Environmental DNA recovers fish composition turnover of the coral reefs of West Indian Ocean islands.

Islands have been used as model systems to study ecological and evolutionary processes, and they provide an ideal set-up for validating new biodiversity monitoring methods. The application of environmental DNA metabarcoding for monitoring marine biodiversity requires an understanding of the spatial scale of the eDNA signal, which is best tested in island systems. Here, we investigated the variation in Actinopterygii and Elasmobranchii species composition recovered from eDNA metabarcoding along a gradient of distance-to-reef in four of the five French Scattered Islands in the Western Indian Ocean. We collected surface water samples at an increasing distance from reefs (0 m, 250 m, 500 m, 750 m). We used a metabarcoding protocol based on the 'teleo' primers to target marine reef fishes and classified taxa according to their habitat types (benthic or pelagic). We investigated the effect of distance-to-reef on ß diversity variation using generalised linear mixed models and estimated species-specific distance-to-reef effects using a model-based approach for community data. Environmental DNA metabarcoding analyses recovered distinct fish species compositions across the four inventoried islands and variations along the distance-to-reef gradient. The analysis of ß-diversity variation showed significant taxa turnover between the eDNA samples on and away from the reefs. In agreement with a spatially localised signal from eDNA, benthic species were distributed closer to the reef than pelagic ones. Our findings demonstrate that the combination of eDNA inventories and spatial modelling can provide insights into species habitat preferences related to distance-to-reef gradients at a small scale. As such, eDNA can not only recover large compositional differences among islands but also help understand habitat selection and distribution of marine species at a finer spatial scale.

Optimizing detectability of the endangered fan mussel using eDNA and ddPCR.

Spatial and temporal monitoring of species threatened with extinction is of critical importance for conservation and ecosystem management. In the Mediterranean coast, the fan mussel (Pinna nobilis) is listed as critically endangered after suffering from a mass mortality event since 2016, leading to 100% mortality in most marine populations. Conventional monitoring for this macroinvertebrate is done using scuba, which is challenging in dense meadows or with low visibility. Here we developed an environmental DNA assay targeting the fan mussel and assessed the influence of several environmental parameters on the species detectability in situ. We developed and tested an eDNA molecular marker and collected 48 water samples in two sites at the Thau lagoon (France) with distinct fan mussel density, depths and during two seasons (summer and autumn). Our marker can amplify fan mussel DNA but lacks specificity since it also amplifies a conspecific species (Pinna rudis). We successfully amplified fan mussel DNA from in situ samples with 46 positive samples (out of 48) using ddPCR, although the DNA concentrations measured were low over almost all samples. Deeper sampling depth slightly increased DNA concentrations, but no seasonal effect was found. We highlight a putative spawning event on a single summer day with much higher DNA concentration compared to all other samples. We present an eDNA molecular assay able to detect the endangered fan mussel and provide guidelines to optimize the sampling protocol to maximize detectability. Effective and non-invasive monitoring tools for endangered species are promising to monitor remaining populations and have the potential of ecological restoration or habitat recolonization following a mass mortality event.

The global depth range of marine fishes and their genetic coverage for environmental DNA metabarcoding.

The bathymetric and geographical distribution of marine species represent a key information in biodiversity conservation. Yet, deep-sea ecosystems are among the least explored on Earth and are increasingly impacted by human activities. Environmental DNA (eDNA) metabarcoding has emerged as a promising method to study fish biodiversity but applications to the deep-sea are still scarce. A major limitation in the application of eDNA metabarcoding is the incompleteness of species sequences available in public genetic databases which reduces the extent of detected species. This incompleteness by depth is still unknown. Here, we built the global bathymetric and geographical distribution of 10,826 actinopterygian and 960 chondrichthyan fish species. We assessed their genetic coverage by depth and by ocean for three main metabarcoding markers used in the literature: teleo and MiFish-U/E. We also estimated the number of primer mismatches per species amplified by in silico polymerase chain reaction which influence the probability of species detection. Actinopterygians show a stronger decrease in species richness with depth than Chondrichthyans. These richness gradients are accompanied by a continuous species turnover between depths. Fish species coverage with the MiFish-U/E markers is higher than with teleo while threatened species are more sequenced than the others. "Deep-endemic" species, those not ascending to the shallow depth layer, are less sequenced than not threatened species. The number of primer mismatches is not higher for deep-sea species than for shallower ones. eDNA metabarcoding is promising for species detection in the deep-sea to better account for the 3-dimensional structure of the ocean in marine biodiversity monitoring and conservation. However, we argue that sequencing efforts on "deep-endemic" species are needed.

Cross-ocean patterns and processes in fish biodiversity on coral reefs through the lens of eDNA metabarcoding.

Increasing speed and magnitude of global change threaten the world's biodiversity and particularly coral reef fishes. A better understanding of large-scale patterns and processes on coral reefs is essential to prevent fish biodiversity decline but it requires new monitoring approaches. Here, we use environmental DNA metabarcoding to reconstruct well-known patterns of fish biodiversity on coral reefs and uncover hidden patterns on these highly diverse and threatened ecosystems. We analysed 226 environmental DNA (eDNA) seawater samples from 100 stations in five tropical regions (Caribbean, Central and Southwest Pacific, Coral Triangle and Western Indian Ocean) and compared those to 2047 underwater visual censuses from the Reef Life Survey in 1224 stations. Environmental DNA reveals a higher (16%) fish biodiversity, with 2650 taxa, and 25% more families than underwater visual surveys. By identifying more pelagic, reef-associated and crypto-benthic species, eDNA offers a fresh view on assembly rules across spatial scales. Nevertheless, the reef life survey identified more species than eDNA in 47 shared families, which can be due to incomplete sequence assignment, possibly combined with incomplete detection in the environment, for some species. Combining eDNA metabarcoding and extensive visual census offers novel insights on the spatial organization of the richest marine ecosystems.

How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?

Quantifying fish species diversity in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, because eDNA concentration is low in marine environments, the reliability of eDNA to detect species diversity can be limited. Using an eDNA metabarcoding approach to identify fish Molecular Taxonomic Units (MOTUs) with a single 12S marker, we aimed to assess how the number of sampling replicates and filtered water volume affect biodiversity estimates. We used a paired sampling design of 30 L per replicate on 68 reef transects from 8 sites in 3 tropical regions. We quantified local and regional sampling variability by comparing MOTU richness, compositional turnover, and compositional nestedness. We found strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non-overlapping assemblages rather than subsets of one another. As a result, non-saturated localized diversity accumulation curves suggest that even 6 replicates (180 L) in the same location can underestimate local diversity (for an area <1 km). However, sampling regional diversity using ~25 replicates in variable locations (often covering 10 s of km) often saturated biodiversity accumulation curves. Our results demonstrate variability of diversity estimates possibly arising from heterogeneous distribution of eDNA in seawater, highly skewed frequencies of eDNA traces per MOTU, in addition to variability in eDNA processing. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes in local assemblages. Avoiding false-negative detections in future biomonitoring efforts requires increasing replicates or sampled water volume to better inform management of marine biodiversity using eDNA.

Use of environmental DNA in assessment of fish functional and phylogenetic diversity.

Assessing the impact of global changes and protection effectiveness is a key step in monitoring marine fishes. Most traditional census methods are demanding or destructive. Nondisturbing and nonlethal approaches based on video and environmental DNA are alternatives to underwater visual census or fishing. However, their ability to detect multiple biodiversity factors beyond traditional taxonomic diversity is still unknown. For bony fishes and elasmobranchs, we compared the performance of eDNA metabarcoding and long-term remote video to assess species' phylogenetic and functional diversity. We used 10 eDNA samples from 30 L of water each and 25 hr of underwater videos over 4 days on Malpelo Island (pacific coast of Colombia), a remote marine protected area. Metabarcoding of eDNA detected 66% more molecular operational taxonomic units (MOTUs) than species on video. We found 66 and 43 functional entities with a single eDNA marker and videos, respectively, and higher functional richness for eDNA than videos. Despite gaps in genetic reference databases, eDNA also detected a higher fish phylogenetic diversity than videos; accumulation curves showed how 1 eDNA transect detected as much phylogenetic diversity as 25 hr of video. Environmental DNA metabarcoding can be used to affordably, efficiently, and accurately census biodiversity factors in marine systems. Although taxonomic assignments are still limited by species coverage in genetic reference databases, use of MOTUs highlights the potential of eDNA metabarcoding once reference databases have expanded.

Uso de ADN Ambiental en la Evaluación de la Diversidad Funcional y Filogenética de los Peces Resumen La evaluación del impacto de los cambios globales y la efectividad de la protección es un paso fundamental para el monitoreo de peces marinos. La mayoría de los métodos tradicionales de censos son demandantes o destructivos, por lo que las estrategias no letales y no intrusivas basadas en videograbaciones y en el ADN ambiental (ADNa) son alternativas a los censos visuales submarinos y a la pesca. Sin embargo, todavía no se conoce la habilidad que tienen estos métodos para detectar diferentes factores de la biodiversidad más allá de la diversidad taxonómica. Para los peces óseos y los elasmobranquios, comparamos el desempeño de la caracterización genética con ADNa y del video remoto de larga duración para evaluar la diversidad funcional y filogenética de las especies. Usamos diez muestras de ADNa tomadas de 30 litros de agua cada una y 25 horas de vídeos submarinos grabados durante cuatro días en la Isla Malpelo (costa del Pacífico de Colombia), un área marina protegida remota. La caracterización genética con el ADNa detectó 66% más unidades taxonómicas moleculares operacionales (UTMOs) que el video. Encontramos 66 y 43 entidades funcionales con un solo marcador de ADNa y con el video, respectivamente, y una riqueza funcional más alta para el ADNa que el video. A pesar de los vacíos en las bases de datos genéticos usadas como referencia, el ADNa también detectó una diversidad filogenética más alta que aquella en los videos; las curvas de acumulación mostraron cómo un solo transecto de ADNa detectó tanta diversidad filogenética como 25 horas de video. La caracterización genética con ADN ambiental puede usarse para censar los factores de biodiversidad de manera asequible, eficiente y certera en los sistemas marinos. Aunque las atribuciones taxonómicas todavía están limitadas por la cobertura de especies en las bases de datos genéticos de referencia, el uso de los UTMOs resalta el potencial que tiene la caracterización genética con ADNa una vez que las bases de datos de referencia sean expandidas.

Detection of the elusive Dwarf sperm whale (Kogia sima) using environmental DNA at Malpelo island (Eastern Pacific, Colombia).

Monitoring large marine mammals is challenging due to their low abundances in general, an ability to move over large distances and wide geographical range sizes.The distribution of the pygmy (Kogia breviceps) and dwarf (Kogia sima) sperm whales is informed by relatively rare sightings, which does not permit accurate estimates of their distribution ranges. Hence, their conservation status has long remained Data Deficient (DD) in the Red list of the International Union for Conservation of Nature (IUCN), which prevent appropriate conservation measures.Environmental DNA (eDNA) metabarcoding uses DNA traces left by organisms in their environments to detect the presence of targeted taxon, and is here proved to be useful to increase our knowledge on the distribution of rare but emblematic megafauna.Retrieving eDNA from filtered surface water provides the first detection of the Dwarf sperm whale (Kogia sima) around the remote Malpelo island (Colombia).Environmental DNA collected during oceanic missions can generate better knowledge on rare but emblematic animals even in regions that are generally well sampled for other taxa.

Accumulation curves of environmental DNA sequences predict coastal fish diversity in the coral triangle.

Environmental DNA (eDNA) has the potential to provide more comprehensive biodiversity assessments, particularly for vertebrates in species-rich regions. However, this method requires the completeness of a reference database (i.e. a list of DNA sequences attached to each species), which is not currently achieved for many taxa and ecosystems. As an alternative, a range of operational taxonomic units (OTUs) can be extracted from eDNA metabarcoding. However, the extent to which the diversity of OTUs provided by a limited eDNA sampling effort can predict regional species diversity is unknown. Here, by modelling OTU accumulation curves of eDNA seawater samples across the Coral Triangle, we obtained an asymptote reaching 1531 fish OTUs, while 1611 fish species are recorded in the region. We also accurately predict (R² = 0.92) the distribution of species richness among fish families from OTU-based asymptotes. Thus, the multi-model framework of OTU accumulation curves extends the use of eDNA metabarcoding in ecology, biogeography and conservation.

Long-Distance Benefits of Marine Reserves: Myth or Reality?

Long-distance (>40-km) dispersal from marine reserves is poorly documented; yet, it can provide essential benefits such as seeding fished areas or connecting marine reserves into networks. From a meta-analysis, we suggest that the spatial scale of marine connectivity is underestimated due to the limited geographic extent of sampling designs. We also found that the largest marine reserves (>1000km2) are the most isolated. These findings have important implications for the assessment of evolutionary, ecological, and socio-economic long-distance benefits of marine reserves. We conclude that existing methods to infer dispersal should consider the up-to-date genomic advances and also expand the spatial scale of sampling designs. Incorporating long-distance connectivity in conservation planning will contribute to increase the benefits of marine reserve networks.

Body reserves mediate trade-offs between life-history traits: new insights from small pelagic fish reproduction.

Limited resources in the environment prevent individuals from simultaneously maximizing all life-history traits, resulting in trade-offs. In particular, the cost of reproduction is well known to negatively affect energy investment in growth and maintenance. Here, we investigated these trade-offs during contrasting periods of high versus low fish size and body condition (before/after 2008) in the Gulf of Lions. Female reproductive allocation and performance in anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) were examined based on morphometric historical data from the 1970s and from 2003 to 2015. Additionally, potential maternal effects on egg quantity and quality were examined in 2014/2015. After 2008, the gonadosomatic index increased for sardine and remained steady for anchovy, while a strong decline in mean length at first maturity indicated earlier maturation for both species. Regarding maternal effects, for both species egg quantity was positively linked to fish size but not to fish lipid reserves, while the egg quality was positively related to lipid reserves. Atresia prevalence and intensity were rather low regardless of fish condition and size. Finally, estimations of total annual numbers of eggs spawned indicated a sharp decrease for sardine since 2008 but a slight increase for anchovy during the last 5 years. This study revealed a biased allocation towards reproduction in small pelagic fish when confronted with a really low body condition. This highlights that fish can maintain high reproductive investment potentially at the cost of other traits which might explain the present disappearance of old and large individuals in the Gulf of Lions.