3rd Biennial Marine Mammal eDNA Workshop

Workshop Presentations

eDNA through an aDNA lense: lessons from a related discipline

Morten Allentoft

Abstract: As a newcomer to the eDNA field with a background in aDNA research, I see many similarities among the two disciplines. Both are deriving information from degraded biomolecules and are thus facing all the same challenges related to substrate selection, sampling, extraction, library prep, sequencing, and analyses. There are also differences; notably the aDNA field has a 40-year history, whereas eDNA research has only really taken off in the past decade. With that in mind it is highly relevant for the eDNA community to reflect on the lessons learned in the aDNA field. Both those related to technical improvements but also more strategical and ethical ones. It has been 40 years characterized by some tremendous victories, such as the Neanderthal genome and a recent Nobel Prize, but also some tremendous failures that we do not want to repeat. We need to learn from both. In this presentation I will therefore look back at the past through an aDNA lens in an attempt to look forward through an eDNA perspective.

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Presentation slides (pdf)

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Biodiversity reporting using eDNA - industrial applications with the element of research 

Dóra Székely

Abstract: The applications of environmental DNA based methods have been diversifying in the recent decade, however the implementation in routine monitoring is a recent development. This shift is now gaining momentum, as updated EU level regulations require companies to report the impacts of their activities on local biodiversity. Many of these industrial applications are related to coastal and offshore construction, shipping, and other prominent anthropogenic activities that affect not only marine mammals but the entire trophic chain. While implementing eDNA in biodiversity surveys is not compulsory, it is becoming a popular tool for private companies and governmental bodies due to its inherent capacity to capture a broad range of taxonomic groups using non-invasive sampling. However, the challenge is to provide a comprehensive and reliable dataset using eDNA methods from a dynamic environment. Here, we present some applications and scientific assessments that can implement eDNA detections. The inference includes biodiversity measures and indices to report on the status, complexity, and functionality of marine ecosystems (BioQ) and help us pinpoint the spatial origin of the detected genetic material (eRewind). We aim to provide a robust and holistic overview of the local marine ecosystem and the impact of anthropogenic activities, striving to aid transparent and regular biodiversity monitoring and reporting. 

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Presentation slides (pdf) | Presentation video

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Application of two novel qPCR assays for quantifying pinniped environmental DNA (eDNA) in coastal environments

Julia Clem

Abstract: Environmental DNA (eDNA) approaches offer minimally invasive methods for collecting genetic material from marine mammals. One such approach, quantitative PCR (qPCR), can be used to detect and quantify small amounts of genetic material from particular taxa of interest. Here, we present novel qPCR assays for gray and harbor seals and demonstrate their use in measuring pinniped eDNA in varied coastal environments in the Northeast United States. Gray seal eDNA was successfully captured and quantified around haul-out sites on Cape Cod, Massachusetts, where large groups of seals are present on sandy beaches or in shallow water. At these aggregations, gray seal eDNA could be detected up to 150 meters from shore, with a consistent detection rate among samples collected as far as 50 meters offshore. The impacts of distance from shore and seal abundance on eDNA concentration were evaluated with generalized linear models. Both distance and abundance were significant predictors of eDNA concentration, and explained approximately 73% of the observed deviance in eDNA concentration, although distance had a larger effect than seal abundance. To test the ability of eDNA sampling to detect seals in environments with fewer individuals and more dynamic transport processes, we also conducted water sampling in the Penobscot River, Maine, where small groups of seals are regularly observed in the water and on rocky outcroppings. In this system, eDNA detection via qPCR was less reliable; while both species were detected, our sampling often missed seals even when they were visually observed. By comparing detection success across these systems, we develop recommendations for sampling pinniped eDNA in dynamic coastal systems and evaluate the utility of these approaches for pinniped monitoring and management. 

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Presentation slides (pdf) | Presentation video

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Improving environmental DNA (eDNA) methods for marine mammal monitoring

Megan Shaffer

Abstract: Collection of environmental DNA (eDNA) offers a promising new approach to complement existing visual and acoustic methods for marine mammal monitoring, especially for enigmatic species that spend little time at the surface and/or vocalize infrequently. Here, we evaluate critical steps of the eDNA workflow to optimize the detection of marine mammals. We present results comparing methods of: (1) collection (filter pore size and volume filtered), (2) DNA preservation and extraction, and (3) metabarcoding primer choice. Leveraging a managed bottlenose dolphin population (Tursiops truncatus) in an open system, we quantitatively examined the effects of varying parameters for collection (1 versus 5 micron; 1 versus 3 L); preservation (desiccation, storage at -80ºC, Longmire's buffer, RNAlater, DNA/RNA Shield); and extraction (phenol-chloroform, Qiagen DNeasy Blood and Tissue Kit, Zymo Mini Prep Plus Kit), using a species-specific digital droplet PCR assay. Based on DNA copy yields, we found that while smaller pore sizes and increased sample volume led to the capture of more total DNA, they did not necessarily yield more target dolphin DNA. Different extraction methods also revealed differences in total DNA recovery, with larger amounts of total DNA in samples extracted with the phenol-chloroform method. Target dolphin DNA concentrations were instead similar among all preservation and extraction combinations. We then examined the relative performance of two common eDNA metabarcoding primers by sequencing over 200 samples collected at various depths (0-500 m) in 2019 along the US West Coast. We metabarcoded these samples using a general mitochondrial DNA (mtDNA) 12S marker that captures fish and marine mammals (MiFish) and a more marine mammal-specific marker for the mtDNA control region (D-loop), and found substantial differences in performance in terms of both sensitivity and species resolution. Together, these findings contribute to our understanding of how to maximize detections of rare cetacean targets using eDNA.

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Presentation slides (pdf) | Presentation video

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The efficacy of using salivary environmental DNA (eDNA) to detect cryptic manatees

Sarah Farinelli

Abstract: Environmental DNA (eDNA) is useful for detecting marine mammals in remote environments or with low population densities, but its effectiveness in aquatic ecosystems can be uncertain due to displacement, rapid degradation, and potential inhibition. We assessed the detection potential of salivary eDNA from manatee bite marks on floating and shoreline vegetation (n = 95), a novel method for manatees, and compared preservation methods using FTA® cards and Longmire’s buffer, testing incubation durations (10 seconds, 12 hours, until eDNA extraction). We validated the methodology by handfeeding lettuce to captive Florida manatees (Trichechus manatus latirostris) at Mote Marine Laboratory & Aquarium and collected water lettuce foraged by Amazonian manatees (T. inunguis) at the Rainforest Awareness, Rescue, and Education Center (RAREC) in Peru. For in-situ testing, we opportunistically collected vegetation likely foraged by African manatees (T. senegalensis) in Badagry and Lagos Lagoons, Nigeria, representing the second in-situ eDNA study for African manatees. Sites had similar median occurrence estimates (ψ = 0.70-0.71 [0.15-0.99]), validating both controlled and in-situ tests. Detection rates were highest at Mote (p = 0.96 [0.90-0.99]), with similar estimates at Badagry (p = 0.74 [0.54-0.87]), RAREC (p = 0.70 [0.56-0.83]), and Lagos (p = 0.65 [0.49-0.78]). Incubation in Longmire’s until eDNA extraction best preserved eDNA, yielding the highest median occurrence estimates (θ = 0.72-1.00) compared to FTA® cards (θ = 5.88x10-10-0.40) at all sites except Mote. In conclusion, salivary eDNA is an effective source for manatees that may improve detectability and spatial inferences, as demonstrated by Nigeria’s 70% occurrence estimates.

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Presentation slides (pdf) | Presentation video

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eDNA metabarcoding for estimating haplotype diversity and population differentiation of social odontocetes

Debbie Steel

Abstract: Environmental (e) DNA metabarcoding has potential for enhancing the estimation of mitochondrial and nuclear diversity from large groups of social odontocetes. Descriptions of genetic diversity and differentiation of these social groups have been dependent on a relatively small number of biopsy samples from any given encounter. In the case of species with high mtDNA diversity and group sizes of several hundred individuals, this is obviously inadequate. Here, we used droplet digital (dd)PCR to quantify eDNA, and Illumina MiSeq to sequence mtDNA haplotypes from seawater collected in the proximity of social odontocetes near Catalina Island, California and the Kailua-Kona coast, Hawai’i. By using a high-throughput sequencing platform, such as the Illumina MiSeq, and primers designed to target the variable region of the cetacean mtDNA control region, we can both identify species and resolve numerous haplotypes (i.e., Amplicon Sequence Variants, ASVs) from each species. For the Catalina Island dataset, a customized bioinformatic pipeline and database of reference sequences were developed for species identification and validation of haplotype identity. Two measures of diversity were calculated for each species: 1) Haplotype Richness, the simple number of unique ASVs and 2) the Effective Number of ASVs, the true measure of diversity, taking into account relative frequency of occurrence. Haplotype Richness and the Effective Number of haplotypes were noticeably higher for the two species of common dolphins and noticeably lower for the Risso’s and bottlenose dolphins, as expected given the population sizes of these species in the region. Preliminary analysis of the Hawai’i dataset shows detection of six of the seven targeted species as well as identification of two species (Indopacetus pacificus and Lagenodelphis hosei) not sighted during the surveys but known to occur in the region. Six water samples collected during one encounter with a mixed group of pilot and melon-headed whales highlight the variability in eDNA sampling and allow for comparison of eDNA differentiation with previously reported differentiation of two melon-headed whale populations from this region.

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Presentation slides (pdf) | Presentation video

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Biting into the Cook Inlet Beluga Buffet with eDNA and Acoustic Monitoring

Sonia Kumar

Abstract: Endangered Cook Inlet belugas (CIB, Delphinapterus leucas) reside year-round in Cook Inlet, Alaska; in 2022, the CIB population estimate was 331, a dramatic decrease from 1300 in the 1970s. Reduction of prey is listed as a threat to the population’s recovery and identifying changes in available prey is vital for management actions. The Kenai and Kasilof rivers have historically been important foraging habitat for CIB from April-November, but in recent years, beluga presence has been truncated to spring and fall. From May-September, these rivers are utilized by commercial, recreational, personal-use, and sport fishers, who primarily target Chinook (Oncorhynchus tshawytscha), sockeye (Oncorhynchus nerka), and coho (Oncorhynchus kisutch) salmon. From May-October of 2022, we used passive acoustic monitoring devices to track CIB presence in the Kenai and Kasilof rivers, while simultaneously collecting weekly water samples for environmental DNA (eDNA) metabarcoding to determine how the fish community composition changed throughout the ice-free season. Our results show that CIB were acoustically absent in the Kenai River from May-August, when there was a high relative proportion of eulachon (May-June) and sockeye salmon (June-August) DNA, despite the high nutritional value of these fish. CIB were acoustically present in the Kenai River from late August-October, when there was a high relative proportion of pink salmon DNA. CIB were acoustically absent in the Kasilof River, despite an abundance of sockeye salmon DNA. Fisheries interactions can drive shifts in the synchrony of predator-prey relationships, and our results suggest that CIB may be displaced from their historic foraging grounds.

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Presentation slides (pdf) | Presentation video

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eDNA Monitoring of Vaquita

Tania Valdivia

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Abstract: TBD

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Presentation slides (pdf) | Presentation video

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CETOPIPE: towards a user-friendly interface for the analysis of marine mammals' eDNA traces using a metabarcoding approach

Alessia Rota

Abstract: The detection of genetic material left by organisms in the environment (eDNA) has rapidly grown as a tool for monitoring marine biodiversity. The possibility to simultaneously identify multiple taxa within a single seawater sample allows to study ecological networks within biological communities. For instance, vertebrate-specific metabarcoding primers consent to detect the presence of cetaceans in association to the qualitative/semi-quantitative distribution of their main prey items (fish). This approach has great potential of applications in the field of marine mammalogy. Yet, a couple of drawbacks still need to be addressed. Firstly, the fact that, when using vertebrate-specific primers, species detection may be biased towards more abundant taxa (e.g. bony fish in the marine environment), to the detriment of the rarer ones (e.g. marine mammals). Secondly there are concerns regarding the bioinformatic analysis: up today a reference database dedicated to marine mammals is missing.  Therefore the procedure of "translating" HTS (High Throughput Sequencing) data into a list of observed species per each sample may not be a trivial process for the ever-increasing number of cetologists due to lack of comprehensive molecular and bioinformatic background. We have been working on the latter aspect. Here we present a novel pipeline, called CETOPIPE, tailored for eDNA projects focusing on marine mammals, developed to run on a user-friendly online platform. Indeed, it can be run as a stand-alone pipeline or through a GALAXY instance on the ELIXIR-IT computation infrastructure, making its usage easy and accessible to cetologists. 

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Presentation slides (pdf) | Presentation video

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New insights into the maternal population structure of Hector's dolphin (Cephalorhynchus hectori) revealed using environmental DNA

Steph Bennington

Abstract: Environmental DNA (eDNA) is frequently used for detecting species and describing biodiversity through metabarcoding techniques. More recently, there has been emerging evidence that eDNA can be used to investigate intraspecific variability, providing novel pathways to explore population genetics questions. However, it can be difficult to distinguish between true intraspecific variation and PCR/sequence error, and the presence of DNA from multiple individuals makes using traditional frequency- based approaches challenging. Here, we explore how eDNA can be used to investigate population structure of Hector's dolphin (Cephalorhynchus hectori), an endemic, endangered, and culturally important (taonga) species. In doing so, we present a simple and effective method to filter out noise due to PCR/sequence error and show how treating haplotype detections equally can pro- vide similar results to frequency-based approaches from traditional sampling methods. Over the 2022/23 Austral summer, we collected 85 water samples close to Hector's dolphins, and three negative controls, across three areas on the east coast of Aotearoa New Zealand's South Island: Banks Peninsula (n = 41), Timaru (n = 33), and Dunedin (n = 14). We targeted a 348 bp region of the cetacean D-loop in the mitochondrial DNA (mtDNA) and obtained positive detections in 68 (77%) water samples, confidently identifying seven haplotypes across the study area. The occurrence of specific haplotypes and the overall frequencies in Banks Peninsula and Timaru matched well with previous tissue-based studies and were similar to other East Coast South Island (ECSI) subpopulations. In Dunedin, however, our results indicate a closer relationship to South Coast populations, suggesting that the membership within the ECSI population be reconsidered, which has implications for how this subpopulation is managed. We show that eDNA sampling can be used to elucidate matrilineal population structure for Hector's dolphin and provide a simple method that could be applied to other eDNA-based studies of any taxa.

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Presentation slides (pdf)