Improving ecological surveys for the detection of cryptic, fossorial snakes using eDNA on and under artificial cover objects

Laura Matthias, Michael J. Allison, Carrina Y. Maslovat, Jared Hobbs, Caren C. Helbing

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. Dur- ing 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.

Meta-analysis shows both congruence and complementarity of DNA and eDNA metabarcoding to traditional methods for biological community assessment

François Keck, Rosetta C. Blackman, Raphael Bossart, Jeanine Brantschen, Marjorie Couton, Samuel Hürlemann, Dominik Kirschner, Nadine Locher, Heng Zhang, Florian Altermatt

DNA metabarcoding is increasingly used for the assessment of aquatic communities, and numerous studies have investigated the consistency of this technique with tradi- tional morpho-taxonomic approaches. These individual studies have used DNA meta- barcoding to assess diversity and community structure of aquatic organisms both in marine and freshwater systems globally over the last decade. However, a systematic analysis of the comparability and effectiveness of DNA-based community assessment across all of these studies has hitherto been lacking. Here, we performed the first meta- analysis of available studies comparing traditional methods and DNA metabarcoding to measure and assess biological diversity of key aquatic groups, including plankton, microphytobentos, macroinvertebrates, and fish. Across 215 data sets, we found that DNA metabarcoding provides richness estimates that are globally consistent to those obtained using traditional methods, both at local and regional scale. DNA metabar- coding also generates species inventories that are highly congruent with traditional methods for fish. Contrastingly, species inventories of plankton, microphytobenthos and macroinvertebrates obtained by DNA metabarcoding showed pronounced differ- ences to traditional methods, missing some taxa but at the same time detecting oth- erwise overseen diversity. The method is generally sufficiently advanced to study the composition of fish communities and replace more invasive traditional methods. For smaller organisms, like macroinvertebrates, plankton and microphytobenthos, DNA metabarcoding may continue to give complementary rather than identical estimates compared to traditional approaches. Systematic and comparable data collection will increase the understanding of different aspects of this complementarity, and increase the effectiveness of the method and adequate interpretation of the results.

Are Environmental DNA Methods Ready for Aquatic Invasive Species Management?

Adam J Sepulveda, Nanette M Nelson, Christopher L Jerde, Gordon Luikart

Multiple studies have demonstrated environmental (e)DNA detections of rare and invasive species. However, invasive species managers struggle with using eDNA results because detections might not indicate species presence. We evaluated whether eDNA methods have matured to a point where they can be widely applied to aquatic invasive species management. We have found that eDNA methods meet legal standards for being admissible as evidence in most courts, suggesting eDNA method reliability is not the problem. Rather, we suggest the interface between results and management needs attention since there are few tools for integrating uncertainty into decision-making. Solutions include decision-support trees based on molecular best practices that integrate the temporal and spatial trends in eDNA positives relative to human risk tolerance.

Tracking an invasion front with environmental DNA

Abigail G. Keller, Emily W. Grason, P. Sean McDonald, Ana Ramón-Laca, Ryan P. Kelly

Data from environmental DNA (eDNA) may revolutionize environmental monitoring and management, providing increased detection sensitivity at reduced cost and survey effort. However, eDNA data are rarely used in decision-making contexts, mainly due to uncertainty around (1) data interpretation and (2) whether and how molecular tools dovetail with existing management efforts. We address these challenges by jointly modeling eDNA detection via qPCR and traditional trap data to estimate the density of invasive European green crab (Carcinus maenas), a species where, historically, baited traps have been used for both detection and control. Our analytical framework simultaneously quantifies uncertainty in both detection methods and provides a robust way of integrating different data streams into management processes. Moreover, the joint model makes clear the marginal information benefit of adding eDNA (or any other) additional data type to an existing monitoring program, offering a path to optimizing sampling efforts for species of management interest. Here, we document green crab eDNA beyond the previously known invasion front and find the value of eDNA data dramatically increases with low population densities and low traditional sampling effort, as is often the case at leading-edge locations. We also highlight the detection limits of the molecular assay used in this study, as well as scenarios under which eDNA sampling is unlikely to improve existing management efforts.

Evaluating bioinformatics pipelines for population-level inference using environmental DNA

Bastien Macé, Régis Hocdé, Virginie Marques, Pierre-Edouard Guerin, Alice Valentini, Véronique Arnal, Loïc Pellissier, Stéphanie Manel

Environmental DNA is mainly not only used at the interspecific level, to quantify spe- cies diversity in ecosystems, but can also be used to quantify intraspecific genetic variability, thus avoiding the need to sample individual tissue. However, errors in the amplification and sequencing of eDNA samples can blur this intraspecific signal and strongly over-estimate genetic diversity. Existing bioinformatics pipelines therefore need to be tested to evaluate whether reliable levels of intraspecific genetic variability can be derived from eDNA samples. Here, we compare the ability of twelve metabar- coding pipelines to detect intraspecific genetic variability combining five programs. All pipelines have common pre-processing steps, a processing data step using pro- grams among obiclean; DADA2; SWARM; and LULU. An additional chimera removal step is also investigated based on two programs (VSEARCH or DADA2). The case study was the natural intraspecific variation within Mullus surmuletus in experimental settings. We developed specific primers for this species, located on the mitochondrial D-loop fragment (barcode MS-DL06). Thirty-nine individuals were collected from the Mediterranean Sea, placed into four aquariums, and their DNA was sequenced on this marker to build an intraspecific reference database. After filtering the aquarium water, DNA was extracted, amplified, and sequenced using the primer pair developed. We then quantified the number of true haplotypes returned by each pipeline and its capacity to eliminate most of the erroneous sequences. We show that the program DADA2 with a two-parent chimeric sequence removal step is the best tool to estimate intraspecific diversity from eDNA. Furthermore, our approach was also able to detect true M. surmuletus haplotypes in two eDNA samples collected in the Mediterranean Sea. We conclude that the combination of an appropriate intrapopulation barcode and a denoising pipeline like DADA2 with a chimeric sequence removal step is promising to make population-level inference using environmental DNA possible.