Population genetic data is often essential to inform conservation management. Understanding the distribution of genetic variants within and between populations can reveal novel insights into genetic connectivity and evolutionary processes. However, obtaining such data using invasive approaches such as tissue sampling may negatively affect the very species we are seeking to protect. Thus, interest in using non-invasive environmental DNA (eDNA) techniques for identifying genetic variation within target species populations has grown. Along with this interest comes the desire to expand the amount of population genetic information that can be obtained from eDNA to increasingly large fragments of the genome, such as entire mitogenomes. Here, we introduce an eDNA hybridisation capture approach to sequencing complete mitochondrial genomes of New Zealand fur seals (Arctocephalus forsteri) (Māori: kekeno) from marine water samples. We show that our approach can recover up to 99% of the fur seal mitogenome. Furthermore, we present a pipeline to extract haplotype diversity from such eDNA population genetic data. Haplotypic variation identified using this approach matches previously identified patterns of intraspecific genetic variation from fur seal tissue samples, suggesting that eDNA methods can accurately identify mitochondrial variation. Our study demonstrates that whole mitogenomes can be recovered using hybridisation capture enrichment of eDNA and indicates that eDNA may be a promising tool for population genetics. Within this context, we discuss some of the key challenges that must be overcome before the promise of eDNA can be fully realized.

Escalating concern regarding the impacts of reduced genetic diversity on the conservation of endangered species has spurred efforts to obtain chromosome-level genomes through consortia such as the Vertebrate Genomes Project. However, assembling reference genomes for many threatened species remains challenging due to difficulties obtaining optimal input samples (e.g., fresh tissue, cell lines) that can characterize long-term conservation collections. Here, we present a pipeline that leverages genome synteny to construct high-quality genomes for species of conservation concern despite less-than-optimal samples and/or sequencing data, demonstrating its use on Hector’s and Māui dolphins. These endemic New Zealand dolphins are threatened by human activities due to their coastal habitat and small population sizes. Hector’s dolphins are classified as endangered by the IUCN, while the Māui dolphin is among the most critically endangered marine mammals. To assemble reference genomes for these dolphins, we created a pipeline combining de novo assembly tools with reference-guided techniques, utilizing chromosome-level genomes of closely related species. The pipeline assembled highly contiguous chromosome-level genomes (scaffold N50: 110 MB, scaffold L50: 9, miniBUSCO completeness scores >96.35%), despite non-optimal input tissue samples. We demonstrate that these genomes can provide insights relevant for conservation, including historical demography revealing long-term small population sizes, with subspecies divergence occurring ~20 kya, potentially linked to the Last Glacial Maximum. Māui dolphin heterozygosity was 40% lower than Hector’s and comparable to other cetacean species noted for reduced genetic diversity. Through these exemplar genomes, we demonstrate that our pipeline can provide high-quality genomic resources to facilitate ongoing conservation genomics research.

Marine sponges have recently emerged as efficient natural environmental DNA (eDNA) samplers. The ability of sponges to accumulate eDNA provides an exciting opportunity to reconstruct contemporary communities and ecosystems with high temporal and spatial precision. However, the use of historical eDNA (heDNA), trapped within the vast number of specimens stored in scientific collections, opens up the opportunity to begin to reconstruct the communities and ecosystems of the past. Here, using a variety of Antarctic sponge specimens stored in an extensive marine invertebrate collection, we were able to recover information on Antarctic fish biodiversity from specimens up to 20 years old. We successfully recovered 64 fish heDNA signals from 27 sponge specimens. Alpha diversity measures did not differ among preservation methods, but sponges stored frozen had a significantly different fish community composition compared to those stored dry or in ethanol. Our results show that we were consistently and reliably able to extract the heDNA trapped within marine sponge specimens, thereby enabling the reconstruction and investigation of communities and ecosystems of the recent past with a spatial and temporal resolution previously unattainable. Future research into heDNA extraction from other preservation methods, as well as the impact of specimen age and collection method will strengthen and expand the opportunities for this novel resource to access new knowledge on ecological change during the last century.

Molecular biosecurity surveillance programs increasingly use environmental DNA (eDNA) for detecting marine non-indigenous species (NIS). However, the current molecular detection workflow is cumbersome, prone to errors and delays, and is limited in providing knowledge about eDNA beyond the spatial and temporal extent of the sampling. These limitations can hinder management efforts and restrict the “opportunity window” for a rapid response to new marine NIS incursions. Emerging innovative field-deployable digital droplet PCR (ddPCR) systems offer improved workflow efficiency by autonomously analyzing targeted free-floating extra-cellular eDNA (free-eDNA) signals. Despite their potential, these systems have not been tested in marine environments. Thus, an aquarium study was conducted with three distinct marine NIS: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analyzing aquarium water samples using an optimized species-specific ddPCR assay, without filtration or DNA extraction, so-called, “direct-ddPCR”. The results demonstrated the consistent detection of Sabella spallanzanii and Bugula neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 hours. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. These results are critical for implementing in-situ nucleic acid automated continuous sensing systems for marine biosurveillance, enabling point-of-need detection and rapid management response to biosecurity threats.

The measurement of biodiversity is an integral aspect of life science research. With the establishment of second- and third-generation sequencing technologies, an increasing amount of metabarcoding data is being generated as we seek to describe the extent and patterns of biodiversity in multiple contexts. The reliability and accuracy of taxonomically assigning metabarcoding sequencing data has been shown to be critically influenced by the quality and completeness of reference databases. Custom, curated, eukaryotic reference databases, however, are scarce, as are the software programs for generating them. Here, we present CRABS (Creating Reference databases for Amplicon-Based Sequencing), a software package to create custom reference databases for metabarcoding studies. CRABS includes tools to download sequences from multiple online repositories (i.e., NCBI, BOLD, EMBL, MitoFish), retrieve amplicon regions through in silico PCR analysis and pairwise global alignments, curate the database through multiple filtering parameters (e.g., dereplication, sequence length, sequence quality, unresolved taxonomy), export the reference database in multiple formats for the immediate use in taxonomy assignment software, and investigate the reference database through implemented visualizations for diversity, primer efficiency, reference sequence length, and taxonomic resolution. CRABS is a versatile tool for generating curated reference databases of user-specified genetic markers to aid taxonomy assignment from metabarcoding sequencing data. CRABS is available for download as a conda package and via GitHub (https://github.com/gjeunen/reference_database_creator).

Aquatic environmental DNA (eDNA) surveys have emerged as an alternative method for monitoring complex and vast marine ecosystems. One-to-one comparisons between existing survey techniques and eDNA approaches are essential to determine biases associated with this novel methodology. To date, such direct comparative studies have been scarce in the context of marine eDNA surveys. In this study, we conducted simultaneous baited remote underwater video (BRUV) and eDNA surveys to describe the fish community in Paterson Inlet, Stewart Island/Rakiura, New Zealand. BRUV detected three distinct families of bony fish (Actinopterygii) and four families of cartilaginous fish (Chondrichthyes). Three different eDNA assays, detected 32 (MiFish-U), 42 (MiFish-E), and 23 (16S-Fish) families, spanning the classes of Actinopterygii, Chondrichthyes, Hyperoartia, Mammalia, and Aves. Our direct comparison identified the need for (i) increased sampling, (ii) spatial pooling, and (iii) multiple targeted eDNA assays, to achieve similar detection rates of a given species in eDNA and BRUV monitoring. Diversity, ordination, and indicator species analyses identified distinct eDNA signals between different habitats in our relatively small sampling area, showcasing the high spatial resolution of eDNA approaches in marine habitats. Our results provide valuable insights into the potential biases associated with eDNA monitoring, as well as highlight the power of eDNA for detecting a broad range of taxa beyond traditional observational approaches, including terrestrial, invasive and migratory organisms.