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Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.

Assessing the impact of human activity on ecosystems often links local biodiversity to disturbances measured within the same locality. However, remote disturbances may also affect local biodiversity. Here, we used environmental DNA metabarcoding to evaluate the relationships between vertebrate biodiversity (fish and mammals) and disturbance intensity in two Amazonian rivers. Measurements of anthropic disturbance -here forest cover losses- were made from the immediate vicinity of the biodiversity sampling sites to up to 90 km upstream. The findings suggest that anthropization had a spatially extended impact on biodiversity. Forest cover losses of <11% in areas up to 30 km upstream from the biodiversity sampling sites were linked to reductions of >22% in taxonomic and functional richness of both terrestrial and aquatic fauna. This underscores the vulnerability of Amazonian biodiversity even to low anthropization levels. The similar responses of aquatic and terrestrial fauna to remote disturbances indicate the need for cross-ecosystem conservation plans that consider the spatially extended effects of anthropization. It is unclear how far the impact of deforestation can spread. Here the authors analyse freshwater eDNA data along two rivers in the Amazon forest, and find that low levels of deforestation are linked to substantial reductions of fish and mammalian diversity downstream.

Environmental DNA (eDNA) metabarcoding is a promising tool to estimate aquatic biodiversity. It is based on the capture of DNA from a water sample. The sampled water volume, a crucial aspect for efficient species detection, has been empirically variable (ranging from few centilitres to tens of tilers). This results in a high variability of sampling effort across studies, making comparisons difficult and raising uncertainties about the completeness of eDNA inventories. Our aim was to determine the sampling effort (filtered water volume) needed to get optimal inventories of fish assemblages in species-rich tropical streams and rivers using eDNA. Ten DNA replicates were collected in six Guianese sites (3 streams and 3 rivers), resulting in sampling efforts ranging from 17 to 340 litres of water. We show that sampling 34 litres of water detected more than 64% of the expected fish fauna and permitted to distinguish the fauna between sites and between ecosystem types (stream versus rivers). Above 68 litres, the number of detected species per site increased slightly, with a detection rate higher than 71%. Increasing sampling effort up to 340 litres provided little additional information, testifying that filtering 34 to 68 litres is sufficient to inventory most of the fauna in highly diverse tropical aquatic ecosystems.

A large number of marker typologies have been developed for the metabarcoding of environmental DNA (eDNA). Generalist markers have been advocated for their ability to amplify many taxa simultaneously and produce exhaustive biodiversity assessments, while more specific markers have been proposed for their higher ability to detect and discriminate taxa within a more restricted group. Quantitative comparisons between generalist and specific markers are needed to assess their relative efficiency depending on study targets. Here we compared the performance of one generalist (Euka02, amplifying all eukaryotic groups), one intermediate (Arth02, amplifying arthropods), and two specific markers (Coll01 and Inse01, amplifying springtails and insects, respectively) for the assessment of springtails and insects diversity using eDNA metabarcoding. The four markers were used to analyze eDNA extracted from > 1200 soil samples collected in recently deglaciated terrains. We then assessed whether the different markers were able to detect community responses to key environmental drivers (soil temperature, time since glacier retreat, plant productivity, and topographic wetness). The two specific markers detected more taxa of both springtails and insects than the generalist markers; still the taxonomic richness and community dissimilarity were well correlated between generalist and specific markers. There was good overlap between the taxa identified by the specific markers and those identified by the generalist ones, but the specific markers often detected the taxa identified by the generalist markers, plus several additional ones. Both generalist and specific markers detected the impact of key environmental drivers on arthropods; still the specific markers showed the strongest power to detect relationships, and the most generalist marker was unable to identify the weaker relationships. Generalist markers efficiently provide an overall view of soil biodiversity; nevertheless complementing them with specific markers allows more detailed biodiversity measures, and provides a clearer picture of its response to environmental stressors. By analyzing > 1200 eDNA samples with different markers amplifying arthropods, we show that generalist and specific markers produce correlated pictures of community variation; still specific markers provide a more accurate measure of biodiversity and of its response to environmental stressors.

Environmental DNA (eDNA) metabarcoding has recently gain much attention to assess aquatic environment biodiversity. A great variety of protocols have been developed to collect, extract, and analyze eDNA, some of which are continuously evolving and optimized with technological improvements. Such technological shifts might deprecate the biological data produced with earlier protocols, leading to a loss of biological knowledge. Here, we investigated the robustness of an aquatic eDNA metabarcoding method through the comparison of two biodiversity datasets generated by two optimized protocols with different collection and extraction steps. To this end, we compared fish community richness and composition of 12 streams and 3 rivers from French Guiana, sampled with two distinct protocols commonly used in aquatic eDNA studies. Although sample collection with each protocol was not achieved the same year, our results show that species richness and species composition were only slightly affected by the protocol choice, both protocols producing similar fish assemblages at each sample site. Both protocols had a higher replicability in streams than in rivers, strengthening the importance to adapt sampling effort to waterbody type as rivers host a larger number of species than small streams. Despite the need for a standardized approach in eDNA metabarcoding studies, testing the robustness of datasets to protocol variations remains crucial to valorize old data, time series, or data collected in difficult to access locations. eDNA metabarcoding protocols are continuously evolving with technological improvements leading to the deprecation of biological data produced with earlier protocols and a loss of biological knowledge. While eDNA protocols are usually compared based on the amount of DNA retrieved, we here took a different approach by directly comparing the biodiversity inventories derived from different protocols. We demonstrated that different protocols provide similar fish inventories and underline the importance to test the robustness of eDNA metabarcoding methods to potentially rescue data.

BackgroundDeforestation is a widespread disturbance for neotropical freshwater ecosystems. While biodiversity declines have been associated with deforestation, its functional consequences for stream and river fish faunas remain poorly understood. In this study, we explored how deforestation affects the different facets of the functional structure of fish communities inventoried using environmental DNA metabarcoding in 64 river and 35 stream sites of French Guiana. Specifically, we investigated how functional richness, divergence, evenness and identity of fish faunas are affected by deforestation.ResultsWe showed that anthropogenic disturbances in French Guiana are modifying the functional diversity of freshwater fish communities. These disturbances not only affected the amount of functional traits held by the communities but also the identity of the traits and the internal structure of the functional space. Consequently, different facets of the functional diversity supported by fish assemblages were altered. In streams, deforestation did not affect the overall diversity of traits but reduced functional redundancy, underlined by a shift in functional identity towards assemblages dominated by pelagic detritivores. In contrast, river fish faunas experienced a decline in functional richness, paired with shifts in functional identity and a loss of fish species with extreme functions.ConclusionsThe response to deforestation differed between streams and rivers, but it supports the hypothesis that deforestation is linked to functional changes in fish assemblages. By diminishing the range of the functions in rivers or by jeopardizing the redundancy of functions in streams, deforestation could severely hamper the functioning and stability of neotropical freshwater ecosystems.

The mechanisms shaping plant succession after glacier retreat are dynamic. Compositional dissimilarity between communities decreases over time, accompanied by a shift in the relative contribution of taxa addition versus replacement. Taxa addition prevailed in early communities, whereas replacement became more important after 50 years of succession.

Environmental DNA [eDNA] metabarcoding has recently emerged as a non-destructive alternative to traditional sampling for characterising species assemblages. We here provide a consistent dataset synthetising all eDNA sampling sites in French Guiana to date. Field collections have been initiated in 2014 and have continued until 2019. This dataset is however a work in progress and will be updated after each collecting campaign. We also provide a taxon by site matrix for fishes presence / absence as inferred from eDNA. Our aim is to allow a transparent communication to the stakeholders and provide the foundation for a monitoring programme based on eDNA. The lastest version of the dataset is publicly and freely accessible through the CEBA geoportal (http://vmcebagn-dev.ird.fr) or through the French Guiana geographic portal (https://www.geoguyane.fr).

Dietary plasticity of populations can be associated to ontogenetic diet preferences and depends on the size-structure of populations. Dietary niche characterizes the functional role of organisms in a food web, as it reflects both resources' diversity used by a consumer and trophic interactions in the system. Dietary niches are controlled both by biotic and abiotic factors, but their interactions in natural systems remain poorly studied. Here, we investigated the variability of dietary niche in salmonid wild populations focusing both on inter-population and intra-population (through time) trophic changes, using marble trout ( Salmo marmoratus ) living in Slovenian headwater streams as a model system. Stable isotope analysis showed high variability of dietary niche and trophic diversity among six of the seven remnant marble trout populations. We observed substantial differences in dietary niche width among populations and within populations through time. Results of partial least square path modelling highlighted opposite effects of immature and mature trout on trophic niche structure. Direct effects of temperature and slope (stream and watershed) were opposite; temperature narrowed dietary niches while slope increased them. Environmental factors (e.g., temperature, stream and watershed slope) had indirect effects on trophic niches after accounting for fish density. Our results showed that size-distribution and sexual maturity are key determinants of the dietary niche width in a population. Increasing density of immature trout tended to widen the dietary niche while increasing density of mature trout tended to narrow it. Environmental factors had direct effects both on resources and consumers densities and indirect effects. Direct and indirect effects were often antagonistic.

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of their biodiversity. DNA metabarcoding is increasingly used to assess nematode biodiversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In-silico analyses have a great potential to assess key parameters of primers, including their taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in-silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, as amplified most of sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in-silico analyses provide new insights for the identification of most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.