No abstract

Metabarcoding of environmental DNA (eDNA) when coupled with high throughput sequencing is revolutionising the way biodiversity can be monitored across a wide range of applications. However, the large number of tools deployed in downstream bioinformatic analyses often places a challenge in configuration and maintenance of a workflow, and consequently limits the research reproducibility. Furthermore, scalability needs to be considered to handle the growing amount of data due to increase in sequence output and the scale of project. Here, we describe eDNAFlow, a fully automated workflow that employs a number of state-of-the-art applications to process eDNA data from raw sequences (single-end or paired-end) to generation of curated and noncurated zero-radius operational taxonomic units (ZOTUs) and their abundance tables. This pipeline is based on Nextflow and Singularity which enable a scalable, portable and reproducible workflow using software containers on a local computer, clouds and high-performance computing (HPC) clusters. Finally, we present an in-house Python script to assign taxonomy to ZOTUs based on user specified thresholds for assigning lowest common ancestor (LCA). We demonstrate the utility and efficiency of the pipeline using an example of a published coral diversity biomonitoring study. Our results were congruent with the aforementioned study. The scalability of the pipeline is also demonstrated through analysis of a large data set containing 154 samples. To our knowledge, this is the first automated bioinformatic pipeline for eDNA analysis using two powerful tools: Nextflow and Singularity. This pipeline addresses two major challenges in the analysis of eDNA data; scalability and reproducibility.

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The effective management of rare and threatened species, especially in areas where population sizes have diminished, relies on knowledge of their population size, threats, and distribution. Robust mapping of distribution presents a particular challenge in aquatic environments for cryptic species, especially those with low abundance.Environmental DNA (eDNA) approaches can offer improved detection rates of many rare and threatened species when compared with traditional sampling approaches.In this study, we developed and optimized a targeted eDNA assay for the critically endangered estuarine pipefish (Syngnathus watermeyeri). eDNA sampling and seine netting were undertaken at 39 sites across the historical range of S. watermeyeri in the Eastern Cape of South Africa in 2019. At each site, five water samples were collected for eDNA analysis (n = 195) along with three seine netting hauls (n = 117). Habitat and environmental data were collected at each location to explore what physical and biotic parameters might correlate with pipefish presence/absence. We successfully detected S. watermeyeri in two estuaries (Kariega and Bushmans) using both survey methods. Importantly, the positive detection rate of eDNA (66.7%) was four times that of seine netting (16.7%), highlighting the value of eDNA as a monitoring tool for rare and cryptic species. Null detections in the Kasouga, East Kleinemonde, and West | 133 NESTER et al.

Antarctic krill (Euphausia superba) is a keystone species in the Southern Ocean ecosystem, and monitoring its distribution and abundance is crucial for the sustainable management of expanding fisheries targeting the species. Environmental DNA (eDNA)‐based monitoring could complement conventional krill surveys, but its applicability is limited by a lack of knowledge on eDNA persistence and decay in the Southern Ocean. We aimed to develop a method that can not only quantify Antarctic krill eDNA, but also estimate a relative time since this eDNA was shed (“recent” vs “older”). Three species‐specific qPCR markers targeting the mitochondrial 16S region were developed, and the eDNA decay characteristics of these markers were determined through tank experiments. Krill eDNA was partially degraded in all samples, even when krill were present. Marker concentrations decreased exponentially at similar rates after krill removal, with initial relative abundances maintained across the three markers. Over time, the concentration of the longest marker decreased faster, changing the relative abundances of the markers, and allowing discrimination of more recent samples from more degraded older samples. We employed this new method to quantify Antarctic krill eDNA collected across a 4800 km Southern Ocean transect, and estimated the age of the eDNA in these samples based on the relative abundance of markers, adding a temporal aspect to a quantitative eDNA survey. We also compared a Euphausiid‐specific metabarcoding marker to the qPCR method to assess sensitivity in detecting Antarctic krill eDNA. While these new eDNA methods should be evaluated against existing non‐molecular survey methods, they could add an important novel, dynamic layer of information to future krill surveys. Our method could not only determine where Antarctic krill eDNA is present but shed light on how they may be using certain habitats, expanding our understanding of this important species’ life cycle and contributing to more accurate abundance and distribution estimates.