Austropallenehalanychi sp. nov., a new species of sea spider (Pycnogonida, Callipallenidae) from the Ross Sea, Antarctica.

Here we present Austropallenehalanychisp. nov., a new species of pycnogonid within the family Callipallenidae (Pycnogonida), collected from the Ross Sea, Antarctica. While retaining key morphological features known for the genus Austropallene Hodgson, 1915a, the new species is distinguished from congeners by its much larger size, along with the combined absence of a denticle on the inner surface of the fixed finger of the chelifore claw along with the presence of small conical outgrowths where the fixed finger of the chelifore claw meets the movable finger on both the dorsal and ventral sides, and also the ability to fully close the chelifore claw. Additionally, the complete mitochondrial genome of A.halanychi is consistent with other members of the genus Austropallene in terms of gene order and directionality. A phylogenetic tree consisting of mitochondrial protein-coding gene data places A.halanychi as sister to Austropallenecornigera (Möbius, 1902). Additionally, a phylogenetic tree constructed using partial COI data from other callipallenids placed the new species in a clade containing the genus Austropallene. The combination of molecular data in addition to key morphological differences from similar species in the genus leaves no doubt that the new taxon is a new Antarctic species of Austropallene.

Integrating invasive species risk assessment into environmental DNA metabarcoding reference libraries.

Environmental DNA (eDNA) metabarcoding has shown promise as a tool for estimating biodiversity and early detection of invasive species. In aquatic systems, advantages of this method include the ability to concurrently monitor biodiversity and detect incipient invasions simply through the collection and analysis of water samples. However, depending on the molecular markers chosen for a given study, reference libraries containing target sequences from present species may limit the usefulness of eDNA metabarcoding. To explore the extent of this issue and how it may be resolved to aid biodiversity and invasive species early detection goals, we focus on fishes in the well-studied Laurentian Great Lakes region. First, we provide a synthesis of species currently known from the region and of non-indigenous species identified as threats by international, national, regional, and introduction pathway-specific fish risk assessments. With these species lists, we then evaluate 23 primer pairs commonly used in fish eDNA metabarcoding with available databases of sequence coverage and species specificity. Finally, we identify established and potentially invasive non-indigenous fish that should be prioritized for genetic sequencing to ensure robust eDNA metabarcoding for the region. Our results should increase confidence in using eDNA metabarcoding for fisheries conservation and management in the Great Lakes region and help prioritize reference sequencing efforts. The ultimate utility of eDNA metabarcoding approaches will come when conservation management of existing fish communities is integrated with early detection efforts for invasive species surveillance to assess total fish biodiversity.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Metagenomics of Antarctic Marine Sediment Reveals Potential for Diverse Chemolithoautotrophy.

The microbial biogeochemical processes occurring in marine sediment in Antarctica remain underexplored due to limited access. Further, these polar habitats are unique, as they are being exposed to significant changes in their climate. To explore how microbes drive biogeochemistry in these sediments, we performed a shotgun metagenomic survey of marine surficial sediment (0 to 3 cm of the seafloor) collected from 13 locations in western Antarctica and assembled 16 high-quality metagenome assembled genomes for focused interrogation of the lifestyles of some abundant lineages. We observe an abundance of genes from pathways for the utilization of reduced carbon, sulfur, and nitrogen sources. Although organotrophy is pervasive, nitrification and sulfide oxidation are the dominant lithotrophic pathways and likely fuel carbon fixation via the reverse tricarboxylic acid and Calvin cycles. Oxygen-dependent terminal oxidases are common, and genes for reduction of oxidized nitrogen are sporadically present in our samples. Our results suggest that the underlying benthic communities are well primed for the utilization of settling organic matter, which is consistent with findings from highly productive surface water. Despite the genetic potential for nitrate reduction, the net catabolic pathway in our samples remains aerobic respiration, likely coupled to the oxidation of sulfur and nitrogen imported from the highly productive Antarctic water column above. IMPORTANCE The impacts of climate change in polar regions, like Antarctica, have the potential to alter numerous ecosystems and biogeochemical cycles. Increasing temperature and freshwater runoff from melting ice can have profound impacts on the cycling of organic and inorganic nutrients between the pelagic and benthic ecosystems. Within the benthos, sediment microbial communities play a critical role in carbon mineralization and the cycles of essential nutrients like nitrogen and sulfur. Metagenomic data collected from sediment samples from the continental shelf of western Antarctica help to examine this unique system and document the metagenomic potential for lithotrophic metabolisms and the cycles of both nitrogen and sulfur, which support not only benthic microbes but also life in the pelagic zone.

Effect of extracellular vesicles from S. aureus-challenged human neutrophils on macrophages.

Staphylococcus aureus enhances neutrophil extracellular vesicle (EV) production. To investigate whether S. aureus viability influences EV biogenesis, EVs were isolated from human neutrophils incubated with viable bacteria (bEVs) or heat-killed bacteria (heat-killed EVs). Protein analysis, nanoparticle tracking and transmission electron microscopy showed comparable EV production between subsets, and both viable and nonviable bacteria were also detected in respective EV subsets. As anticipated, S. aureus, as well as bEVs with viable bacteria, were proinflammatory, and killing bacteria with gentamicin reduced cytokine production to baseline levels. Although heat-killed bacteria induced macrophage IL-6 production, heat-killed EVs did not. Additionally, we found that human and bacterial DNA associated with bEVs, but not heat-killed EVs, and that the DNA association could be partially decreased by disrupting electrostatic interactions. We investigated the potential for DNA isolated from EVs (EV-DNA) or EVs to cause inflammation. Although liposomal encapsulation of EV-DNA increased IL-6 production from baseline by 7.5-fold, treatment of bEVs with DNase I had no effect on IL-6 and IL-1ß production, suggesting that the DNA did not contribute to the inflammatory response. Filtered EVs, which lacked DNA and associated bacteria, exhibited less proinflammatory activity relative to bEVs, and enhanced macrophage expression of CD86 and HLA-DR. Ultimately, we show that bEVs isolated by differential centrifugation co-purify with bacteria and DNA, and studying their concerted activity and relative contribution to immune response is important to the study of host-pathogen interactions.

Use of environmental DNA (eDNA) in streams to detect feral swine (Sus scrofa).

Invasive feral swine can damage ecosystems, disrupt plant and animal populations, and transmit diseases. Monitoring of feral swine populations requires expensive and labor-intensive techniques such as aerial surveys, field surveys for sign, trail cameras, and verifying landowner reports. Environmental DNA (eDNA) provides an alternative method for locating feral swine. To aid in detection of this harmful invasive species, a novel assay was developed incorporating molecular methods. From August 2017 to April 2018, water samples and stream data were collected along 400 m transects in two different stream types where swine DNA was artificially introduced to investigate potential factors affecting detection. A generalized linear model (family binomial) was used to characterize environmental conditions affecting swine DNA detection; detection was the dependent variable and stream measurements included stream type, distance downstream, water temperature, velocity, turbidity, discharge, and pH as independent variables. Parameters from the generalized linear model were deemed significant if 95% confidence intervals did not overlap 0. Detection probability for swine DNA negatively related to water temperature (ß =  - 0.21, 95% CI [-0.35 to -0.09]), with the highest detection probability (0.80) at 0 °C and lowest detection probability (0.05) at 17.9 °C water temperature. Results indicate that sampling for swine eDNA in free-flowing stream systems should occur at lower water temperatures to maximize detection probability. This study provides a foundation for further development of field and sampling techniques for utilizing eDNA as a viable alternative to monitoring a terrestrial invasive species in northern regions of the United States.

Phylogenomics of the longitarsal Colossendeidae: The evolutionary history of an Antarctic sea spider radiation.

Sea spiders (Pycnogonida) constitute a group of marine benthic arthropods that has a particularly high species diversity in the Southern Ocean. The "longitarsal" group of the sea spider family Colossendeidae is especially abundant in this region. However, this group also includes some representatives from other oceans, which raises the question where the group originates from. Therefore, we here investigated the phylogeny of the group with a hybrid enrichment approach that yielded a dataset of 1607 genes and over one million base pairs. We obtained a well-resolved phylogeny of the group, which is mostly consistent with morphological data. The data support an Antarctic origin of the longitarsal Colossendeidae and multiple dispersal events to other regions, which occurred at different timescales. This scenario is consistent with evidence found in other groups of marine invertebrates and highlights the role of the Southern Ocean as a source for non-Antarctic biota, especially of the deep sea. Our results suggest an initially slow rate of diversification followed by a more rapid radiation possibly correlated with the mid-Miocene cooling of Antarctica, similar to what is found in other taxa.

Conservation of mitochondrial genome arrangements in brittle stars (Echinodermata, Ophiuroidea).

Brittle stars are conspicuous members of benthic ecosystems, fill many ecological niches and are the most speciose of all classes of echinoderms. With high levels of biodiversity, elucidating the evolutionary history of this group is important. Understanding of higher-level relationships within Ophiuroidea has been aided by multilocus nuclear data and DNA barcoding. However, the degree of consistency between mitochondrial and nuclear data within ophiuroids remains unclear and deserves further assessment. In this study, 17 mitochondrial genomes spanning the taxonomic breadth of Ophiuroidea were utilized to explore evolutionary relationships through maximum likelihood analyses, Bayesian inference and comparative assessment of gene order. Our phylogenetic analyses, based on both nucleotide and amino acid residues, support recent findings based on multilocus nuclear data and morphology, in that the brittle star clades Ophintegrida and Euryophiurida were recovered as monophyletic with the latter comprising Euyalida, Ophiuridae and Ophiopyrgidae. Only three different arrangements of the 13 protein coding and 2 ribosomal RNA genes were observed. As expected, tRNA genes were more likely to have undergone rearrangement but the order of all 37 genes was found to be conserved in all sampled Euryalida and Ophiuridae. Both Euryalida and the clade comprised of Ophiuridae and Ophiopyrgidae, each had their own conserved rearrangement of protein coding genes and ribosomal genes, after divergence from their last common ancestor. Euryalida has a rearrangement of the two ribosomal RNA genes, rrnS and rrnL, in contrast to Ophiuridae and Ophiopyrgidae, which had an inversion of the genes nad1, nad2, and cob relative to Ophintegrida. Further, our data support the gene order found in all sampled Euryalida as the most likely ancestral order for all Ophiuroidea.

Investigating diversity of pathogenic microbes in commercial bait trade water.

The recreational bait trade is a potential pathway for pathogen introduction and spread when anglers dump bait shop sourced water into aquatic systems. Despite this possibility, and previous recognition of the importance of the bait trade in the spread of aquatic invasive species (AIS), to date there has been no region wide survey documenting pathogens in retail bait shops. In this study, we analyzed 96 environmental DNA samples from retail bait shops around the Great Lakes region to identify pathogens, targeting the V4 hypervariable region of the 16S rRNA gene. Additionally, we used samples from one site in Lake Michigan as a comparison to pathogen diversity and abundance in natural aquatic systems. Our results identified nine different groups of pathogens in the bait shop samples, including those that pose risks to both humans and fish species. Compared to wild sourced samples, the bait shops had higher relative abundance and greater taxonomic diversity. These findings suggest that the bait trade represents a potentially important pathway that could introduce and spread pathogens throughout the Great Lakes region. Improving pathogen screening and angler outreach should be used in combination to aid in preventing the future spread of high risk pathogens.

Genomic analyses of Northern snakehead (Channa argus) populations in North America.

BACKGROUND: The introduction of northern snakehead (Channa argus; Anabantiformes: Channidae) and their subsequent expansion is one of many problematic biological invasions in the United States. This harmful aquatic invasive species has become established in various parts of the eastern United States, including the Potomac River basin, and has recently become established in the Mississippi River basin in Arkansas. Effective management of C. argus and prevention of its further spread depends upon knowledge of current population structure in the United States. METHODS: Novel methods for invasive species using whole genomic scans provide unprecedented levels of data, which are able to investigate fine scale differences between and within populations of organisms. In this study, we utilize 2b-RAD genomic sequencing to recover 1,007 single-nucleotide polymorphism (SNP) loci from genomic DNA extracted from 165 C. argus individuals: 147 individuals sampled along the East Coast of the United States and 18 individuals sampled throughout Arkansas. RESULTS: Analysis of those SNP loci help to resolve existing population structure and recover five genetically distinct populations of C. argus in the United States. Additionally, information from the SNP loci enable us to begin to calculate the long-term effective population size ranges of this harmful aquatic invasive species. We estimate long-term Ne to be 1,840,000-18,400,000 for the Upper Hudson River basin, 4,537,500-45,375,000 for the Lower Hudson River basin, 3,422,500-34,225,000 for the Potomac River basin, 2,715,000-7,150,000 for Philadelphia, and 2,580,000-25,800,000 for Arkansas populations. DISCUSSION AND CONCLUSIONS: This work provides evidence for the presence of more genetic populations than previously estimated and estimates population size, showing the invasive potential of C. argus in the United States. The valuable information gained from this study will allow effective management of the existing populations to avoid expansion and possibly enable future eradication efforts.

Crossing the Divide: Admixture Across the Antarctic Polar Front Revealed by the Brittle Star Astrotoma agassizii.

The Antarctic Polar Front (APF) is one of the most well-defined and persistent oceanographic features on the planet and serves as a barrier to dispersal between the Southern Ocean and lower latitudes. High levels of endemism in the Southern Ocean have been attributed to this barrier, whereas the accompanying Antarctic Circumpolar Current (ACC) likely promotes west-to-east dispersal. Previous phylogeographic work on the brittle star Astrotoma agassizii Lyman, 1875 based on mitochondrial genes suggested isolation across the APF, even though populations in both South American waters and the Southern Ocean are morphologically indistinguishable. Here, we revisit this finding using a high-resolution 2b-RAD (restriction-site-associated DNA) single-nucleotide polymorphism (SNP)-based approach, in addition to enlarged mitochondrial DNA data sets (16S rDNA, COI, and COII), for comparison to previous work. In total, 955 biallelic SNP loci confirmed the existence of strongly divergent populations on either side of the Drake Passage. Interestingly, genetic admixture was detected between South America and the Southern Ocean in five individuals on both sides of the APF, revealing evidence of recent or ongoing genetic contact. We also identified two differentiated populations on the Patagonian Shelf with six admixed individuals from these two populations. These findings suggest that the APF is a strong but imperfect barrier. Fluctuations in location and strength of the APF and ACC due to climate shifts may have profound consequences for levels of admixture or endemism in this region of the world.

Early detection monitoring for aquatic non-indigenous species: Optimizing surveillance, incorporating advanced technologies, and identifying research needs.

Following decades of ecologic and economic impacts from a growing list of nonindigenous and invasive species, government and management entities are committing to systematic early- detection monitoring (EDM). This has reinvigorated investment in the science underpinning such monitoring, as well as the need to convey that science in practical terms to those tasked with EDM implementation. Using the context of nonindigenous species in the North American Great Lakes, this article summarizes the current scientific tools and knowledge - including limitations, research needs, and likely future developments - relevant to various aspects of planning and conducting comprehensive EDM. We begin with the scope of the effort, contrasting target-species with broad-spectrum monitoring, reviewing information to support prioritization based on species and locations, and exploring the challenge of moving beyond individual surveys towards a coordinated monitoring network. Next, we discuss survey design, including effort to expend and its allocation over space and time. A section on sample collection and analysis overviews the merits of collecting actual organisms versus shed DNA, reviews the capabilities and limitations of identification by morphology, DNA target markers, or DNA barcoding, and examines best practices for sample handling and data verification. We end with a section addressing the analysis of monitoring data, including methods to evaluate survey performance and characterize and communicate uncertainty. Although the body of science supporting EDM implementation is already substantial, research and information needs (many already actively being addressed) include: better data to support risk assessments that guide choice of taxa and locations to monitor; improved understanding of spatiotemporal scales for sample collection; further development of DNA target markers, reference barcodes, genomic workflows, and synergies between DNA-based and morphology-based taxonomy; and tools and information management systems for better evaluating and communicating survey outcomes and uncertainty.

Controls on eDNA movement in streams: Transport, Retention, and Resuspension.

Advances in detection of genetic material from species in aquatic ecosystems, including environmental DNA (eDNA), have improved species monitoring and management. eDNA from target species can readily move in streams and rivers and the goal is to measure it, and with that infer where and how abundant species are, adding great value to delimiting species invasions, monitoring and protecting rare species, and estimating biodiversity. To date, we lack an integrated framework that identifies environmental factors that control eDNA movement in realistic, complex, and heterogeneous flowing waters. To this end, using an empirical approach and a simple conceptual model, we propose a framework of how eDNA is transported, retained, and resuspended in stream systems. Such an understanding of eDNA dispersal in streams will be essential for designing optimized sampling protocols and subsequently estimating biomass or organismal abundance. We also discuss guiding principles for more effective use of eDNA methods, highlighting the necessity of understanding these parameters for use in future predictive modeling of eDNA transport.

Geographic structure in the Southern Ocean circumpolar brittle star Ophionotus victoriae (Ophiuridae) revealed from mtDNA and single-nucleotide polymorphism data.

Marine systems have traditionally been thought of as "open" with few barriers to gene flow. In particular, many marine organisms in the Southern Ocean purportedly possess circumpolar distributions that have rarely been well verified. Here, we use the highly abundant and endemic Southern Ocean brittle star Ophionotus victoriae to examine genetic structure and determine whether barriers to gene flow have existed around the Antarctic continent. Ophionotus victoriae possesses feeding planktotrophic larvae with presumed high dispersal capability, but a previous study revealed genetic structure along the Antarctic Peninsula. To test the extent of genetic differentiation within O. victoriae, we sampled from the Ross Sea through the eastern Weddell Sea. Whereas two mitochondrial DNA markers (16S rDNA and COI) were employed to allow comparison to earlier work, a 2b-RAD single-nucleotide polymorphism (SNP) approach allowed sampling of loci across the genome. Mitochondrial data from 414 individuals suggested three major lineages, but 2b-RAD data generated 1,999 biallelic loci that identified four geographically distinct groups from 89 samples. Given the greater resolution by SNP data, O. victoriae can be divided into geographically distinct populations likely representing multiple species. Specific historical scenarios that explain current population structure were examined with approximate Bayesian computation (ABC) analyses. Although the Bransfield Strait region shows high diversity possibly due to mixing, our results suggest that within the recent past, dispersal processes due to strong currents such as the Antarctic Circumpolar Current have not overcome genetic subdivision presumably due to historical isolation, questioning the idea of large open circumpolar populations in the Southern Ocean.

Estimating species richness using environmental DNA.

The foundation for any ecological study and for the effective management of biodiversity in natural systems requires knowing what species are present in an ecosystem. We assessed fish communities in a stream using two methods, depletion-based electrofishing and environmental DNA metabarcoding (eDNA) from water samples, to test the hypothesis that eDNA provides an alternative means of determining species richness and species identities for a natural ecosystem. In a northern Indiana stream, electrofishing yielded a direct estimate of 12 species and a mean estimated richness (Chao II estimator) of 16.6 species with a 95% confidence interval from 12.8 to 42.2. eDNA sampling detected an additional four species, congruent with the mean Chao II estimate from electrofishing. This increased detection rate for fish species between methods suggests that eDNA sampling can enhance estimation of fish fauna in flowing waters while having minimal sampling impacts on fish and their habitat. Modern genetic approaches therefore have the potential to transform our ability to build a more complete list of species for ecological investigations and inform management of aquatic ecosystems.

Using Environmental DNA for Invasive Species Surveillance and Monitoring.

The method employed for environmental DNA (eDNA) surveillance for detection and monitoring of rare species in aquatic systems has evolved dramatically since its first large-scale applications. Both active (targeted) and passive (total diversity) surveillance methods provide helpful information for management groups, but each has a suite of techniques that necessitate proper equipment training and use. The protocols described in this chapter represent some of the latest iterations in eDNA surveillance being applied in aquatic and marine systems.

Influence of Stream Bottom Substrate on Retention and Transport of Vertebrate Environmental DNA.

While environmental DNA (eDNA) is now being regularly used to detect rare and elusive species, detection in lotic environments comes with a caveat: The species being detected is likely some distance upstream from the point of sampling. Here, we conduct a series of seminatural stream experiments to test the sensitivity of new digital droplet PCR (ddPCR) to detect low concentrations of eDNA in a lotic system, measure the residence time of eDNA compared to a conservative tracer, and we model the transport of eDNA in this system. We found that while ddPCR improves our sensitivity of detection, the residence time and transport of eDNA does not follow the same dynamics as the conservative tracer and necessitates a more stochastic framework for modeling eDNA transport. There was no evidence for differences in the transport of eDNA due to substrate type. The relatively large amount of unexplained variability in eDNA transport reveals the need for uncovering mechanisms and processes by which eDNA is transported downstream leading to species detections, particularly when inferences are to be made in natural systems where eDNA is being used for conservation management.

Biogeochemical and Microbial Variation across 5500 km of Antarctic Surface Sediment Implicates Organic Matter as a Driver of Benthic Community Structure.

Western Antarctica, one of the fastest warming locations on Earth, is a unique environment that is underexplored with regards to biodiversity. Although pelagic microbial communities in the Southern Ocean and coastal Antarctic waters have been well-studied, there are fewer investigations of benthic communities and most have a focused geographic range. We sampled surface sediment from 24 sites across a 5500 km region of Western Antarctica (covering the Ross Sea to the Weddell Sea) to examine relationships between microbial communities and sediment geochemistry. Sequencing of the 16S and 18S rRNA genes showed microbial communities in sediments from the Antarctic Peninsula (AP) and Western Antarctica (WA), including the Ross, Amundsen, and Bellingshausen Seas, could be distinguished by correlations with organic matter concentrations and stable isotope fractionation (total organic carbon; TOC, total nitrogen; TN, and δ(13)C). Overall, samples from the AP were higher in nutrient content (TOC, TN, and NH4 (+)) and communities in these samples had higher relative abundances of operational taxonomic units (OTUs) classified as the diatom, Chaetoceros, a marine cercozoan, and four OTUs classified as Flammeovirgaceae or Flavobacteria. As these OTUs were strongly correlated with TOC, the data suggests the diatoms could be a source of organic matter and the Bacteroidetes and cercozoan are grazers that consume the organic matter. Additionally, samples from WA have lower nutrients and were dominated by Thaumarchaeota, which could be related to their known ability to thrive as lithotrophs. This study documents the largest analysis of benthic microbial communities to date in the Southern Ocean, representing almost half the continental shoreline of Antarctica, and documents trophic interactions and coupling of pelagic and benthic communities. Our results indicate potential modifications in carbon sequestration processes related to change in community composition, identifying a prospective mechanism that links climate change to carbon availability.

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding.

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206-L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina-sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.