Aquatic environmental DNA: A review of the macro-organismal biomonitoring revolution.

Environmental DNA (eDNA) is the fastest growing biomonitoring tool fuelled by two key features: time efficiency and sensitivity. Technological advancements allow rapid biodiversity detection at both species and community levels with increasing accuracy. Concurrently, there has been a global demand to standardise eDNA methods, but this is only possible with an in-depth overview of the technological advancements and a discussion of the pros and cons of available methods. We therefore conducted a systematic literature review of 407 peer-reviewed papers on aquatic eDNA published between 2012 and 2021. We observed a gradual increase in the annual number of publications from four (2012) to 28 (2018), followed by a rapid growth to 124 publications in 2021. This was mirrored by a tremendous diversification of methods in all aspects of the eDNA workflow. For example, in 2012 only freezing was applied to preserve filter samples, whereas we recorded 12 different preservation methods in the 2021 literature. Despite an ongoing standardisation debate in the eDNA community, the field is seemingly moving fast in the opposite direction and we discuss the reasons and implications. Moreover, by compiling the largest PCR-primer database to date, we provide information on 522 and 141 published species-specific and metabarcoding primers targeting a wide range of aquatic organisms. This works as a user-friendly 'distillation' of primer information that was hitherto scattered across hundreds of papers, but the list also reflects which taxa are commonly studied with eDNA technology in aquatic environments such as fish and amphibians, and reveals that groups such as corals, plankton and algae are under-studied. Efforts to improve sampling and extraction methods, primer specificity and reference databases are crucial to capture these ecologically important taxa in future eDNA biomonitoring surveys. In a rapidly diversifying field, this review synthetises aquatic eDNA procedures and can guide eDNA users towards best practice.

Comparison of materials for rapid passive collection of environmental DNA.

Passive collection is an emerging sampling method for environmental DNA (eDNA) in aquatic systems. Passive eDNA collection is inexpensive and efficient, and requires minimal equipment, making it suited to high-density sampling and remote deployment. Here, we compare the effectiveness of nine membrane materials for passively collecting fish eDNA from a 3-million-litre marine mesocosm. We submerged materials (cellulose, cellulose with 1% and 3% chitosan, cellulose overlayed with electrospun nanofibres and 1% chitosan, cotton fibres, hemp fibres, and sponge with either zeolite or active carbon) for intervals between 5 and 1080 min. We show that for most materials, with as little as 5 min of submersion, mitochondrial fish eDNA measured with qPCR, and fish species richness measured with metabarcoding, was comparable to that collected by conventional filtering. Furthermore, PCR template DNA concentrations and species richness were generally not improved significantly by longer submersion. Species richness detected for all materials ranged between 11 and 37 species, with a median of 27, which was comparable to the range for filtered eDNA (19-32). Using scanning electron microscopy, we visualized biological matter adhering to the surface of materials, rather than entrapped, with images also revealing a diversity in size and structure of putative eDNA particles. eDNA can be collected rapidly from seawater with a passive approach and using a variety of materials. This will suit cost- and time-sensitive biological surveys, and where access to equipment is limited.

Passive eDNA collection enhances aquatic biodiversity analysis.

Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach for species detection and biodiversity assessment. The most common eDNA collection method in aquatic systems is actively filtering water through a membrane, which is time consuming and requires specialized equipment. Ecological studies investigating species abundance or distribution often require more samples than can be practically collected with current filtration methods. Here we demonstrate how eDNA can be passively collected in both tropical and temperate marine systems by directly submerging filter membranes (positively charged nylon and non-charged cellulose ester) in the water column. Using a universal fish metabarcoding assay, we show that passive eDNA collection can detect fish as effectively as active eDNA filtration methods in temperate systems and can also provide similar estimates of total fish biodiversity. Furthermore, passive eDNA collection enables greater levels of biological sampling, which increases the range of ecological questions that eDNA metabarcoding can address.

Zone specific trends in coral cover, genera and growth-forms in the World-Heritage listed Ningaloo Reef.

On coral reefs, changes in the cover and relative abundance of hard coral taxa often follow disturbance. Although the ecological responses of common coral taxa have been well documented, little is known about the ecological responses of uncommon coral taxa or of coral morphological groups across multiple adjacent reef zones. We used Multivariate Auto-Regressive State-Space modelling to assess the rate and direction of change of hard coral cover across a variety of coral genera, growth forms, and susceptibility to bleaching and physical damage covering multiple reef zones at northern Ningaloo Reef in Western Australia. Trends were assessed between 2007 and 2016, during which multiple episodic disturbances occurred including cyclones and a heatwave. We provide evidence of zone specific trends, not only in total hard coral cover, but also in taxonomic and morphological groups of corals at Ningaloo Reef. Declines in total coral cover on the reef flat corresponded with declines in fast growing corals, particularly Acropora. In contrast, total coral cover on the reef slope and inshore (lagoon) did not undergo significant change, despite divergent trajectories of individual genera. Importantly, we also show that changes in the composition of coral assemblages can be detected using a morphological based approach when changes are not evident using a taxonomic approach. Therefore, we recommend that future assessments of coral reef trends incorporate not just standard metrics such as total coral cover, but also metrics that provide for detailed descriptions of trends in common and uncommon taxa and morphological groups across multiple reef zones.

Too hot to handle: Unprecedented seagrass death driven by marine heatwave in a World Heritage Area.

The increased occurrence of extreme climate events, such as marine heatwaves (MHWs), has resulted in substantial ecological impacts worldwide. To date, metrics of thermal stress within marine systems have focussed on coral communities, and less is known about measuring stress relevant to other primary producers, such as seagrasses. An extreme MHW occurred across the Western Australian coastline in the austral summer of 2010-2011, exposing marine communities to summer seawater temperatures 2-5°C warmer than average. Using a combination of satellite imagery and in situ assessments, we provide detailed maps of seagrass coverage across the entire Shark Bay World Heritage Area (ca. 13,000 km2 ) before (2002 and 2010) and after the MHW (2014 and 2016). Our temporal analysis of these maps documents the single largest loss in dense seagrass extent globally (1,310 km2 ) following an acute disturbance. Total change in seagrass extent was spatially heterogeneous, with the most extensive declines occurring in the Western Gulf, Wooramel Bank and Faure Sill. Spatial variation in seagrass loss was best explained by a model that included an interaction between two heat stress metrics, the most substantial loss occurring when degree heating weeks (DHWm) was ≥10 and the number of days exposed to extreme sea surface temperature during the MHW (DaysOver) was ≥94. Ground truthing at 622 points indicated that change in seagrass cover was predominantly due to loss of Amphibolis antarctica rather than Posidonia australis, the other prominent seagrass at Shark Bay. As seawater temperatures continue to rise and the incidence of MHWs increase globally, this work will provide a basis for identifying areas of meadow degradation, or stability and recovery, and potential areas of resilience.

DNA metabarcoding assays reveal a diverse prey assemblage for Mobula rays in the Bohol Sea, Philippines.

Diet studies provide base understanding of trophic structure and are a valuable initial step for many fields of marine ecology, including conservation and fisheries biology. Considerable complexity in marine trophic structure can exist due to the presence of highly mobile species with long life spans. Mobula rays are highly mobile, large, planktivorous elasmobranchs that are frequently caught either directly or as bycatch in fisheries, which, combined with their conservative life history strategy, makes their populations susceptible to decline in intensely fished regions. Effective management of these iconic and vulnerable species requires an understanding of the diets that sustain them, which can be difficult to determine using conventional sampling methods. We use three DNA metabarcode assays to identify 44 distinct taxa from the stomachs (n = 101) of four sympatric Mobula ray species (Mobula birostris, Mobula tarapacana, Mobula japanica, and Mobula thurstoni) caught over 3 years (2013-2015) in a direct fishery off Bohol in the Philippines. The diversity and incidence of bony fishes observed in ray diets were unprecedented. Nevertheless, rays showed dietary overlap, with krill (Euphausia) dominating their diet. Our results provide a more detailed assessment of sympatric ray diets than was previously described and reveal the complexity that can exist in food webs at critical foraging habitats.

Drying method has no substantial effect on δ(15)N or δ(13)C values of muscle tissue from teleost fishes.

RATIONALE: Stable isotope analysis (SIA) is a powerful tool in many fields of research that enables quantitative comparisons among studies, if similar methods have been used. The goal of this study was to determine if three different drying methods commonly used to prepare samples for SIA yielded different δ(15)N and δ(13)C values. METHODS: Muscle subsamples from 10 individuals each of three teleost species were dried using three methods: (i) oven, (ii) food dehydrator, and (iii) freeze-dryer. All subsamples were analysed for δ(15)N and δ(13)C values, and nitrogen and carbon content, using a continuous flow system consisting of a Delta V Plus mass spectrometer and a Flush 1112 elemental analyser via a Conflo IV universal interface. The δ(13)C values were normalized to constant lipid content using the equations proposed by McConnaughey and McRoy. RESULTS: Although statistically significant, the differences in δ(15)N values between the drying methods were small (mean differences ≤0.21‰). The differences in δ(13)C values between the drying methods were not statistically significant, and normalising the δ(13)C values to constant lipid content reduced the mean differences for all treatments to ≤0.65‰. A statistically significant difference of ~2% in C content existed between tissues dried in a food dehydrator and those dried in a freeze-dryer for two fish species. There was no significant effect of fish size on the differences between methods. CONCLUSIONS: No substantial effect of drying method was found on the δ(15)N or δ(13)C values of teleost muscle tissue.