperature variation, the reduced light penetration due to platform structures makes difficult the algal growth necessary for browsing herbivorous fish. Thereby, the effect of sunlight penetration may be reflected in the vertical distribution of herbivorous fish, which are rare in deeper layers. Planktivores are densely grouped within and just below the thermocline, which is located around 18 m during summertime. The study demonstrates that oil platforms represent a hotspot of fish diversity and interesting sites for studying fish communities, abundance and behaviour.

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.

Insect responses to recent climate change are well documented, but the role of resource specialization in determining species vulnerability remains poorly understood. Uncovering local ecological effects of temperature change with high-quality, standardized data provides an important first opportunity for predictions about responses of resource specialists, and long-term time series are essential in revealing these responses. Here, we investigate temperature-related changes in local insect communities, using a sampling site with more than a quarter-million records from two decades (1992-2009) of full-season, quantitative light trapping of 1543 species of moths and beetles. We investigated annual as well as long-term changes in fauna composition, abundance and phenology in a climate-related context using species temperature affinities and local temperature data. Finally, we explored these local changes in the context of dietary specialization. Across both moths and beetles, temperature affinity of specialists increased through net gain of hot-dwelling species and net loss of cold-dwelling species. The climate-related composition of generalists remained constant over time. We observed an increase in species richness of both groups. Furthermore, we observed divergent phenological responses between cold- and hot-dwelling species, advancing and delaying their relative abundance, respectively. Phenological advances were particularly pronounced in cold-adapted specialists. Our results suggest an important role of resource specialization in explaining the compositional and phenological responses of insect communities to local temperature increases. We propose that resource specialists in particular are affected by local temperature increase, leading to the distinct temperature-mediated turnover seen for this group. We suggest that the observed increase in species number could have been facilitated by dissimilar utilization of an expanded growing season by cold- and hot-adapted species, as indicated by their oppositely directed phenological responses. An especially pronounced advancement of cold-adapted specialists suggests that such phenological advances might help minimize further temperature-induced loss of resource specialists. Although limited to a single study site, our results suggest several local changes in the insect fauna in concordance with expected change of larger-scale temperature increases.

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.

Species ’ dispersal abilities have been considered a major driving force in establishment and maintenance of large range sizes. However, recent studies question the general validity of this relationship because the relationship between dispersal ability and range size might in some cases be less important than species phylogeny or local spatial attributes. In this study we used the water beetle Graphoderus bilineatus a philopatric species of conservation concern in Europe as a model to explain large range size and to support eff ective conservation measures for such species that also have limited dispersal. We recorded the presence/absence of G. bilineatus and measured 14 habitat and 20 landscape variables at 228 localities in Estonia, Poland and Sweden within the core range of the species . Using information theory and average multivariate logistic regression models we determined that presence of G. bilineatus depended on landscape connectivity, distance to a possible source habitat, and stability of the site; however, specifi city of habitat characteristics was not vital for the species. We reason that the large range of G. bilineatus is best explained by the historical combination of lakes, river systems and wetlands which used to be highly connected throughout the central plains of Europe. Our data suggest that a broad habitat niche can prevent landscape elements from becoming barriers for species like G. bilineatus . Th erefore, we question the usefulness of site protection as conservation measures for G. bilineatus and similar philopatric species. Instead, conservation actions should be focused at the landscape level to ensure a long-term viability of such species across their range.

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.

Aquatic ecosystems across the globe are under significant threat, suffering from various forms of anthropogenic disturbances, which is greatly impacting global biodiversity, economy and human health. Reliable monitoring of species is crucial for data-driven management actions in this context, but remains a challenge owing to non-standardized and selective methods relaying on physical identification of species by visual surveys and counting of individuals. However, traditional monitoring techniques remain problematic due to difficulties associated with correct identification of cryptic species or juvenile life stages, These traditional methods depend on practical and taxonomic expertise, which is steadily declining. In an attempt to come up with new objective solutions for monitoring biodiversity, recent and ongoing studies in Qatari waters, uses and further develops novel environmental DNA (eDNA) methods for Monitoring aquatic biodiversity in the Gulf and elsewhere.Previous studies have shown that diversity of rare and threatened European freshwater animals - representing amphibians, fish, mammals, insects and crustaceans - can be detected and quantified based on environmental DNA (eDNA) obtained directly from small water samples of lakes, ponds and streams (Thomsen et al. 2012a).Subsequently, for the first time, we investigated the potential of using metabarcoding of eDNA obtained directly from seawater samples to account for marine fish and mammal biodiversity. We show that such marine eDNA can account for fish biodiversity using high-throughput sequencing. Promisingly, eDNA covered the fish diversity better than any of 9 methods, conventionally used in marine fish surveys. Additionally, we show that even short fish eDNA sequences in seawater degrades beyond detectable levels within days, in accordance with results obtained from freshwater eDNA (Thomsen et al. 2012b). Controlled mesocosm experiments have also shown that eDNA becomes undetectable within 2 weeks after removal of animals, indicating that eDNA traces are near contemporary with species presence. Our findings underpin the ubiquitous nature of eDNA traces in the environment and support the use of eDNA as a tool for monitoring rare, threatened and economically important species across a wide range of taxonomic groups.A particularly interesting study focuses on the large whale shark aggregation at the Al Shaheen Oil Field, located in the offshore area called “Block 5”, in the NE of the Exclusive Economic Zone (EEZ) of Qatar. Maersk Oil Qatar is currently operating several oil and gas production platforms within this field, and also take active part in a large whale shark research project studying many aspect about the aggregation. Quantitative PCR (qPCR) and high-throughput sequencing of sea water from the area have successfully been employed to gain new information about the occurrence, abundance and biology of the aggregation. More specifically, data from 2013 and 2014 showed that whale shark eDNA concentrations follows visual observations of abundance, and that degradation of eDNA occur rapidly, supporting that the obtained genetic material is of local origin. Our findings confirm that seawater samples can contain key information on populations of oceanic species, and demonstrate a general potential of eDNA for studying populations of marine organisms.Although further studies are needed to validate the eDNA approach under varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of aquatic biodiversity and resources in the Gulf.ReferencesThomsen PF, Kielgast J, Iversen LL, Wiuf C, Rasmussen M, Gilbert MTP, Orlando L, Willerslev E (2012a). Monitoring Endangered Freshwater Biodiversity using Environmental DNA. Molecular Ecology 21, 2565–2573.Thomsen PF, Kielgast J, Iversen LL, Møller PR, Rasmussen M, Willerslev E (2012b). Detection of a Diverse Marine Fish Fauna using Environmental DNA from Seawater Samples. PLOS ONE 7(8), e41732.

Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data-driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals—representing amphibians, fish, mammals, insects and crustaceans—can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high-throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.

The European stag beetle, Lucanus cervus, is thought to be widely dis- tributed across its range, but a detailed description of its occurrence is lacking. 2. Researchers in 41 countries were contacted and information sought on various life history characteristics of the insect. Data on adult body size were collected from seven countries. 3. Habitat associations differ between the United Kingdom and mainland Europe. Larvae are most commonly associated with oak, but the duration of the larval stage and the number of instars varies by up to 100% across Europe. 4. Adult size also varies; beetles from Spain, Germany, and the Netherlands are larger than those from Belgium or the UK. In the former countries, populations are composed mainly of large individuals, while in the UK, the majority of individuals are relatively small. Allometric relations between mandible size and total body length differ in Germany compared with the rest of Europe. 5. Distribution maps of the insect, split into records pre- and post-1970, from 24 countries are presented. While these inevitably suffer from recorder bias, they indi- cate that in only two countries, Croatia and Slovakia, does the insect seem to be increasing in range. 6. Our data suggest that the insect may be in decline across Europe, most likely due to habitat loss, and that conservation plans need to be produced that focus on the biology of the insect in the local area.

The fish ectoparasites Branchiura (Crustacea) display two different ways of attachment to the fish surface as adults: the first maxillae are either hooks (Dolops) or suction discs (Argulus, Chonopeltis, and Dipteropeltis). In larval Argulus foliaceus the first maxillae are hooks. With the first molecular phylogeny of the Branchiura as a background, the present paper discusses the evolutionary scenarios leading to hooks versus suction discs. Specific homologies exist between larval Argulus foliaceus hooks and adult Dolops ranarum hooks. These include the presence of a comparable number of segments/portions and a distal segment terminating in a double structure: a distal two-part hook (in Argulus) or one hook and an associate spine-like structure (in Dolops). In the phylogenetic reconstruction based on three molecular markers (mitochondrial 16S rRNA, nuclear 18S and 28S rRNA), Dolops ranarum is found to be in a sister group position to all other Branchiura, which in this analysis include six Argulus and one Chonopeltis sequences. Based on the molecular phylogeny a likely evolutionary scenario is that the ancestral branchiuran used hooks (on the first maxilla) for attachment, as seen in Dolops, of which the proximal part was subsequently modified into suction discs in Argulus and Chonopeltis (and Dipteropeltis). The sister group relationship of the Branchiura and Pentastomida is confirmed based on the most comprehensive taxon sampling until now. No evidence was found for a branchiuran in-group position of the Pentastomida.

Within parasitic barnacles of the family Sacculinidae, the genus Heterosaccus is the third largest, with 12 species infesting various brachyuran hosts throughout the world. As part of an effort to reconstruct rhizocephalan phylogeny we performed an analysis of four species of Heterosaccus and of selected sacculinid and non-sacculinid rhizocephalans. We used both molecular sequence data (16s rDNA and 18s rDNA) and morphological characters from an SEM analysis of the cypris larvae. Using Bayesian methods we obtained a highly supported tree in which the four species of Heterosaccus form a monophylum, whereas both the genus Sacculina and the family Sacculinidae are polyphyletic. The sistergroup to Heterosaccus is a clade consisting of the sacculinids Loxothylacus panopaei and the "classical" rhizocephalan Sacculina carcini. The molecular results found support in cypris morphology, where we identified two distinct synapomorphies for Heterosaccus, both present in male cyprids only: A large flap extending posteriorly from the third antennular segment, and the male-specific aesthetasc on the third segment being reduced to a rudiment or lacking completely. Female cyprids didn't show generic level apomorphies but resembled those of other sacculinid species. No morphological synapomorphies were identified between Heterosaccus, L. panopaei and S. carcini. While larval characters proved to be informative, we conclude that future studies on rhizocephalan taxonomy must rely primarily on molecular data, both to provide an overall phylogenetic framework and to assure an accurate identification of species for biogeographical and other biological purposes.