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Species inventories are the building blocks of our assessment of biodiversity patterns and human impact. Yet, historical inventories based on visual observations are often incomplete, impairing subsequent analyses of ecological mechanisms, extinction risk and management success. Environmental DNA (eDNA) metabarcoding is an emerging tool that can provide wider biodiversity assessments than classical visual‐based surveys. However, eDNA‐based inventories remain limited by sampling effort and reference database incompleteness. In this study, we propose a new framework coupling eDNA surveys and sampling‐theory methods to estimate species richness in under‐sampled and hyper‐diverse regions where some species remain absent from the checklist or undetected by visual surveys. We applied this framework to the coastal fish diversity in the heart of the coral triangle, the richest marine biodiversity hotspot worldwide. Combining data from 279 underwater visual censuses, 92 eDNA samples and an extensive custom genetic reference database, we show that eDNA metabarcoding recorded 196 putative species not detected by underwater visual census including 37 species absent from the regional checklist. We provide an updated checklist of marine fishes in the ‘Raja Ampat Bird's Head Peninsula' ecoregion with 2534 species including 1761 confirmed and 773 highly probable presences. The Chao lower‐bound diversity estimator, based on the incidence of rare species, shows that the region potentially hosts an additional 123 fish species, including pelagic, cryptobenthic and vulnerable species. The extended and hidden biodiversity along with their asymptotic estimates highlight the ability of eDNA to expand regional inventories and species distributions to better guide conservation strategies.

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 \\textless1 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.

Assessing species geographic distributions is critical to approximate their ecological niches, understand how global change may reshape their occurrence patterns, and predict their extinction risks. Yet, species records are over-aggregated across taxonomic, geographic, environmental, and anthropogenic dimensions. The under-sampling of remote locations biases the quantification of species geographic distributions and ecological niche for most species. Here, we used nearly one thousand environmental DNA (eDNA) samples across the world’s oceans, including polar regions and tropical remote islands, to determine the extent to which the geographic and ecological niche ranges of marine fishes are underestimated through the lens of global occurrence records based on conventional surveys. Our eDNA surveys revealed that the known geographic ranges for 93% of species and the ecological niche ranges for 7% of species were underestimated, and contributed to filling them. We show that the probability to detect a range filling for a given species is primarily shaped by the GBIF/OBIS sampling effort in a cell, but also by the number of occurrences available for the species. Most gap fillings were achieved by addressing a methodological sampling bias, notably when eDNA facilitated the detection of small fishes in previously sampled locations using conventional methods. Using a machine learning model, we found that a local effort of 10 eDNA samples would detect 24 additional fish species on average and a maximum of 98 species in previously unsampled tropical areas. Yet, a null model revealed that only half of ecological niche range fillings would be due to eDNA surveys, beyond a random allocation of classical sampling effort. Altogether, our results suggest that sampling in remote areas and performing eDNA surveys in over-sampled areas may both increase fish ecological niche ranges toward unexpected values with consequences in biodiversity modeling, management, and conservation.

Environmental DNA (eDNA) analysis is a revolutionary method to monitor marine biodiversity from animal DNA traces. Examining the capacity of eDNA to provide accurate biodiversity measures in species‐rich ecosystems such as coral reefs is a prerequisite for their application in long‐term monitoring. Here, we surveyed two Colombian tropical marine reefs, the island of Providencia and Gayraca Bay near Santa Marta, using eDNA and underwater visual census (UVC) methods. We collected a large quantity of surface water (30 L per filter) above the reefs and applied a metabarcoding protocol using three different primer sets targeting the 12S mitochondrial DNA, which are specific to the vertebrates Actinopterygii and Elasmobranchii. By assigning eDNA sequences to species using a public reference database, we detected the presence of 107 and 85 fish species, 106 and 92 genera, and 73 and 57 families in Providencia and Gayraca Bay, respectively. Of the species identified using eDNA, 32.7% (Providencia) and 18.8% (Gayraca) were also found in the UVCs. We further found congruence in genus and species richness and abundance between eDNA and UVC approaches in Providencia but not in Gayraca Bay. Mismatches between eDNA and UVC had a phylogenetic and ecological signal, with eDNA detecting a broader phylogenetic diversity and more effectively detecting smaller species, pelagic species and those in deeper habitats. Altogether, eDNA can be used for fast and broad biodiversity surveys and is applicable to species‐rich ecosystems in the tropics, but improved coverage of the reference database is required before this new method could serve as an effective complement to traditional census methods. We examined the capacity of eDNA to provide accurate biodiversity measures in species‐rich ecosystems with a case study in two Colombian tropical marine reefs. We compared the results of eDNA analyses with historical data of underwater visual census monitoring. We found that eDNA allows a fast and broader detection of biodiversity and that it is applicable to species‐rich ecosystems in the tropic, but requires to increase the coverage of the reference database before replacing traditional census methods.

Through the development of environmental DNA (eDNA) metabarcoding, in situ monitoring of organisms is becoming easier and promises a revolution in our approaches to detect changes in biodiversity over space and time. A cornerstone of eDNA approach is the development of primer pairs that allow amplifying the DNA of specific taxonomic groups, which is then used to link the DNA sequence to taxonomic identification. Here, we propose a framework for comparing primer pairs regarding (a) their capacity to bind and amplify a broad coverage of species within the target clade using in silico PCR, (b) their capacity to not only discriminate between species but also genera or families, and (c) their in situ specificity and efficiency across a variety of environments. As a case study, we focus on two mitochondrial 12S primer pairs, MiFish‐U and teleo, which were designed to amplify fishes. We found that the performance of in silico PCRs were high for both primer pairs, but teleo amplified more genera across Actinopterygii, Chondrichthyes, and Petromyzontomorphi than MiFish‐U. In contrast, the discriminatory power for species, genera, and families were higher for MiFish‐U than teleo, likely associated with the greater length of the amplified DNA fragments. The evaluation of their in situ efficiency showed a higher recovered species richness of teleo compared to MiFish‐U in tropical and temperate freshwater environments, but that generally both teleo and MiFish‐U primers pairs perform well to monitor fish species. Since more species were detected when used together, those primer pairs are best used in combination to increase the ability of species detection. We propose a framework for comparing the performance of primer pairs, which we applied to the comparison of two fish eDNA primer pairs, teleo and MiFish‐U. We showed how those primer pairs could be best used together to increase diversity coverage of eDNA studies.

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.

The link between biodiversity and ecosystem functioning has been the topic of considerable research, but it remains unclear how biodiversity decline is compromising ecosystem functionality, particularly in the pelagic realm. Here, we explore how pelagic fish species diversity relates to functional diversity by sampling two locations, which, on the basis of biogeography, environmental conditions and human pressures, were expected to host pronounced differences in species composition and abundances and therefore functionality. Strings of five drifting mid-water Baited Remote Underwater Video Systems were used to survey pelagic vertebrate diversity and abundance in two isolated oceanic island systems, the Malpelo Fauna and Flora Sanctuary—a large, 25-year-old marine protected area—and an unprotected area in Cape Verde. Functional diversity, which offers insight into a community’s resilience against disturbance, was analysed using six key functional traits of marine fishes. Abundance was recorded as MaxN, the maximum number of individuals of a given species in a single frame during the 2-h deployment time. Cape Verde showed high overall abundance (Total MaxN 873) and low biomass (3559 kg), with a predominance of smaller fishes. Malpelo showed high biomass (7839 kg) but lower abundance (Total MaxN 465), with a predominance of large species. Species and functional diversity were marginally different between locations. Multivariate analysis of species relative abundances showed significant divergence between locations, although community functional traits overlapped strongly, suggesting that both communities share a similar structure and vulnerability. The existence of a common functional ‘backbone’ in diverging species communities across the oceans, under different productivity regimes, and under different protection levels, suggests that although pelagic communities may differ considerably in terms of species composition, this does not translate into a differing functional structure and resilience potential. Whether this vulnerability is a common feature of pelagic communities and how this contrasts with benthic systems warrants further research.

Environmental DNA is mainly not only used at the interspecific level, to quantify species diversity in ecosystems, but can also be used to quantify intraspecific genetic variability, thus avoiding the need to sample individual tissue. However, errors in the amplification and sequencing of eDNA samples can blur this intraspecific signal and strongly over‐estimate genetic diversity. Existing bioinformatics pipelines therefore need to be tested to evaluate whether reliable levels of intraspecific genetic variability can be derived from eDNA samples. Here, we compare the ability of twelve metabarcoding pipelines to detect intraspecific genetic variability combining five programs. All pipelines have common pre‐processing steps, a processing data step using programs among obiclean; DADA2; SWARM; and LULU. An additional chimera removal step is also investigated based on two programs (VSEARCH or DADA2). The case study was the natural intraspecific variation within Mullus surmuletus in experimental settings. We developed specific primers for this species, located on the mitochondrial D‐loop fragment (barcode MS‐DL06). Thirty‐nine individuals were collected from the Mediterranean Sea, placed into four aquariums, and their DNA was sequenced on this marker to build an intraspecific reference database. After filtering the aquarium water, DNA was extracted, amplified, and sequenced using the primer pair developed. We then quantified the number of true haplotypes returned by each pipeline and its capacity to eliminate most of the erroneous sequences. We show that the program DADA2 with a two‐parent chimeric sequence removal step is the best tool to estimate intraspecific diversity from eDNA. Furthermore, our approach was also able to detect true M. surmuletus haplotypes in two eDNA samples collected in the Mediterranean Sea. We conclude that the combination of an appropriate intrapopulation barcode and a denoising pipeline like DADA2 with a chimeric sequence removal step is promising to make population‐level inference using environmental DNA possible. We compared 12 pipelines in their ability to analyze eDNA barcode data at population level. We found that removing chimera sequences is an important bioinformatics filter for population eDNA inference.

Environmental DNA (eDNA) metabarcoding is changing the way biodiversity is surveyed in many types of ecosystems. eDNA surveys are now commonly performed and integrated into biodiversity monitoring programs and public databases. Although it is widely recognized that eDNA records require interpretation in light of taxonomy and biogeography, there remains a range of perceptions about how thoroughly records should be evaluated and which ones should be reported. Here, we present a modular procedure, available as an R script, that uses a set of five steps to assess the confidence of species‐level eDNA records by assigning them a score from 0 to 5. This procedure includes evaluations of the known geographic distribution of each taxon, the taxonomic resolution of the marker used, the regional completeness of the reference database, the diversification rate, and the range map of each taxon. We tested the procedure on a large‐scale marine fish eDNA dataset (572 samples) covering 15 ecoregions worldwide, from the poles to the tropics, using the teleo marker on the mitochondrial 12S ribosomal gene. Our analysis revealed broad variation in the average confidence score of eDNA records among regions, with the highest scores occurring along the European and Eastern Atlantic coasts. Generalized linear models applied to record covariates highlighted the significant influences of latitude and species richness on low confidence scores (< 2.5). The polar regions notably displayed high proportions of low confidence scores, probably due to the limited completeness of the regional reference databases and the taxonomic resolution of the teleo marker. We conclude that only records with high confidence scores (> 2.5) should be integrated into biodiversity databases. The medium (2.5) to relatively low‐confidence (< 2.5) records correspond to species that require further investigation and may be integrated after inspection to ensure high‐quality species records. Metabarcoding of environmental DNA (eDNA) is transforming biodiversity detection and surveys across ecosystems and is becoming part of biodiversity monitoring programs. However, interpretations of eDNA records vary widely, from treating them as standard biodiversity records to requiring careful validation against taxonomic and biogeographic data. A new five‐step procedure assesses the confidence of species‐level eDNA records, revealing that confidence varies by region due to incomplete reference databases and taxonomic resolution issues.

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 ( R ² = 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.