Aim Environmental DNA metabarcoding has recently emerged as a non‐invasive tool for aquatic biodiversity inventories, frequently surpassing traditional methods for detecting a wide range of taxa in most habitats. The major limitation currently impairing the large‐scale application of eDNA‐based inventories is the lack of species sequences available in public genetic databases. Unfortunately, these gaps are still unknown spatially and taxonomically, hindering targeted future sequencing efforts. Innovation We propose GAPeDNA, a user‐friendly web interface that provides a global overview of genetic database completeness for a given taxon across space and conservation status. As an application, we synthetized data from regional checklists for marine and freshwater fishes along with their IUCN conservation status to provide global maps of species coverage using the European Nucleotide Archive public reference database for 19 metabarcoding primers. This tool automatizes the scanning of gaps in these databases to guide future sequencing efforts and support the deployment of eDNA inventories at larger scale. This tool is flexible and can be expanded to other taxa and primers upon data availability. Main conclusions Using our global fish case study, we show that gaps increase towards the tropics where species diversity and the number of threatened species are the highest. It highlights priority areas for fish sequencing like the Congo, the Mekong and the Mississippi freshwater basins which host more than 60 non‐sequenced threatened fish species. For marine fishes, the Caribbean and East Africa host up to 42 non‐sequenced threatened species. By presenting the global genetic database completeness for several primers on any taxa and building an open‐access, updatable and flexible tool, GAPeDNA appears as a valuable contribution to support any kind of eDNA metabarcoding study.

As fish communities are a major concern in rivers ecosystems, we investigated if their environmental (e)DNA signals vary according to the sampling period or hydromorphological conditions. Three rivers were studied over a year using eDNA metabarcoding approach. The majority of the species (c. 80%) were detected all year round in two rivers having similar hydromorphological conditions, whereas in the river affected by an upstream lake waterflow, more species were detected sporadically (42%). For all the rivers, in more than 98% of the occasional detections, the reads abundance represented <0.4% of the total reads per site and per sampling session. Even if the majority of the fish communities remained similar over the year for each of the three rivers, specific seasonal patterns were observed. We studied if the waterflow or the reproduction period had an effect on the observed dynamics. Waterflow, which influences eDNA downstream transportation, had a global influence in taxonomic richness, while the fishes' reproductive period had only an influence on certain species. Our results may help selecting the best sampling strategy according to research objectives. To study fish communities at local scale, seasons of low waterflow periods are recommended. This particularly helps to restraint effects of external eDNA coming from connections with other aquatic environment (tributaries, lakes, wetlands, sewage effluents, etc.). To obtain a more integrative overview of the fish community living in a river basin, high waterflow or breeding seasons are preferable for enhancing species detection probability, especially for rare species.

1. To mitigate the ongoing threats to coastal ecosystems, and the biodiversity erosion they are causing, marine-protected areas (MPAs) have emerged as powerful and widespread conservation tools. Strictly no-take MPAs, also called marine reserves, undeniably promote fish biomass and density, but it remains unclear how biodiversity responds to protection. Identifying which facets of biodiversity respond to protection is critical for the management of MPAs and the development of relevant conservation strategies towards the achievement of biodiversity targets.2. We collected 99 environmental DNA (eDNA) samples inside and outside nine marine reserves in the Mediterranean Sea to assess the effect of protection on 11 biodiversity indicators based on fish traits, phylogeny and vulnerability to fishing. We controlled for the effect of environmental heterogeneity (habitat, bathymetry, productivity, temperature and accessibility) using a principal component analysis, and for spatial autocorrelation due to potential unmeasured factors.3. We found a positive and significant effect of protection on only 3 out of 11 indicators: functional and phylogenic diversity but also the ratio between demersopelagic and benthic species richness. Rather, total fish richness responded significantly and negatively to protection. We did not detect any significant effect of protection on threatened and elasmobranch species richness, probably due to their large home range compared to the size of Mediterranean marine reserves.

Environmental DNA (eDNA) metabarcoding is a reliable method to assess taxonomic diversity, but the incompleteness of genetic reference databases prevents the assignation of many sequences to a given species. Functional diversity (FD) is a key biodiversity facet to monitor, but it requires the identification of all species within communities to account for their trait values. So, the ability of eDNA‐based inventories to estimate the “true” level of FD is unknown. Here, using fish surveys in two representative temperate and tropical rivers, with a quasi‐exhaustive genetic and trait database, we measured the bias and variability of different FD indices when calculated with uncertainty in taxonomic assignment at the genus and family level. Our results show that when measuring FD indices with species randomly chosen within genera and families, the bias cannot exceed 30% from real observed FD values. The variability is higher for species‐poorer communities and when those communities are composed of genera and families with high functional heterogeneity. Despite taxonomic uncertainty, our results demonstrate the potential of eDNA surveys to estimate reliable FD values.

Environmental DNA metabarcoding has recently emerged as a non-invasive tool for aquatic biodiversity inventories, frequently surpassing traditional methods for detecting a wide range of taxa in most habitats. One of the major limitations currently impairing the large-scale application of DNA-based inventories, such as eDNA or bulk-sample analysis is the lack of species sequences available in public genetic databases. These gaps are still largely unknown spatially and taxonomically for most regions of the world, which can hinder targeted future sequencing efforts. We propose GAPeDNA, a user-friendly web-interface (Fig. 1) that provides a global overview of genetic database completeness for a given taxon across space and conservation status. As an initial application, we synthetized data from regional checklists for marine and freshwater fishes along with their IUCN conservation status to provide global maps of species coverage using the European Nucleotide Archive public reference database for 19 metabarcoding primers. This tool automatizes the scanning of gaps in these databases to guide future sequencing efforts and support the deployment of DNA-based inventories at larger scale. It is flexible and can be expanded to other taxa and primers upon data availability. Using our global fish case study, we show that gaps increase toward the tropics where species diversity and the number of threatened species were the highest. It highlights priority areas for fish sequencing like the Congo, the Mekong and the Mississippi freshwater basins which host more than 60 non-sequenced threatened fish species. For marine fishes, the Caribbean and East Africa host up to 42 non-sequenced threatened species. As an open-acces, updatable and flexible tool, GAPeDNA can be used to evaluate the completeness of sequence reference libraries of various markers and for any taxonomic group.

The cover image relates to the Biodiversity Methods https://doi.org/10.1111/ddi.13142 “GAPeDNA: Assessing and mapping global species gaps in genetic databases for eDNA metabarcoding” by Marques et al. Photo credit: Virginie Marques. A couple of butterflyfish (Chaetodontidae) on the coral reef lagoon of the Fakarava atoll in French Polynesia.