A vision for global eDNA-based monitoring in a changing world.

Environmental DNA (eDNA) has opened promising avenues for establishing standardized, cost-efficient monitoring of biodiversity. However, comprehensive and systematic implementation is urgently needed to address the current biodiversity crisis. Here, we envision a global eDNA biomonitoring scheme, which could potentially revolutionize the understanding and conservation of life on Earth.

Formation of the Holarctic Fauna: Dated molecular phylogenetic and biogeographic insights from the Quedius-lineage of Ground-Dwelling Rove Beetles (Coleoptera, Staphylinidae).

Although the Holarctic fauna has been explored for centuries, many questions on its formation are still unanswered. For example, i) what was the impact of the uplift of the Himalaya and Tibetan Plateau?, ii) what were the timings and climate of the faunal bridges connecting the Nearctic and Palearctic regions?, and iii) how did insect lineages respond to the late Paleogene global cooling and regional aridification? To answer these, we developed a phylogenetic dataset of 1229 nuclear loci for a total of 222 species of rove beetles (Staphylinidae) with emphasis in the tribe Quediini, especially Quedius-lineage and its subclade Quedius sensu stricto. Using eight fossils for calibrating molecular clock, we estimated divergence times and then analysed in BioGeoBEARS paleodistributions of the most recent common ancestor for each target lineage. For each species we generated climatic envelopes of the temperature and precipitation and mapped them across the phylogeny to explore evolutionary shifts. Our results suggest that the warm and humid Himalaya and Tibetan Plateau acted as an evolutionary cradle for the Quedius-lineage originating during the Oligocene from where, in the Early Miocene, the ancestor of the Quedius s. str. dispersed into the West Palearctic. With the climate cooling from the Mid Miocene onwards, new lineages within Quedius s. str. emerged and gradually expanded distributions across the Palearctic. In Late Miocene, a member of the group dispersed to the Nearctic region via Beringia before the closure of this land bridge 5.3 Ma. Paleogene global cooling and regional aridification largely shaped the current biogeographic pattern for Quedius s. str. species, many of them originating during the Pliocene and shifting or contracting their ranges during Pleistocene.

Contrasting seasonal patterns in diet and dung-associated invertebrates of feral cattle and horses in a rewilding area.

Trophic rewilding is increasingly applied in restoration efforts, with the aim of reintroducing the ecological functions provided by large-bodied mammals and thereby promote self-regulating, biodiverse ecosystems. However, empirical evidence for the effects of megafauna introductions on the abundance and richness of other organisms such as plants and invertebrates, and the mechanisms involved still need strengthening. In this study, we use environmental DNA (eDNA) metabarcoding of dung from co-existing feral cattle and horses to assess the seasonal variation in plant diet and dung-associated arthropods and nematodes. We found consistently high diet richness of horses, with low seasonal variability, while the generally lower dietary diversity of cattle increased substantially during summer. Intriguingly, season-specific diets differed, with a greater proportion of trees in the horses' diet during winter, where cattle relied more on shrubs. Graminoids were predominantly found in the diet of horses, but were generally underrepresented compared to previous studies, possibly due to the high prevalence of forbs in the study area. Dung-associated arthropod richness was higher for cattle, largely due to a high richness of flies during summer. Several species of dung-associated arthropods were found primarily in dung from one of the two herbivores, and our data confirmed known patterns of seasonal activity. Nematode richness was constantly higher for horses, and nematode communities were markedly different between the two species. Our results demonstrate complementary effects of cattle and horses through diet differences and dung-associated invertebrate communities, enhancing our understanding of large herbivore effects on vegetation and associated biodiversity. These results are directly applicable for decision-making in rewilding projects, suggesting biodiversity-benefits by inclusion of functionally different herbivores.

Individual haplotyping of whale sharks from seawater environmental DNA.

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.

Environmental DNA metabarcoding of cow dung reveals taxonomic and functional diversity of invertebrate assemblages.

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.

Genome-scale target capture of mitochondrial and nuclear environmental DNA from water samples.

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.

Population-level inferences from environmental DNA-Current status and future perspectives.

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.

Using vertebrate environmental DNA from seawater in biomonitoring of marine habitats.

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.

Pancrustacean Evolution Illuminated by Taxon-Rich Genomic-Scale Data Sets with an Expanded Remipede Sampling.

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 prioritized 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.

Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods.

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.

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.

Resource specialists lead local insect community turnover associated with temperature - analysis of an 18-year full-seasonal record of moths and beetles.

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.

Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) vs. 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

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.

Ancient and modern environmental DNA.

DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field.

Detection of a diverse marine fish fauna using environmental DNA from seawater samples.

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.

Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals.

The exploitation of non-invasive samples has been widely used in genetic monitoring of terrestrial species. In aquatic ecosystems, non-invasive samples such as feces, shed hair or skin, are less accessible. However, the use of environmental DNA (eDNA) has recently been shown to be an effective tool for genetic monitoring of species presence in freshwater ecosystems. Detecting species in the marine environment using eDNA potentially offers a greater challenge due to the greater dilution, amount of mixing and salinity compared with most freshwater ecosystems. To determine the potential use of eDNA for genetic monitoring we used specific primers that amplify short mitochondrial DNA sequences to detect the presence of a marine mammal, the harbor porpoise, Phocoena phocoena, in a controlled environment and in natural marine locations. The reliability of the genetic detections was investigated by comparing with detections of harbor porpoise echolocation clicks by static acoustic monitoring devices. While we were able to consistently genetically detect the target species under controlled conditions, the results from natural locations were less consistent and detection by eDNA was less successful than acoustic detections. However, at one site we detected long-finned pilot whale, Globicephala melas, a species rarely sighted in the Baltic. Therefore, with optimization aimed towards processing larger volumes of seawater this method has the potential to compliment current visual and acoustic methods of species detection of marine mammals.