Basin-Scale Surveys of Stream-Associated Amphibians in Intensively Managed Forests

Abstract: Conservation and management of native species on landscapes managed for intensive wood production represents an ongoing challenge to forest managers. Previous research suggests that impacts of forest practices on stream-associated amphibians (SAA; giant [Dicamptodon spp.], torrent [Rhyacotriton spp.], and plethodontid [Plethodon spp.] salamanders and coastal tailed frogs [Ascaphus truei]) in Oregon and Washington, USA, vary spatially and temporally as a result of biotic and abiotic factors, some of which can be influenced by management treatments. Although individual harvest units can encompass multiple stream reaches and entire second-order basins, nearly all published research studies used stream reaches of various lengths as sample units. To address this discrepancy between research and operational scales, we sampled first-, second-, and third-order streams in 70 randomly selected third-order basins in Oregon and Washington in 2007 and 2008 to estimate detection and occupancy parameters for SAA and to develop basin-level density estimates for different species and genera. We estimated occupancy probabilities of 0.99 (95% CL = 0.96–1.00) for torrent and giant salamanders, 0.93 (95% CL = 0.76–0.92) for Dunn's salamanders (Plethodon dunni), and 0.60 (95% CL = 0.46–0.72) for tailed frogs. Our estimates can be compared with estimates for unmanaged third-order basins in Oregon and Washington to provide a relative measure of potential impacts of forest management on these taxa. In addition, our estimates provide baseline information with which to assess potential effects of future environmental changes on the 4 genera.     

Factors influencing detection of eDNA from a stream‐dwelling amphibian

Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream‐dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full‐sun and shaded treatments degraded exponentially to <1% of the original concentration after 3 days. eDNA was no longer detectable in full‐sun samples after 8 days, whereas eDNA was detected in 20% of shaded samples after 11 days and 100% of refrigerated control samples after 18 days. When translocated into unoccupied streams, salamanders were detectable after 6 h, but only when densities were relatively high (0.2481 individuals/m²) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 h after introduction, reaching 0.0022 ng/L. Within 1 h of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.     

The distribution of the Rocky Mountain tailed frog (Ascaphus montanus) in relation to the fluvial system: implications for management and conservation

The mating, egg-laying, and larval development of tailed frogs occur in dynamic mountain streams. During the lengthy (up to 5 years) aquatic residency these species are vulnerable to channel disturbances that can be exacerbated by land uses. Researchers have highlighted specific tailed frog habitat associations but never in the context of fluvial system processes. Based on an extensive regional study with a watershed-wide sampling strategy, we demonstrate that the Rocky Mountain tailed frog (Ascaphus montanus) is limited to contributing basins of roughly 0.3–100 km² in size, with peak numbers in basins up to 35 km². We conclude that the primary determinant of tailed frog distribution patterns in a watershed is basin area, a proximate variable for channel process domain and regional stream discharge: tailed frogs are adapted to cascade and step-pool channel morphologies that characterize these small basins, presumably because they afford more bedform stability and pore-space refugia than do smaller, colluvial headwaters, or larger, floodplain-forming plane bed and pool-riffle bedforms of mainstem rivers. Secondarily, climate and physiography interact to influence occurrence and abundance at the watershed level by controlling such variables as runoff, water temperature, and sedimentation regime. This point has important management implications because it forces us to recognize that in complex ecosystems, wildlife habitat associations are contingent on site-specific interactions amongst fluvial system control variables: significance levels of any one variable to tailed frog distribution will not necessarily be consistent among basins. The study clearly shows that case studies can produce conflicting results when they lack a process-based understanding of ecological response.     

Influences of Wildfire and Channel Reorganization on Spatial and Temporal Variation in Stream Temperature and the Distribution of Fish and Amphibians

Abstract Wildfire can influence a variety of stream ecosystem properties. We studied stream temperatures in relation to wildfire in small streams in the Boise River Basin, located in central Idaho, USA. To examine the spatio-temporal aspects of temperature in relation to wildfire, we employed three approaches: a pre–post fire comparison of temperatures between two sites (one from a burned stream and one unburned) over 13 years, a short-term (3 year) pre–post fire comparison of a burned and unburned stream with spatially extensive data, and a short-term (1 year) comparative study of spatial variability in temperatures using a “space for time” substitutive design across 90 sites in nine streams (retrospective comparative study). The latter design included streams with a history of stand-replacing wildfire and streams with severe post-fire reorganization of channels due to debris flows and flooding. Results from these three studies indicated that summer maximum water temperatures can remain significantly elevated for at least a decade following wildfire, particularly in streams with severe channel reorganization. In the retrospective comparative study we investigated occurrence of native rainbow trout (Oncorhynchus mykiss) and tailed frog larvae (Ascaphus montanus) in relation to maximum stream temperatures during summer. Both occurred in nearly every site sampled, but tailed frog larvae were found in much warmer water than previously reported in the field (26.6°C maximum summer temperature). Our results show that physical stream habitats can remain altered (for example, increased temperature) for many years following wildfire, but that native aquatic vertebrates can be resilient. In a management context, this suggests wildfire may be less of a threat to native species than human influences that alter the capacity of stream-living vertebrates to persist in the face of natural disturbance. Electronic supplementary material: The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of seasonal dynamics of fungal DNA assemblages in a flow-regulated stream in a restored forest using eDNA metabarcoding

Investigation of seasonal variation in fungal communities is essential for understanding biodiversity and ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community composition, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis has been explored for its utility in biodiversity surveys. In this study, we assessed whether the seasonality of fungal communities can be detected by monitoring eDNA in a forest stream. We conducted monthly water sampling in a forest stream over 2 years and used DNA metabarcoding to identify fungal eDNA. The stream water contained DNA from functionally diverse aquatic and terrestrial fungi, such as plant decomposers, parasites and mutualists. The variation in the fungal assemblage showed a regular annual periodicity, meaning that the assemblages in a given season were similar, irrespective of the year or sampling. Furthermore, the strength of the annual periodicity varied among functional groups. Our results suggest that forest streams may act as a ‘trap’ for terrestrial fungal DNA derived from different habitats, allowing the analysis of fungal DNA in stream water to provide information about the temporal variation in fungal communities in both the aquatic and the surrounding terrestrial ecosystems.     

Comparison of Two Techniques for Surveying Headwater Stream Amphibians

Abstract: We compared rubble-rousing versus light-touch stream amphibian survey techniques in multiple 1-m plots across 10 streams in southwest Washington, USA. Specifically, we wanted to determine if light-touch surveys provide unbiased estimates of abundance (i.e., provide counts correlated with rubble-rousing counts) and which method would provide more cost-effective presence or absence information. Rubble-rousing, a common technique for surveying stream-associated amphibians in the Pacific Northwest, took 12 times as long as light-touch to apply. Abundance estimates and standard errors for rubble-rousing were consistently higher than those for light-touch for all life stages for the coastal tailed frog (Ascaphus truei) and Columbia torrent salamander (Rhyacotriton kezeri). Except for eggs, light-touch detected all life stages found during rubble-rousing. For frogs, only some rubble-rousing abundance estimates, mostly involving second-year larvae, were highly correlated with their light-touch counterparts, whereas for salamanders, similar comparisons generated high correlations across most life stages. Correlations between methods were consistently greater for salamanders than for frogs. However the smaller tailed frog sample sizes and the cryptozoic nature of some life stages may have contributed to this pattern. Depending on the degree to which researchers can tolerate false-negative error rates, light-touch may prove less costly than rubble-rousing for detecting species presence. For the cost of obtaining one rubble-rousing sample, many light-touch samples can be used across a range of habitats for detecting species patchily distributed.     

Implementation of Novel Design Features for qPCR-Based eDNA Assessment

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.     

基于环境DNA宏条形码技术的辽东湾典型围海养殖池塘内水母多样性研究

研究使用环境DNA宏条形码技术(eDNA metabarcoding)检测辽东湾东北部河口区围海养殖池塘水母种类多样性,探索适用于水母种类物种鉴定和监测的新方法。利用环境DNA宏条形码技术,分别基于18S rDNA和COI宏条形码检测了辽东湾东北部河口区围海养殖池塘水母种类多样性,通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从围海养殖池塘7个采样点中获得可检测的采样点数据。结果显示,基于18S rDNA宏条形码检测出8种水母种类,其中钵水母纲大型水母2种、水螅水母总纲小型水母6种;基于COI宏条形码技术共检测出19种水母种类,其中钵水母纲大型水母5种、水螅水母总纲小型水母14种;两种DNA条形码标记都显示养殖种类海蜇(Rhopilema esculentum)为优势种。研究结果表明,环境DNA宏条形码技术作为一种新兴的生物多样性监测手段可用于快速检测水母种类多样性,在水母类物种鉴定、监测及早期预警中有较大的应用潜能。     

Using stable isotopes to test for trophic niche partitioning: a case study with stream salamanders and fish

1. Stream salamanders and fish often co‐occur even though fish prey on and outcompete salamanders. However, the mechanisms that allow palatable salamanders to coexist with fish are unknown. 2. We tested mechanisms in the field that promote coexistence between Idaho giant salamanders (Dicamptodon aterrimus) and stream salmonid fishes in headwater streams. Previous research in this system indicated that salamander dispersal did not promote coexistence with fish. We tested the hypothesis that D. aterrimus shift their diet when they occur with fish, facilitating coexistence through local niche partitioning. 3. We used nitrogen and carbon stable isotopes to describe the trophic niche of D. aterrimus and fish in three co‐occurring populations of salamanders and fish and three populations of salamanders without fish. We used two approaches to quantify trophic niche partitioning with stable isotopes: 95% kernel density estimators and isotopic mixing models. 4. We found that salamanders and fish were generalists that consumed aquatic invertebrates primarily, but both species were also cannibalistic and predatory on one another. We also found no support for trophic niche partitioning as a coexistence mechanism because there were no differences in the trophic niche metrics among salamander populations with and without fish. 5. Although we did not identify mechanisms that facilitate salamander and fish coexistence, our empirical data and use of novel approaches to describe the trophic niche did yield important insights on the role of predator–prey interactions and cannibalism as alternative coexistence mechanisms. In addition, we found that 95% kernel estimators are a simple and robust method to describe population‐level measure of trophic structure.     

An enigmatic heteronemertean from the Gulf of Mexico

The effects of acid (HNO3) on drift and survival of benthic invertebrate communities were assessed in stream microcosms over a 7-day exposure period. Communities were obtained from the Cache la Poudre River, Colorado, using artificial substrates colonized in the stream for 30 days and then transferred to stream microcosms. Streams receiving the highest acid concentration (pH 4.0) contained significantly fewer individuals ( F = 378.42, p < 0.0001) and taxa ( F = 7.8, p = 0.0123) at the end of the experiment compared to the other two treatments (pH 5.5, 6.5) and the control (pH 7.4). Reduced macroinvertebrate abundance resulted primarily from reduced abundance of mayflies (Ephemeroptera) which were particularly sensitive. Comparisons of Plecoptera, Trichoptera, and Diptera abundances showed no statistically significant differences among treatments. Analysis of invertebrate drift samples, collected after 2, 6, 18, and 42 h exposure, revealed that percent drift in the most acidic streams was nine times that of control streams. Ephemeroptera was the only aquatic insect order to exhibit a significant drift response, and timing and magnitude of responses varied among mayfly taxa. Differences in sensitivity to acid among aquatic insect orders observed in our experimental streams were similar to those reported from field studies in other regions. Effects of acid on drift and survival of benthic invertebrate communities were also similar to effects of heavy metals, one of the primary water quality concerns in the Rocky Mountain region. These results suggest a general pattern of responses to chemical stressors in benthic communities from Rocky Mountain streams. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluating the effects of urbanisation on salamander abundances using a before‐after control‐impact design

1. Urbanisation represents a significant threat to semi‐aquatic amphibian populations, especially stream‐dwelling salamanders. Although studies of urbanisation effects on amphibians have been conducted, there is an urgent need to follow populations over longer time periods, account for imperfect detection and determine the response time to urbanisation. Consequently, we used a before‐after control‐impact (BACI) study design to estimate changes in abundances of larval and adult salamanders in streams affected by urbanisation. 2. From 2005 to 2009, we used standard sampling techniques to obtain a count of salamanders in 13 first‐order streams that underwent urbanisation of their catchments after the first year of sampling. Simultaneously, we counted salamanders in 17 streams that experienced no disturbance within stream catchments. Additionally, we measured environmental variables at each stream. 3. We used Royle’s binomial mixture model to estimate annual mean abundances and individual detection probabilities, and Bayesian inference was used to estimate population parameters for each stage and species. 4. Although mean abundance estimates varied among years in control and urbanised streams, we found that urbanisation had a negative effect on larval and adult salamander abundances. Larval salamander abundances at sites 1 year after urbanisation were significantly lower than abundances from control sites. Abundances of adult two‐lined salamanders (Eurycea cirrigera) at urbanised sites were lower than abundances at control sites 2 years post‐urbanisation, and adult dusky salamander (Desmognathus fuscus) abundances at urbanised sites were lower than abundances at control sites 3 years post‐urbanisation. Maximum conductivity, sedimentation level and maximum stream channel width differed between urban and non‐urban streams. 5. Our results suggest that stream‐dwelling salamanders exhibit little resistance to urbanisation. Our study also highlights the use of the BACI design to study how urbanisation affects populations in semi‐aquatic habitats. We emphasise that inferences regarding urbanisation effects on population response may be compromised unless urban populations are compared to populations in control sites, especially for species in which populations fluctuate.     

Residual eDNA detection sensitivity assessed by quantitative real‐time PCR in a river ecosystem

Several studies have demonstrated that environmental DNA (eDNA) can be used to detect the presence of aquatic species, days to weeks after the target species has been removed. However, most studies used eDNA analysis in lentic systems (ponds or lakes), or in controlled laboratory experiments. While eDNA degrades rapidly in all aquatic systems, it also undergoes dilution effects and physical destruction in flowing systems, complicating detection in rivers. However, some eDNA (i.e. residual eDNA) can be retained in aquatic systems, even those subject to high flow regimes. Our goal was to determine residual eDNA detection sensitivity using quantitative real‐time polymerase chain reaction (qRT–PCR), in a flowing, uncontrolled river after the eDNA source was removed from the system; we repeated the experiment over 2 years. Residual eDNA had the strongest signal strength at the original source site and was detectable there up to 11.5 h after eDNA source removal. Residual eDNA signal strength decreased as sampling distance downstream from the eDNA source site increased, and was no longer detectable at the source site 48 h after the eDNA source water was exhausted in both experiments. This experiment shows that residual eDNA sampled in surface water can be mapped quantitatively using qRT–PCR, which allows a more accurate spatial identification of the target species location in lotic systems, and relative residual eDNA signal strength may allow the determination of the timing of the presence of target species.     

Spatial and temporal patterns of environmental DNA detection to inform sampling protocols in lentic and lotic systems

The development of efficient sampling protocols for the capture of environmental DNA (eDNA) could greatly help improve accuracy of occupancy monitoring for species that are difficult to detect. However, the process of developing a protocol in situ is complicated for rare species by the fact that animal locations are often unknown. We tested sampling designs in lake and stream systems to determine the most effective eDNA sampling protocols for two rare species: the Sierra Nevada yellow‐legged frog (Rana sierrae) and the foothill yellow‐legged frog (Rana boylii). We varied water volume, spatial sampling, and seasonal timing in lakes and streams; in lakes we also tested multiple filter types. We found that filtering 2 L versus 1 L increased the odds of detection in streams 5.42X (95% CI: 3.2–9.19X) in our protocol, from a probability of 0.51–0.85 per technical replicate. Lake sample volumes were limited by filter clogging, and we found no effect of volume or filter type. Sampling later in the season increased the odds of detection in streams by 1.96X for every 30 days (95% CI: 1.3–2.97X) but there was no effect for lakes. Spatial autocorrelation of the quantity of yellow‐legged frog eDNA captured in streams ceased between 100 and 200 m, indicating that sampling at close intervals is important.     

Distance,flow and PCR inhibition: eDNA dynamics in two headwater streams

Environmental DNA (eDNA) detection has emerged as a powerful tool for monitoring aquatic organisms, but much remains unknown about the dynamics of aquatic eDNA over a range of environmental conditions. DNA concentrations in streams and rivers will depend not only on the equilibrium between DNA entering the water and DNA leaving the system through degradation, but also on downstream transport. To improve understanding of the dynamics of eDNA concentration in lotic systems, we introduced caged trout into two fishless headwater streams and took eDNA samples at evenly spaced downstream intervals. This was repeated 18 times from mid‐summer through autumn, over flows ranging from approximately 1–96 L/s. We used quantitative PCR to relate DNA copy number to distance from source. We found that regardless of flow, there were detectable levels of DNA at 239.5 m. The main effect of flow on eDNA counts was in opposite directions in the two streams. At the lowest flows, eDNA counts were highest close to the source and quickly trailed off over distance. At the highest flows, DNA counts were relatively low both near and far from the source. Biomass was positively related to eDNA copy number in both streams. A combination of cell settling, turbulence and dilution effects is probably responsible for our observations. Additionally, during high leaf deposition periods, the presence of inhibitors resulted in no amplification for high copy number samples in the absence of an inhibition‐releasing strategy, demonstrating the necessity to carefully consider inhibition in eDNA analysis.     

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.     

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.     

Environmental DNA detection of aquatic invasive plants in lab mesocosm and natural field conditions

Aquatic invasive plant species cause negative impacts to economies and ecosystems worldwide. Traditional survey methods, while necessary, often do not result in timely detections of aquatic invaders, which can be cryptic, difficult to identify, and exhibit very rapid growth and reproduction rates. Environmental DNA (eDNA) is a relatively new method that has been used to detect multiple types of animals in freshwater and marine ecosystems through tissues naturally shed from the organism into the water column or sediment. While eDNA detection has proven highly effective in the detection of aquatic animals, we know less about the efficacy of eDNA as an effective surveillance tool for aquatic plants. To address this disparity, we designed mesocosm experiments with Elodea species to determine the ability to detect accumulation and degradation of the DNA signal for aquatic plants, followed by field surveillance of the highly invasive Hydrilla verticillata in freshwaters across several U.S. geographic regions. In both lab and field experiments, we designed a high sensitivity quantitative PCR assay to detect the aquatic plant species. In both experiments, plant eDNA detection was successful; we saw accumulation of DNA when plants were introduced to tanks and a decrease in DNA over time after plants were removed. We detected eDNA in the field in areas of known Hydrilla distribution. Employing eDNA detection for aquatic plants will strengthen efforts for early detection and rapid response of invaders in global freshwater ecosystems.     

Environmental DNA for the enumeration and management of Pacific salmon

Pacific salmon are a keystone resource in Alaska, generating annual revenues of well over ~US$500 million/year. Due to their anadromous life history, adult spawners distribute amongst thousands of streams, posing a huge management challenge. Currently, spawners are enumerated at just a few streams because of reliance on human counters and, rarely, sonar. The ability to detect organisms by shed tissue (environmental DNA, eDNA) promises a more efficient counting method. However, although eDNA correlates generally with local fish abundances, we do not know if eDNA can accurately enumerate salmon. Here we show that daily, and near‐daily, flow‐corrected eDNA rate closely tracks daily numbers of returning sockeye and coho spawners and outmigrating sockeye smolts. eDNA thus promises accurate and efficient enumeration, but to deliver the most robust numbers will need higher‐resolution stream‐flow data, at‐least‐daily sampling, and a focus on species with simple life histories, since shedding rate varies amongst jacks, juveniles, and adults.     

Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi‐aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water‐borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus‐specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.     

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.