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.
•eDNA and fishing tools were used to monitor the weather loach in Denmark.•Two localities were positive for loach eDNA, and eight were negative.•Results of eDNA were supported by fishing ...surveys.•Monitoring effort and costs were lower with the eDNA method.•eDNA provides a reliable, cost-efficient method for monitoring rare fishes.
The European weather loach (Misgurnus fossilis) represents one of many European freshwater fishes in decline. Efficient monitoring is essential if conservation efforts are to be successful, but due to the species’ cryptic biology, traditional monitoring methods currently in use are inefficient, time consuming and likely prone to non-detection error. Here, we investigate the usefulness of environmental DNA (eDNA) monitoring as an alternative or supplementary method for surveying the Danish weather loach population, which is presumed to consist primarily of a single group of no more than 50 individuals. In 2008, the majority of historical Danish localities were surveyed, using traditional fishing techniques. We then applied eDNA methods to a number of these, as well as other potential localities. We successfully detected the weather loach at multiple sites in the single known remaining locality; a result that was later confirmed when local managers caught eight live specimens. Furthermore, the eDNA method indicated presence of the weather loach in another historical locality, where the species has not been observed since 1995. At the remaining localities, weather loach eDNA was not detected, providing further evidence for its absence. Importantly, the eDNA survey required less effort in person-hours and lower costs than the traditional fishing survey. This study confirms that eDNA monitoring is a valid supplement to traditional monitoring methods currently applied to monitor rare freshwater fishes. We propose that by providing reliable distribution data at lower cost and limited effort, the eDNA method can allow for increased management efficiency of endangered freshwater species such as the European weather loach.
Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. ...Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.
Buoyancy and balance are important parameters for slow-moving, low-metabolic, aquatic organisms. The extant coelacanths have among the lowest metabolic rates of any living vertebrate and can afford ...little energy to keep station. Previous observations on living coelacanths support the hypothesis that the coelacanth is neutrally buoyant and in close-to-perfect hydrostatic balance. However, precise measurements of buoyancy and balance at different depths have never been made. Here we show, using non-invasive imaging, that buoyancy of the coelacanth closely matches its depth distribution. We found that the lipid-filled fatty organ is well suited to support neutral buoyancy, and due to a close-to-perfect hydrostatic balance, simple maneuvers of fins can cause a considerable shift in torque around the pitch axis allowing the coelacanth to assume different body orientations with little physical effort. Our results demonstrate a close match between tissue composition, depth range and behavior, and our collection-based approach could be used to predict depth range of less well-studied coelacanth life stages as well as of deep sea fishes in general.
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 ~235× (90× − 352× 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 earliest Eocene (Ypresian) Fur Formation (Denmark) is globally renowned for its exceptionally well-preserved fossils, including birds, sea turtles, insects, plants, and fishes. Fishes, albeit ...abundant and diverse, however, are only superficially known and very few detailed, taxonomic studies have been realized to date. A new polymixiiform fish, Polyspinatus fluere gen. et sp. nov., from the Fur Formation is described based on seven well-preserved, nearly complete specimens. All the specimens were studied by traditional stereomicroscopy, and by micro-X-ray fluorescence-element mapping. Digital 2D-element images of strontium-, phosphorus-, and calcium distributions of each specimen were directly applied for the taxonomic descriptions presented herein. Polyspinatus fluere gen. et sp. nov. is the first known Eocene record of the family Polymixiidae based on articulated skeletal remains. Polyspinatus gen. nov. exhibits a unique combination of characters that support its recognition as a new genus of the Polymixiidae. Its specialized hyoid apparatus with the first and second branchiostegal rays being sinuous and parallel to each other, and the third being wide and plate-like, followed by four saber-like elements, is strikingly similar to that of Polymixia, supporting a possible sister-group relationship with extant beardfishes.
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.
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.
ADN Ambiental de Vertebrados Tomado del Agua Marina para Realizar Biomonitoreos de los Hábitats Marinos
Resumen
La conservación y el manejo de la biodiversidad marina depende del biomonitoreo de los hábitats marinos, pero las estrategias actuales requieren de muchos recursos y de diferentes estrategias para diferentes organismos. El ADN ambiental (ADNa) extraído de muestras de agua es una estrategia eficiente y versátil para detectar animales acuáticos. En el océano, la composición del ADNa refleja la fauna local a escalas espaciales finas, pero se sabe poco sobre la efectividad del monitoreo basado en el ADNa de las comunidades marinas a grandes escalas. Investigamos el potencial del ADNa para caracterizar y distinguir las comunidades marinas a escalas espaciales grandes mediante una comparación de la composición de especies de vertebrados entre los hábitats marinos de Qatar, en el Golfo Arábigo (también conocido como el Golfo Persa), con base en el meta‐código de barras del ADNa extraído de muestras de agua de mar. Realizamos análisis de acumulación de especies para estimar cuánta de la diversidad de vertebrados logramos detectar. Obtuvimos secuencias de ADNa de diversos ensamblajes de vertebrados marinos, los cuales abarcaron 191 taxones de 73 familias. Estos taxones incluyeron a especies raras y en peligro de extinción y cubrieron el 36% de los géneros de peces óseos previamente registrados en el golfo. Los sitios con tipos similares de hábitat también fueron similares en cuanto a la composición del ADNa. Los análisis de acumulación de especies mostraron que el número de réplicas de muestras fue insuficiente para algunos sitios de muestreo, pero sugieren que unos cientos de muestras de ADNa podrían capturar potencialmente >90% de la diversidad de vertebrados marinos en el área de estudio. Nuestros resultados confirman que las muestras de agua marina contienen firmas moleculares características del hábitat y que el monitoreo de ADNa puede cubrir eficientemente la diversidad de vertebrados a escalas relevantes para la conservación y el manejo nacional y regional.
摘要
海洋生物多样性的保护和管理依赖于对海洋生境的生物监测, 而目前的监测方法都属于资源密集型, 且对不同生物需要采取不同的方法。从水样中提取环境 DNA (eDNA) 是一种调查水生动物高效通用的方法。在海洋中, eDNA 的组成反映了区域精细空间尺度上动物群的情况, 但对于 eDNA 在更大尺度上调查海洋生物群落的有效性尚有待探究。本研究基于 eDNA 条形码技术在海水样品中的应用, 比较了卡塔尔和阿拉伯湾 (又称波斯湾) 海洋生境的脊椎动物组成, 以分析 eDNA 在大空间尺度上描述和分辨海洋生物群落的能力。我们通过物种累积分析估计了我们检测到的脊椎动物多样性。结果显示, 我们获得的 eDNA 序列来自包含 73 个科 191 个类群的海洋脊椎动物群集。这些动物中包括稀有物种和濒危物种, 覆盖了该海湾现有记录中 36% 的硬骨鱼属。生境类型相近的位点检测到的 eDNA 组成也相似。物种累积分析还表明一些采样位点的样品重复个数不足, 但几百个 eDNA 样本可能足够捕获该研究地区 90% 以上的海洋脊椎动物多样性。我们的研究结果证实了海水样本中含有具有生境特点的分子信号, 且 eDNA 检测可以在与国家和区域保护管理所涉及的尺度上有效覆盖脊椎动物多样性。【翻译: 胡怡思; 审校: 聂永刚】
Article impact statement: Environmental DNA provides habitat‐characteristic molecular signatures and can be used efficiently to map marine biodiversity at large spatial scales.
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.
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.
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.
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.
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.
Sammendrag
Europeisk edelkreps (Astacus astacus) trues av krepsepest som forårsakes av eggsporesoppen Aphanomyces astaci. Smitten spres av fremmed nordamerikansk ferskvannskreps (f.eks. signalkreps; Pacifastacus leniusculus). Overvåking av krepsepest i Norge har tradisjonelt basert seg på burforsøk, en etisk problematisk metode hvor dødelighet hos edelkreps i bur overvåkes ved relevante lokaliteter. Overvåking av edelkrepsbestander blir gjort ved bruk av teiner. Vi har testet om overvåking basert på innsamling av miljø‐DNA (eDNA) kan være et egnet alternativ til burforsøk, og et supplement til teinefangst.
Etter en ulovlig introduksjon av smittebærende signalkreps i en innsjø med edelkreps, utnyttet vi vissheten om et kommende krepsepestutbrudd til å initiere eDNA‐overvåking og burforsøk samtidig, supplert med teinefangst. Tilsammen ble 304 vannprøver filtrert fra ulike prøvetakingsstasjoner over en fire‐års periode. eDNA data (arts‐spesifikk qPCR) for tilstedeværelse av A. astaci, edelkreps og signalkreps i vannprøver ble sammenlignet med dødelighet i burforsøk og teinefangst.
Dette er den første studien som sammenligner eDNA‐overvåkning og burforsøk under et naturlig krepsepestutbrudd. Vi viser at eDNA‐overvåking korresponderer godt med biologisk status målt i form av dødelighet hos burkreps og resultater fra teinefangst. eDNA‐analyser avslører også tilstedeværelsen av A. astaci smittestoff i vannet opptil 2,5 uker før edelkreps dør i burforsøk. Mengdeestimater av eDNA fra A. astaci og edelkreps i vannet økte markant under dødelighet, og forsvant deretter raskt. Uansett årstid gir eDNA et øyeblikksbilde av tilstedeværelse, fravær eller bortfall av edelkreps, og utgjør derfor også et verdifullt supplement til teinefiske, som avhenger av sesong og nasjonal lovgivning.
Syntese og bruksområder. Parallell eDNA‐overvåkning av Aphanomyces astaci (krepsepest agens) og relevante stedegne og fremmede arter av ferskvannskreps er velegnet for tidlig varsling av invasjon og smitte, risikovurderinger, evaluering av habitatstatus, og overvåking av status for smittestoff og fremmed/stedegen ferskvannskreps. Metoden er dyrevelferdsvennlig, og utelukker behovet for burforsøk med levende kreps i sykdomsovervåking. Videre gir eDNA‐overvåkning mindre sannsynlighet for å spre Aphanomyces astaci smitte enn tradisjonelle metoder. Denne studien har bidratt til å implementere eDNA‐overvåking i norsk overvåkning av krepsepest, edelkreps og signalkreps, og vi tror at andre land også kan forbedre sine forvaltningsstrategier for ferskvannskreps ved hjelp av en lignende tilnærming.
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.
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