Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R²) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

Environmental DNA (eDNA) analysis has advanced conservation biology and biodiversity management. However, accurate estimation of age and origin of eDNA is complicated by particle transport and the presence of legacy genetic material, which can obscure accurate interpretation of eDNA detection and quantification. To understand the state of genomic material within the environment, we investigated the degradation relationships between (a) size of fragments (long vs short), (b) genomic origins (mitochondrial vs nuclear), (c) nucleic acids (eDNA vs eRNA), and (d) RNA types (messenger (m)RNA vs ribosomal (r)RNA) from non-indigenous Dreissena mussels. Initial concentrations of eRNA followed expected transcriptional trends, with rRNAs found at > 1000 × that of eDNA, and a mitosis-associated mRNA falling below detection limits within 24 h. Furthermore, the ratio of eRNA:eDNA significantly decreased throughout degradation, potentially providing an estimate for the age of genomic material. Thus, eRNA quantification can increase detection due to the high concentrations of rRNAs. Furthermore, it may improve interpretation of positive detections through the eRNA:eDNA ratio and/or by detecting low abundant mitosis-associated mRNAs that degrade within ~ 24 h.

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high‐throughput sequencing (HTS) metabarcode assays offers powerful and cost‐effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorphaandD. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter‐ and intra‐specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50–100 tagged field‐collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed‐species/haplotype compositions of adults, and (c) field‐collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population‐level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species‐ and population‐level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.

Species compositions and diversity levels of aquatic macroinvertebrate communities provide important indicators of ecosystem health. However, such community analyses typically are limited by time and effort of sampling, sorting, and identifications, as well as morphological character uncertainty for some taxa, especially at early life stages. Our objective was to evaluate a suite of targeted metabarcode high‐throughput sequencing assays to characterize the macroinvertebrate communities (specifically targeting Annelida, Bryozoa, Crustacea, Insecta, and Mollusca) from environmental (e)DNA water samples along 160 km of the Maumee River, OH (a major Lake Erie, Laurentian Great Lakes tributary). Multiple alpha (richness, Shannon Index, and Simpson's Index) and beta (Bray–Curtis and Sørensen dissimilarities) diversity metrics from the metabarcode assays were compared with an Invertebrate Community Index (ICI) metric calculated from traditional morphological sampling surveys conducted by the Ohio Environmental Protection Agency. The 15 Maumee River sites varied in their ICI scores (ranging from 4 to 46), with seven sites rated as “poor” or “very poor” and eight scoring “fair” or “good.” Metabarcoding assays yielded greater gamma richness, delineating 181 Operational Taxonomic Units versus 172 taxa from morphology (the latter often limited to family or genus‐level identifications). Both datasets supported similar river‐wide trends, with comparable gamma, alpha, and beta diversity patterns and community compositions across habitat types and habitat quality scores. Metabarcode assays revealed similar detection of important bioindicator Insecta, but missed most Trichoptera (caddisflies). eDNA identified eight aquatic invasive species on the GLANSIS (Great Lakes Aquatic Nonindigenous Species Information System) list, including three missed by the morphological surveys (Branchiura sowerbyi, Potamothrix bedoti, and Skistodiaptomus pallidus; with Lophopodella carteri, Faxonius rusticus, Corbicula fluminea, Dreissena polymorpha, and Valvata piscinalis in both datasets). Findings illustrate the utility of eDNA sampling and targeted metabarcode assays to enhance and complement environmental assessments of aquatic ecosystems.