Environmental DNA (eDNA) metabarcoding is increasingly applied to a variety of questions and challenges across basic and applied ecology. Although streams and rivers (i.e., lotic ecosystems) can serve as conveyor belts of both aquatic and terrestrial eDNA from upstream or riparian areas, precipitation can dilute eDNA due to increasing discharge and/or mobilize eDNA into rivers from adjacent terrestrial ecosystems. Previous research has examined eDNA detectability of single species after high flow events, but no studies have compared aquatic and terrestrial communities recovered by eDNA metabarcoding together in response to rainfall. For this study, we used eDNA metabarcoding to sample three rivers before and after precipitation over six sampling events to evaluate if terrestrial eDNA exhibits a mobilization effect and aquatic eDNA exhibits a dilution effect after rainfall. We found that as rainfall increased, terrestrial taxa richness significantly increased and aquatic taxa richness decreased but not significantly. As such, researchers using eDNA metabarcoding from lotic ecosystems to characterize terrestrial communities might not need to avoid, and could even seek out, precipitation events in their sampling design. However, our study should be replicated over more lotic ecosystems and ecoregions and larger gradients of precipitation events.

ABSTRACT Multi‐metric indices like the Index of Biotic Integrity (IBI) are important biomonitoring tools for Clean Water Act compliance in the USA. Environmental DNA (eDNA) metabarcoding could complement IBIs by increasing detection sensitivity for rare taxa while also reducing monitoring costs. To date, there is a lack of studies that have examined the efficacy of using eDNA metabarcoding to calculate IBIs in the USA. Here, we used eDNA metabarcoding to calculate a fish‐based IBI for streams and rivers of the Tennessee River Basin in northern Alabama, USA. We collected water samples from 50 stream and river sites across a gradient of land use intensity, extracted eDNA from these samples, and sequenced the eDNA using vertebrate‐specific primers. We compared our eDNA‐IBI to a previous fish‐IBI implemented by the state of Alabama using conventional sampling, as well as predicted biological condition of these streams from the US Environmental Protection Agency (EPA) based on benthic macroinvertebrates. We found a significant, positive relationship between the eDNA‐IBI and fish‐IBI, as well as shared relationships to a measure of in‐stream habitat quality, but a weaker, non‐significant positive relationship to predicted biological condition from benthic macroinvertebrates. Notably, the former was recovered despite eDNA‐IBI sampling being conducted 9 years after the conventional fish sampling at slightly different locations (although distance did not explain variation), the fish‐IBI having used greater sampling effort throughout the year (including spring and autumn rather than only summer sampling), and a lack of reference DNA sequences that prevented eDNA detection for some species detected by conventional fish sampling. Our study provides a baseline for how an eDNA‐IBI may work relative to multi‐metric indices calculated from conventional sampling, which can be improved through future directions identified and discussed in our paper.

Species distribution models (SDMs) have become highly popular across applications in ecology, including predicting the potential establishment of nonnative invasive species. However, SDMs can underpredict where invasive species may establish for reasons including violation of the equilibrium assumption of the ecological niche (e.g., distributions of organisms may be determined by dispersal constraints rather than environmental conditions) or model extrapolation to novel environmental conditions (e.g., the nonnative range is too different from the native range where a model was developed). These problems especially affect SDM applications to freshwater invasive species because watershed boundaries, rather than abiotic conditions, determine distributions for many freshwater organisms. Here I make recommendations for applying SDMs to emerging freshwater invasive species, emphasizing that researchers should 1) use freshwater-specific environmental predictors, 2) build models within regions accessible to their focal species, 3) favor simpler over more complex models, 4) require models to pass transferability tests prior to prediction to a new region, and 5) know and acknowledge when a model is extrapolated to novel conditions. Despite these recommendations, researchers will still need to periodically extrapolate SDMs to novel or non-analog environmental conditions. In these cases, researchers may consider using supraspecific modeling units, which borrow ecological information from sister taxa, organisms farther along in the invasion process, or co-occurring native species. More research is needed on supraspecific modeling approaches to evaluate whether they improve on predictions of more conventional SDMs and, if so, to synthesize recommendations for their implementation in invasive species policy and management.

The red swamp crayfish Procambarus clarkii (Girard, 1852) was collected from southwestern Idaho, United States in 1975, but went undetected in this region over more recent decades. In 2023 and 2024, multiple P. clarkii individuals were collected from the same watershed as the 1975 population but approximately 13 km downstream. We sequenced the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA to compare museum-vouchered P. clarkii from 1975 to the contemporary specimens. Modifications to our extraction protocol enabled retrieval of usable DNA from museum specimens stored in ethanol (EtOH) for a half century. We found that four (100%) of the 1975 specimens and four of five (80%) of the 2023 and 2024 specimens shared an identical COI haplotype, distinct from the known haplotype of the nearest P. clarkii population approximately 320 km north in Idaho. As such, P. clarkii may have persisted in the study region over time, although it is also possible that southwestern Idaho has been reinvaded by P. clarkii from a similar or shared source population between 1975 and the present day.

Recent phylogenetic analyses have suggested that the Signal Crayfish, Pacifastacus leniusculus (Dana, 1852), contains two highly distinct lineages that merit recognition as species. We further investigate these lineages here using genome skimming to conduct phylogenetic analyses on mitogenomes and highly repetitive 18S, 28S, and H3 nuclear markers. We also analyze morphological characters of these putative species to identify traits that may facilitate their identification in the field. Phylogenetic trees of mitogenomes support these lineages as species in the family Astacidae, and phylogenetic trees based on concatenated nuclear markers return comparable topologies. We describe these crayfishes as the Misfortunate Crayfish, Pacifastacus malheurensis sp. nov., which occurs in central and eastern Oregon, United States, and the Okanagan Crayfish, Pacifastacus okanaganensis sp. nov., which occurs in south central British Columbia, Canada and north central Washington, United States. Both of these species of Pacifastacus face conservation risks from displacement by non-native invasive crayfishes, but P. malheurensis sp. nov. is especially vulnerable to the rapidly spreading Rusty Crayfish, Faxonius rusticus (Girard, 1852), in central Oregon.

The conversion of natural ecosystems to agriculture is a leading cause of habitat loss and threatens global biodiversity. For the past two centuries, the Midwestern United States has experienced agricultural intensification and expansion, resulting in losses of natural ecosystems like forests. Forest cover in states like Illinois has increased over the last several decades, partially due to agricultural conservation practices like riparian buffers. However, does this increasing forest cover, intended to reduce nutrient and soil loss and benefit in‐stream biota, also benefit terrestrial biodiversity? To evaluate how forest cover influences and potentially benefits terrestrial vertebrates in agricultural landscapes, we collected environmental DNA (eDNA) samples from 47 low‐order streams over a riparian forest cover gradient and conducted eDNA metabarcoding of vertebrate communities using 12S and COI primers. Sites with complete (100%) riparian buffers supported three times the terrestrial vertebrate taxa richness compared to sites lacking buffers entirely (0%). Increased vertebrate taxa richness at highly forested sites was driven by the turnover of communities to forest‐associated species like southern two‐lined salamander ( Eurycea cirrigera ) and red‐eyed vireo ( Vireo olivaceus ), whereas unforested sites were dominated by common rodent taxa. Synthesis and applications. We demonstrate that eDNA metabarcoding has the potential to rapidly quantify the benefits of agricultural conservation to terrestrial wildlife across taxa. Notably, we found that riparian buffers along streams in the Midwestern United States return an increase of one taxon for every 10% increase in forest cover, an important potential benefit of this conservation action in an intensive agricultural landscape.

Invasive species often experience phenotypic change during the invasion process. For example, many invasive species are larger in their non-native than native ranges, but some invasive species experience body size declines with time since invasion. Mechanisms of these phenotypic changes are poorly known, likely due to a paucity of long-term datasets and complex interactions among abiotic and biotic factors that affect body size over time. We use a long-term dataset (1980-2020) in 17 lakes of Wisconsin, United States to investigate trends in body size for an invasive species, the rusty crayfish (Faxonius rusticus). We relate F. rusticus body size to crayfish relative abundance as catch-per-unit effort (CPUE) from baited trapping, as well as modelled lake temperatures and pelagic primary productivity, using a structural equation model (SEM). Our use of an SEM allowed us to investigate the direct effect of time on F. rusticus on body size, as well as indirect effects through factors like warming of lakes due to climate change. We found that F. rusticus body size has declined by 10% over the past four decades in our study lakes, from a mean of 36.6 mm total carapace length in 1980 to 32.8 mm in 2020. Faxonius rusticus individuals were larger when lakes were warmer, but the overall effect of time on declining F. rusticus body size was stronger than this predictor. Relative abundance as CPUE had no effect on F. rusticus body size, rejecting a role for density dependence in explaining adult body size of this invasive crayfish. Declining F. rusticus body sizes have accompanied population declines of this invasive crayfish in some Wisconsin lakes, and both trends provide potential for post-invasion ecosystem recovery. As one example, smaller F. rusticus individuals may be less effective or active predators on fish nests. By contrast, declining F. rusticus body size may also create opportunities for serial or over-invasion by future crayfish invaders, sustaining a need to discourage crayfish introductions to this region.

Sparse distributional data, taxonomic uncertainty, and cryptic diversity present significant barriers to building reliable species-level ecological niche models (ENMs). Supraspecific ENMS hold potential to address these challenges by combining data across multiple species to capture shared habitat associations. This study investigated the application of supraspecific ENMs to predict the distribution of primary burrowing crayfish, a freshwater faunal group with taxonomic ambiguity and limited historical data. We developed four Maxent ENMs for three of the most widespread burrowing crayfish species in the United States-Creaserinus fodiens (Cottle 1863), Procambarus gracilis (Bundy 1876), and Lacunicambarus ludovicianus (Faxon 1884)-and a combined supraspecific dataset using spatially filtered historical records of all three species. We conducted field validation across 96 semi-randomly selected sites in Missouri, United States, stratified by habitat suitability quartiles. Model performance was assessed using a suite of threshold dependent and independent metrics to evaluate discrimination and calibration. Modelling above the species level successfully identified suitable habitat for burrowing crayfish as a group. Field validation confirmed the model's predictive ability, with crayfish detected at 35 locations, including 11 sites where Lacunicambarus species not used in model training were found. Our Maxent models performed significantly better than expected by chance and showed that broadly suitable burrowing crayfish habitat consisted of shallow water tables, low elevation, low average maximum daily temperatures, and moderate annual precipitation. Our findings suggest that burrowing crayfish species may exhibit shared habitat associations, making them strong candidates for supraspecific modelling approaches, especially in regions with limited historical records or lineages with unresolved taxonomy and cryptic diversity. Further, by using species from three different genera, we demonstrated that supraspecific ENMs based on ecological traits, rather than phylogeny, offer a promising tool for extending distributional knowledge and informing conservation planning for data-poor taxa.

MotivationHere, we make available a second version of the BioTIME database, which compiles records of abundance estimates for species in sample events of ecological assemblages through time. The updated version expands version 1.0 of the database by doubling the number of studies and includes substantial additional curation to the taxonomic accuracy of the records, as well as the metadata. Moreover, we now provide an R package (BioTIMEr) to facilitate use of the database.Main Types of Variables IncludedThe database is composed of one main data table containing the abundance records and 11 metadata tables. The data are organised in a hierarchy of scales where 11,989,233 records are nested in 1,603,067 sample events, from 553,253 sampling locations, which are nested in 708 studies. A study is defined as a sampling methodology applied to an assemblage for a minimum of 2 years.Spatial Location and GrainSampling locations in BioTIME are distributed across the planet, including marine, terrestrial and freshwater realms. Spatial grain size and extent vary across studies depending on sampling methodology. We recommend gridding of sampling locations into areas of consistent size.Time Period and GrainThe earliest time series in BioTIME start in 1874, and the most recent records are from 2023. Temporal grain and duration vary across studies. We recommend doing sample-level rarefaction to ensure consistent sampling effort through time before calculating any diversity metric.Major Taxa and Level of MeasurementThe database includes any eukaryotic taxa, with a combined total of 56,400 taxa.Software Formatcsv and. SQL.

The application of environmental DNA (eDNA) methods to simultaneously study vertebrate diversity holds promise to accelerate conservation efforts, especially in freshwater systems which are among the most imperiled in the world. Here, using eDNA sampling, we identify patterns of vertebrate biodiversity across different habitats of the Kankakee River watershed, one of the most diverse lotic systems in Illinois, USA. Our eDNA metabarcoding analyses identified 147 different taxa, including 77 fishes, 38 birds, 24 mammals, five amphibians and three reptiles at 11 locations in the watershed, including tributaries and mainstem stretches upstream and within the Kankakee River State Park protected area. When compared to seining, eDNA sampling consistently detected more fish species, including non-native and imperiled species. We also found that vertebrate communities among the different habitats significantly varied in taxonomic composition, showing an upstream-downstream shift along the mainstem river as well as tributary-specific assemblages. Our study demonstrates the ability of single-marker eDNA metabarcoding to simultaneously document aquatic and terrestrial communities across large temperate lotic ecosystems and to monitor diversity patterns across protected areas.

Aquatic invasive species (AIS) are a global threat to freshwater biodiversity and ecosystem services. Documenting AIS prevalence at broad spatial scales is critical to effective management and early detection. However, conventional monitoring for AIS is costly and is rarely applied at the resolution and scale required for effective management. Monitoring of AIS using environmental DNA (eDNA) has the potential to enable broadscale surveillance at a fraction of the cost of conventional methods, but key questions must first be addressed related to how eDNA detection probability varies among environments, seasons, and multiple species with different life histories. To quantify spatiotemporal variation in the detection probability of AIS using eDNA sampling, we surveyed 20 lakes with known populations of four aquatic invasive species: common carp (Cyprinus carpio), rusty crayfish (Faxonius rusticus), spiny waterflea (Bythotrephes longimanus), and zebra mussels (Dreissena polymorpha). We collected water samples at 10 locations per lake, five times throughout the open water season resulting in a total of 1,000 water samples. Quantitative polymerase chain reaction was used with species-specific assays to determine the presence of each species' eDNA in water samples. With Bayesian occupancy models, we quantified the effects of lake and site characteristics and Julian date on eDNA detection probability. The probability of eDNA detection varied seasonally, and the seasonal variation was species-specific and related to species life histories. Zebra mussel eDNA was generally the most detectable among the species we targeted, and detection probability peaked in midsummer when only six water samples were required to achieve a 95% probability of detection (80% Bayesian credible interval: 3-12 samples). Spiny waterflea eDNA detections also peaked in mid to late summer, but were overall the most difficult species to detect, requiring 160 samples for a 95% probability of detection (80% Bayesian credible interval: 67-1,616 samples). Common carp eDNA was most detectable in the spring and rusty crayfish eDNA was most detectable in the early autumn, corresponding to key life history events. Sampling for eDNA during the optimal time of the year for each species decreased the number of samples required to reach a 95% probability of detection by an order of magnitude or more. Our results are relevant for decision makers interested in using eDNA as a multi-species monitoring tool and highlight the importance of life history in the efficacy of eDNA monitoring.

AbstractEnvironmental DNA (eDNA) refers to genetic material released by organisms into their surrounding environment. Collecting and identifying eDNA has gained popularity for monitoring and surveillance of aquatic invasive species. Invasive species management is most successful when an invasion is identified early while population size is likely to be low, highlighting the importance of eDNA detection sensitivity. Various factors influence DNA yield recovered from environmental samples. Environmental DNA storage and extraction methods, for example, can be adjusted to maximize DNA yield, thereby improving detectability. In this study, we compared the performance of two eDNA storage and extraction methods in detecting three common aquatic invasive species (Bythotrephes longimanus, Dreissena polymorpha, and Faxonius rusticus) across five natural ecosystems of Minnesota, United States. One method involved storing filters in 95% ethanol (EtOH) and extracting DNA using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), whereas the other method used cetyl trimethylammonium bromide (CTAB) for storage and a phenol–chloroform–isoamyl (PCI) procedure for DNA extraction. We also investigated the effect of DNA extract volume (1 μL relative to 3 μL) in qPCR reactions on eDNA detections for the commercial kit method. The CTAB‐PCI method yielded significantly more positive detections, across all three species, compared to the EtOH‐Qiagen method. Moreover, we found that using 1 μL of DNA extract in qPCR reactions was equally effective as using 3 μL. To improve detections of aquatic invasive species, we recommend that researchers store eDNA sample filters in CTAB or a similar lysis buffer such as Longmire's solution and extract with PCI when feasible, but note that lower extract volumes might be used without negative effect when either increasing technical replicates or repurposing samples for the detection of multiple species.

Researchers, managers, and policymakers have historically neglected non-game fishes relative to game fishes, and this oversight has extended to invasive non-game fishes in the United States. One such fish—the Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817)—has established and rapidly spread in Lake Michigan and connected waters since 2000. Here, we assess potential drivers of the successful invasion of Eastern Banded Killifish, as well as their potential to disrupt native communities and food webs. Specifically, we compare the trophic niche breadth and diet composition between Eastern Banded Killifish and a native subspecies, Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877), using stable isotope and gut content metabarcoding analyses. Stable isotope analysis showed that Eastern Banded Killifish had a higher variance in littoral dependence and trophic position than Western Banded Killifish, but both stable isotope and gut content metabarcoding analyses revealed an overlap in the diet composition and trophic position between the subspecies. Eastern Banded Killifish may successfully establish outside its native range due to higher feeding variability than Western Banded Killifish, including in habitats historically unused by the native subspecies, but the trophic niche between these two subspecies was similar overall. This study provides insights into the successful invasion of a potentially overlooked non-game fish—Eastern Banded Killifish—while also comparing stable isotope and gut content metabarcoding analyses for an invasive freshwater fish for the first time.

AbstractEnvironmental DNA (eDNA) analysis is an effective and non‐invasive technique for surveying and monitoring rare, threatened, or endangered (RTE) species. Compared to conventional capture‐based sampling, eDNA analysis may offer a more cost‐effective approach for surveying RTE species, yet few studies have compared their cost‐efficiency—a critical consideration for conservation planning. We compared the costs, effort, and relative performance of aquatic eDNA sampling and conventional trapping for detecting the Alligator Snapping Turtle, Macrochelys temminckii Troost, 1835, in southwest Louisiana, United States. Environmental DNA was sampled quarterly over 1 year (2018–2019) at 19 streams, including three streams where M. temminckii presence had been previously confirmed via conventional trapping efforts (2012–2013). Water samples from each stream were analyzed using quantitative polymerase chain reaction (qPCR) to assess M. temminckii eDNA presence/absence. Time and costs (i.e., labor, travel, wages, and supplies) per detection via eDNA analysis and trapping were calculated and compared. Environmental DNA analysis documented the presence of M. temminckii DNA at two of the three streams where individuals had previously been trapped and yielded detections (qPCR amplifications) at 16 additional streams not previously sampled, expanding M. temminckii's documented distribution at our study sites by 84%. Environmental DNA analysis returned a detection rate (per site) 5.55 times higher than conventional trapping and was 18.7% less expensive. Our results provide evidence that strategically deployed eDNA surveys may be an effective and cost‐efficient approach for detecting freshwater RTE species. With eDNA analysis, additional resources can be invested toward expanding survey coverage and increasing sampling frequency, allowing managers to more effectively target subsequent intensive monitoring efforts.

Three novel crayfish-infecting nudiviruses from crayfish in North America represent the first genomic confirmation of nudiviruses in crayfish: Faxonius propinquus nudivirus (FpNV), Faxonius rusticus nudivirus (FrNV), and Faxonius virilis nudivirus (FvNV). Histopathology and electron microscopy revealed nuclear infections, including nuclear hypertrophy in hepatopancreatic epithelial cells and the presence of membrane-bound bacilliform virions. Metagenomic sequencing resulted in complete circular genome assembly, and phylogenetic analyses (based on nudivirus core genes) placed these viruses within the unofficial Epsilonnudivirus genus. One of the nudiviruses was detected in the antennal gland of its host, and another is correlated with invasive crayfish decline in one infected lake ecosystem - suggesting a potential route for viral transmission through water, and possible population level impact. This study highlights the importance of genomic and ecological data in elucidating the diversity and evolutionary relationships of the Nudiviridae, while expanding their known diversity and range of host species.

“Sleeper” invaders are non-native populations that experience time-lags post-establishment before subsequent spread or negative impacts, challenging managers to differentiate harmless non-native species from invasive species. In lakes of northern Wisconsin, United States, Rusty Crayfish (Faxonius rusticus Girard, 1852) has dominated as an invasive species for decades, but this species has recently experienced population declines. Following these F. rusticus declines, we rediscovered in 2020 a population of non-native Calico Crayfish (Faxonius immunis Hagen, 1870) that had not been documented since the 1970s. Declining F. rusticus populations may create opportunities for F. immunis spread to other lakes and impacts as a sleeper invader. We conducted additional sampling in summer 2021 that suggests F. immunis remains isolated in only one lake within this watershed. We used mitochondrial DNA barcoding to confirm these crayfish were F. immunis and had not been misidentified as a congener. Next, we investigated whether biotic interactions with F. rusticus may have prevented F. immunis spread over the past several decades. We measured agonistic behaviors using F. immunis and F. rusticus pairs in the laboratory, and then modeled differences in aggression between species while controlling for size and reproductive form. We found that F. rusticus were consistently dominant over F. immunis, suggesting that competition with an established hyper-abundant invasive species may have restricted past spread by F. immunis. Managers and policy makers should consider whether precautionary actions against F. immunis are warranted while the population of this species remains small and localized, especially in the context of F. rusticus declines.

Hybridisation can be an important driver of evolutionary change, but hybridisation with invasive species can have adverse effects on native biodiversity. While hybridisation has been documented across taxa, there is limited understanding of ecological factors promoting patterns of hybridisation and the spatial distribution of hybrid individuals. We combined the results of ecological niche modelling (ENM) and restriction site-associated DNA sequencing to test theories of niche conservatism and biotic resistance on the success of invasion, admixture, and extent of introgression between native and non-native fishes. We related Maxent predictions of habitat suitability based on the native ranges of invasive Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817) and native Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877) to admixture indices of individual Banded Killifish. We found that Eastern Banded Killifish predominated at sites predicted as suitable from their ENM, consistent with niche conservatism. Admixed individuals were more common as Eastern Banded Killifish habitat suitability declined. We also found that Eastern Banded Killifish were most common at sites closest to the presumed source of this invasion, whereas the proportion of admixed individuals increased with distance from the source of invasion. Lastly, we found little evidence that habitat suitability for Western Banded Killifish provides biotic resistance from either displacement by, or admixture with, invasive Eastern Banded Killifish. Our study demonstrates that ENMs can inform conservation-relevant outcomes between native and invasive taxa while emphasising the importance of protecting isolated Western Banded Killifish populations from invasive conspecifics.