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[Display omitted] • Cryptic, fossorial snake distribution is challenging to determine. • Environmental DNA (eDNA) may solve this but there are few terrestrial applications. • A new sharp-tailed snake eDNA assay was developed and validated. • eDNA was measured from swabs and soil under artificial cover objects. • Detection rate was an order of magnitude better than conventional surveys. Performing ecological surveys for secretive, fossorial snakes is challenging. Traditional survey methods involve visual observation under artificial cover objects (ACOs); this is labor-intensive and requires multiple consistent surveys of suitable habitats. Detection of snake DNA deposited under ACOs represents an innovative method for species detection. However, for terrestrial species, common issues with soil-based methods include the challenges of adequately removing enzyme inhibitors that reduce environmental DNA (eDNA) detection and potential photodegradation of DNA taken from surface samples. These issues may be circumvented by obtaining swabs and soil samples directly from the underside of ACOs for eDNA analysis. We demonstrate the application of this method in surveys of sharp-tailed snake (Contia tenuis), an endangered species under the Canadian Species at Risk Act. We describe the design and validation of a new quantitative real-time polymerase chain reaction (qPCR)-based eDNA eCOTE3 assay with high specificity and sensitivity for sharp-tailed snake. We developed a practical and robust protocol for obtaining eDNA samples by swabbing the underside of ACOs and collecting soil samples under ACOs. Traditional surveys were conducted over two successive years (2018–19) on 220 paired ACOs at 110 sites monitored between 12 and 30 times each. Of the 6,060 ACO visits, only 24 resulted in sharp-tailed snake observations (0.4% success rate) illustrating the considerable difficulty in detecting these snakes. During this same time, 109 swabs were taken directly from the undersides of ACOs and 78 soil samples were collected from a subset of these ACOs. Of the 24 occurrences where sharp-tailed snakes were visually observed, 13 of 23 ACO swabs (57%) and nine of 20 soil samples (45%) tested positive for DNA. eDNA deposition is likely low because of the small size and behavior of this cryptic species, yet DNA was detected from soil exposed to captured snakes for only 10 min. Nevertheless, sharp-tailed snake eDNA was detected at eight sites (9%) from ACO swabs (n = 86) and seven sites (13%) from soil samples (n = 56) where snakes were not observed. This is an overall detection rate of 25% (14/56) for swab and soil samples testing positive in sites where both were tested, representing a substantial reduction in the effort required for detection of this species. Given the time-consuming nature of traditional surveys, eDNA holds great promise as a complementary survey tool for this terrestrial species. While further work is needed to delineate DNA deposition rates, this work represents a significant advance in monitoring a challenging species. [ABSTRACT FROM AUTHOR]

Combined use of sedimentary DNA (sedDNA) and Indigenous Knowledge in reconstructing historical fish records. [Display omitted] • Indigenous peoples have a long-term link with Cowpar Lake in the Canadian Oil Sands. • Sedimentary DNA (sedDNA) from sediment core acts as an archive of lake history. • qPCR-based environmental DNA methods reconstructed historical fish occupation. • SedDNA results aligned with Indigenous Knowledge (IK) on fish communities. • Combined sedDNA and IK give powerful insight into natural and human-mediated impact. Lake sediment serves as a natural archive of historical biological information. The use of sedimentary DNA (sedDNA), a form of environmental DNA (eDNA) shed by aquatic organisms and preserved in sediment, has been instrumental in reconstructing past faunal composition in aquatic communities. However, the low abundance of fish sedDNA and the often humic-rich nature of lake sediments create methodological challenges for the accurate detection of target sedDNA using quantitative polymerase chain reaction (qPCR)-based approaches. Herein, we present a consolidated qPCR-based eDNA workflow to reconstruct past and current fish fauna in Cowpar Lake located in the Oil Sands region in Alberta (Canada), which were then validated using Indigenous Knowledge from Chipewyan Prairie First Nation community members. The present study highlights the importance of combining column- and precipitation-based PCR inhibitor clean-up, nucleic acid concentration, incorporating endogenous chloroplast DNA as a sample integrity control. Robust qPCR-based eDNA assays were also useful in preventing the false-negative detection of low copies of target fish DNA. The presence of Northern pike (1905 to 2019) and Cisco (1919 to 1942) in Cowpar Lake was confirmed based on detected sedDNA from sediment core. The reconstructed fish records from sedDNA-inferred data aligned with the Indigenous accounts of natural and human-mediated changes in land use around the lake. Overall, the present study addresses common methodological concerns in processing lake sediment samples for fish eDNA detection and demonstrates the great potential of combined eDNA-inferred data and Indigenous Knowledge in reconstructing historical fish records in aquatic communities. [ABSTRACT FROM AUTHOR]

• Forage fish are keystones of the coastal marine food web. • Pacific Sand Lance and Surf Smelt habitat use data are difficult to obtain. • Environmental DNA (eDNA) may improve data from visual egg surveys from sand samples. • Novel eDNA assays for both species were developed and validated. • Detection rate shows promise for broad application of eDNA for forage fish. Many coastal forage fish species spend most of their life history using the water column, but some species such as Pacific Sand Lance (Ammodytes personatus) and Surf Smelt (Hypomesus pretiosus) have a requirement for intertidal sediments for spawning and or burying. Detection of suitable but typically uncommon intertidal habitats for spawning has historically relied on extensive visual surveys for eggs from large sand samples. In the present study, we developed and validated two new non-destructive qPCR-based tools for detecting Sand Lance and Surf Smelt eDNA from small sand samples. A total of 279 composite transect sand samples were collected from 101 beaches and were tested for eDNA sample integrity using the IntegritE-DNATM test. Initial testing showed that all but 42 of 46 samples passed after an inhibitor clean-up step. Of the 101 beaches sampled 275 times, there were 111 detections of Sand Lance on 52 beaches, and there were 13 detections of Surf Smelt on 10 beaches. One hundred and eighty-one samples were paired with visual egg counts and 76 (42%) of these tested positive for Sand Lance while 9 of 132 (7%) samples tested positive for Surf Smelt. For the 27 paired samples with two or more Sand Lance eggs visually detected there was a significant positive correlation (r = 0.694, p = 0.0001) with eDNA copies per liter suggesting that a larger Sand Lance presence is easily and reliably detected using eDNA. The methods and approaches described herein will serve as a foundation for broad application of eDNA approaches for forage fish species that utilize intertidal benthic habitats. [ABSTRACT FROM AUTHOR]