A large scale temporal and spatial environmental DNA biodiversity survey of marine vertebrates in Brazil following the Fundão tailings dam failure.

Seawater contains a wealth of genetic information, representing the biodiversity of numerous species residing within a particular marine habitat. Environmental DNA (eDNA) metabarcoding offers a cost effective, non-destructive method for large scale monitoring of environments, as diverse taxonomic groups are detected using metabarcoding assays. A large-scale eDNA monitoring program of marine vertebrates was conducted across three sampling seasons (Spring 2018, Autumn 2019; Spring 2019) in coastal waters of Brazil. The program was designed to investigate eDNA as a testing method for long term monitoring of marine vertebrates following the Fundão tailings dam failure in November 2015. While no baseline samples were available prior to the dam failure there is still value in profiling the taxa that use the impacted area and the trajectory of recovery. A total of 40 sites were sampled around the mouths of eight river systems, covering approximately 500 km of coastline. Metabarcoding assays targeting the mitochondrial genes 16S rRNA and COI were used to detect fish, marine mammals and elasmobranchs. We detected temporal differences between seasons and spatial differences between rivers/estuaries sampled. Overall, the largest eDNA survey in Brazil to date revealed 69 families from Class Actinopterygii (fish), 15 species from Class Chondrichthyes (sharks and rays), 4 species of marine and estuarine mammals and 23 species of conservation significance including 2 species of endangered dolphin. Our large-scale study reinforces the value eDNA metabarcoding can bring when monitoring the biodiversity of coastal environments and demonstrates the importance of collection of time-stamped environmental samples to better understand the impacts of anthropogenic activities.

Marine environmental DNA biomonitoring reveals seasonal patterns in biodiversity and identifies ecosystem responses to anomalous climatic events.

Marine ecosystems are changing rapidly as the oceans warm and become more acidic. The physical factors and the changes to ocean chemistry that they drive can all be measured with great precision. Changes in the biological composition of communities in different ocean regions are far more challenging to measure because most biological monitoring methods focus on a limited taxonomic or size range. Environmental DNA (eDNA) analysis has the potential to solve this problem in biological oceanography, as it is capable of identifying a huge phylogenetic range of organisms to species level. Here we develop and apply a novel multi-gene molecular toolkit to eDNA isolated from bulk plankton samples collected over a five-year period from a single site. This temporal scale and level of detail is unprecedented in eDNA studies. We identified consistent seasonal assemblages of zooplankton species, which demonstrates the ability of our toolkit to audit community composition. We were also able to detect clear departures from the regular seasonal patterns that occurred during an extreme marine heatwave. The integration of eDNA analyses with existing biotic and abiotic surveys delivers a powerful new long-term approach to monitoring the health of our world's oceans in the context of a rapidly changing climate.

Ecosystem biomonitoring with eDNA: metabarcoding across the tree of life in a tropical marine environment.

Effective marine management requires comprehensive data on the status of marine biodiversity. However, efficient methods that can document biodiversity in our oceans are currently lacking. Environmental DNA (eDNA) sourced from seawater offers a new avenue for investigating the biota in marine ecosystems. Here, we investigated the potential of eDNA to inform on the breadth of biodiversity present in a tropical marine environment. Directly sequencing eDNA from seawater using a shotgun approach resulted in only 0.34% of 22.3 million reads assigning to eukaryotes, highlighting the inefficiency of this method for assessing eukaryotic diversity. In contrast, using 'tree of life' (ToL) metabarcoding and 20-fold fewer sequencing reads, we could detect 287 families across the major divisions of eukaryotes. Our data also show that the best performing 'universal' PCR assay recovered only 44% of the eukaryotes identified across all assays, highlighting the need for multiple metabarcoding assays to catalogue biodiversity. Lastly, focusing on the fish genus Lethrinus, we recovered intra- and inter-specific haplotypes from seawater samples, illustrating that eDNA can be used to explore diversity beyond taxon identifications. Given the sensitivity and low cost of eDNA metabarcoding we advocate this approach be rapidly integrated into biomonitoring programs.

DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea).

The analysis of apex predator diet has the ability to deliver valuable insights into ecosystem health, and the potential impacts a predator might have on commercially relevant species. The Australian sea lion (Neophoca cinerea) is an endemic apex predator and one of the world's most endangered pinnipeds. Given that prey availability is vital to the survival of top predators, this study set out to understand what dietary information DNA metabarcoding could yield from 36 sea lion scats collected across 1,500 km of its distribution in southwest Western Australia. A combination of PCR assays were designed to target a variety of potential sea lion prey, including mammals, fish, crustaceans, cephalopods, and birds. Over 1.2 million metabarcodes identified six classes from three phyla, together representing over 80 taxa. The results confirm that the Australian sea lion is a wide-ranging opportunistic predator that consumes an array of mainly demersal fauna. Further, the important commercial species Sepioteuthis australis (southern calamari squid) and Panulirus cygnus (western rock lobster) were detected, but were present in <25% of samples. Some of the taxa identified, such as fish, sharks and rays, clarify previous knowledge of sea lion prey, and some, such as eel taxa and two gastropod species, represent new dietary insights. Even with modest sample sizes, a spatial analysis of taxa and operational taxonomic units found within the scat shows significant differences in diet between many of the sample locations and identifies the primary taxa that are driving this variance. This study provides new insights into the diet of this endangered predator and confirms the efficacy of DNA metabarcoding of scat as a noninvasive tool to more broadly define regional biodiversity.

Real-time PCR detection of Didemnum perlucidum (Monniot, 1983) and Didemnum vexillum (Kott, 2002) in an applied routine marine biosecurity context.

Prevention and early detection are well recognized as the best strategies for minimizing the risks posed by nonindigenous species (NIS) that have the potential to become marine pests. Central to this is the ability to rapidly and accurately identify the presence of NIS, often from complex environmental samples like biofouling and ballast water. Molecular tools have been increasingly applied to assist with the identification of NIS and can prove particularly useful for taxonomically difficult groups like ascidians. In this study, we have developed real-time PCR assays suited to the specific identification of the ascidians Didemnum perlucidum and Didemnum vexillum. Despite being recognized as important global pests, this is the first time specific molecular detection methods have been developed that can support the early identification and detection of these species from a broad range of environmental sample types. These fast, robust and high-throughput assays represent powerful tools for routine marine biosecurity surveillance, as detection and confirmation of the early presence of species could assist in the timely establishment of emergency responses and control strategies. This study applied the developed assays to confirm the ability to detect Didemnid eDNA in water samples. While previous work has focused on detection of marine larvae from water samples, the development of real-time PCR assays specifically aimed at detecting eDNA of sessile invertebrate species in the marine environment represents a world first and a significant step forwards in applied marine biosecurity surveillance. Demonstrated success in the detection of D. perlucidum eDNA from water samples at sites where it could not be visually identified suggests value in incorporating such assays into biosecurity survey designs targeting Didemnid species.