Environmental DNA: The next chapter.

Molecular tools are an indispensable part of ecology and biodiversity sciences and implemented across all biomes. About a decade ago, the use and implementation of environmental DNA (eDNA) to detect biodiversity signals extracted from environmental samples opened new avenues of research. Initial eDNA research focused on understanding population dynamics of target species. Its scope thereafter broadened, uncovering previously unrecorded biodiversity via metabarcoding in both well-studied and understudied ecosystems across all taxonomic groups. The application of eDNA rapidly became an established part of biodiversity research, and a research field by its own. Here, we revisit key expectations made in a land-mark special issue on eDNA in Molecular Ecology in 2012 to frame the development in six key areas: (1) sample collection, (2) primer development, (3) biomonitoring, (4) quantification, (5) behaviour of DNA in the environment and (6) reference database development. We pinpoint the success of eDNA, yet also discuss shortfalls and expectations not met, highlighting areas of research priority and identify the unexpected developments. In parallel, our retrospective couples a screening of the peer-reviewed literature with a survey of eDNA users including academics, end-users and commercial providers, in which we address the priority areas to focus research efforts to advance the field of eDNA. With the rapid and ever-increasing pace of new technical advances, the future of eDNA looks bright, yet successful applications and best practices must become more interdisciplinary to reach its full potential. Our retrospect gives the tools and expectations towards concretely moving the field forward.

Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land-use and geology.

Despite being the most important source of liquid freshwater on the planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It is thus extensively monitored for pollutants and declines in quantity. The organisms living in groundwater, however, are rarely the target of surveillance programmes and little is known about the fauna inhabiting underground habitats. The difficulties accessing groundwater, the lack of expertise, and the apparent scarcity of these organisms challenge sampling and prohibit adequate knowledge on groundwater fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome these limitations but is largely unexplored. Here, we sampled water in 20 communal spring catchment boxes used for drinking water provisioning in Switzerland, with a high level of replication at both filtration and amplification steps. We sequenced a portion of the COI mitochondrial gene, which resulted in 4917 ASVs, yet only 3% of the reads could be assigned to a species, genus, or family with more than 90% identity. Careful evaluation of the unassigned reads corroborated that these sequences were true COI sequences belonging mostly to diverse eukaryotic groups, not present in the reference databases. Principal component analyses showed a strong correlation of the community composition with the surface land-use (agriculture vs. forest) and geology (fissured rock vs. unconsolidated sediment). While incomplete reference databases limit the assignment of taxa in groundwater eDNA metabarcoding, we showed that taxonomy-free approaches can reveal large hidden diversity and couple it with major land-use drivers, revealing their imprint on chemical and biological properties of groundwater.

Meta-analysis shows both congruence and complementarity of DNA and eDNA metabarcoding to traditional methods for biological community assessment.

DNA metabarcoding is increasingly used for the assessment of aquatic communities, and numerous studies have investigated the consistency of this technique with traditional morpho-taxonomic approaches. These individual studies have used DNA metabarcoding to assess diversity and community structure of aquatic organisms both in marine and freshwater systems globally over the last decade. However, a systematic analysis of the comparability and effectiveness of DNA-based community assessment across all of these studies has hitherto been lacking. Here, we performed the first meta-analysis of available studies comparing traditional methods and DNA metabarcoding to measure and assess biological diversity of key aquatic groups, including plankton, microphytobentos, macroinvertebrates, and fish. Across 215 data sets, we found that DNA metabarcoding provides richness estimates that are globally consistent to those obtained using traditional methods, both at local and regional scale. DNA metabarcoding also generates species inventories that are highly congruent with traditional methods for fish. Contrastingly, species inventories of plankton, microphytobenthos and macroinvertebrates obtained by DNA metabarcoding showed pronounced differences to traditional methods, missing some taxa but at the same time detecting otherwise overseen diversity. The method is generally sufficiently advanced to study the composition of fish communities and replace more invasive traditional methods. For smaller organisms, like macroinvertebrates, plankton and microphytobenthos, DNA metabarcoding may continue to give complementary rather than identical estimates compared to traditional approaches. Systematic and comparable data collection will increase the understanding of different aspects of this complementarity, and increase the effectiveness of the method and adequate interpretation of the results.

Water eDNA metabarcoding is effective in detecting non-native species in marinas, but detection errors still hinder its use for passive monitoring.

Marinas are high-priority targets for marine non-indigenous species (NIS), where they compose a large portion of the biofouling communities. The practicality of water samples collection makes environmental DNA (eDNA) metabarcoding an interesting tool for routine NIS surveys. Here the effectiveness of water-eDNA-metabarcoding to identify biofouling NIS, in 10 marinas from western France, was examined. Morphological identification of specimens collected in quadrats brought out 18 sessile benthic NIS beneath floating pontoons. Water-eDNA-metabarcoding detected two thirds of them, failing to detect important NIS. However, sampling and bioinformatics filtering steps can be optimized to identify more species. In addition, this method allowed the detection of additional NIS from neighboring micro-habitats. Caution should, however, be taken when reporting putative novel NIS, because of errors in species assignment. This work highlights that water-eDNA-metabarcoding is effective for active (targeted) NIS surveys and could be significantly improved for its further use in marine NIS passive surveys.

Navigating the seven challenges of taxonomic reference databases in metabarcoding analyses.

Assessment of biodiversity using metabarcoding data, such as from bulk or environmental DNA sampling, is becoming increasingly relevant in ecology, biodiversity sciences and monitoring. Thereby, the taxonomic identification of species from their DNA sequences relies strongly on reference databases that link genetic sequences to taxonomic names. These databases vary in completeness and availability, depending on the taxonomic group studied and the genetic region targeted. The incompleteness of reference databases is an important argument to explain the nondetection by metabarcoding of species supposedly present. However, there exist further and generally overlooked problems with reference databases that can lead to false or inaccurate inferences of taxonomic assignment. Here, we synthesize all possible problems inherent to reference databases. In particular, we identify a complete, mutually nonexclusive list of seven classes of challenges when it comes to selecting, developing and using a reference database for taxonomic assignment. These are: (i) mislabelling, (ii) sequencing errors, (iii) sequence conflict, (iv) taxonomic conflict, (v) low taxonomic resolution, (vi) missing taxa and (vii) missing intraspecific variants. For each problem identified, we provide a description of possible consequences on the taxonomic assignment process. We illustrate the respective problem with examples taken from the literature or obtained by quantitative analyses of public databases, such as GenBank or BOLD. Finally, we discuss possible solutions to the identified problems and how to navigate them. Only by raising users' awareness of the limitations of metabarcoding data and DNA reference databases will adequate interpretations of these data be achieved.

Integrating citizen science and environmental DNA metabarcoding to study biodiversity of groundwater amphipods in Switzerland.

Groundwater is the physically largest freshwater ecosystem, yet one of the least explored habitats on earth, both because of accessing difficulties and the scarcity of the organisms inhabiting it. Here, we demonstrate how a two-fold approach provides complementary information on the occurrence and diversity of groundwater amphipods. Firstly, we used a citizen science approach in collaboration with municipal water providers who sampled groundwater organisms in their spring catchment boxes over multiple weeks, followed by DNA barcoding. Secondly, we collected four 10 L water samples at each site, in one sampling event, for environmental DNA (eDNA) metabarcoding. We found that citizen science was very effective in describing the distribution and abundance of groundwater amphipods. Although the single time-point of eDNA sampling did not detect as many amphipods, it allowed the assessment of the entire groundwater community, including microorganisms. By combining both methods, we found different amphipod species co-occurring with distinct sequences from the eDNA-metabarcoding dataset, representing mainly micro-eukaryotic species. We also found a distinct correlation between the diversity of amphipods and the overall biodiversity of groundwater organisms detected by eDNA at each site. We thus suggest that these approaches can be used to get a better understanding of subterranean biodiversity.

High-throughput sequencing on preservative ethanol is effective at jointly examining infraspecific and taxonomic diversity, although bioinformatics pipelines do not perform equally.

High-throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices.We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding).Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual-based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (HS and FST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes.This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera-removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus.

BACKGROUND: Ixodes ricinus is the most important vector of tick-borne diseases in Europe. A better knowledge of its genome and transcriptome is important for developing control strategies. Previous transcriptomic studies of I. ricinus have focused on gene expression during the blood meal in specific tissues. To obtain a broader picture of changes in gene expression during the blood meal, our study analysed the transcriptome at the level of the whole body for both nymphal and adult ticks. Ixodes ricinus ticks from a highly inbred colony at the University of Neuchâtel were used. We also analysed previously published RNAseq studies to compare the genetic variation between three wild strains and three laboratory strains, including the strain from Neuchâtel. RESULTS: RNA was extracted from whole tick bodies and the cDNA was sequenced, producing 162,872,698 paired-end reads. Our reference transcriptome contained 179,316 contigs, of which 31% were annotated using Trinotate. Gene expression was compared between ticks that differed by feeding status (unfed vs partially fed). We found that blood-feeding in nymphs and female adult ticks increased the expression of cuticle-associated genes. Using a set of 3866 single nucleotide polymorphisms to calculate the heterozygosity, we found that the wild tick populations of I. ricinus had much higher levels of heterozygosity than the three laboratory populations. CONCLUSION: Using high throughput strand-oriented sequencing for whole ticks in different stages and feeding conditions, we obtained a de novo assembly that significantly increased the genomic resources available for I. ricinus. Our study illustrates the importance of analysing the transcriptome at the level of the whole body to gain additional insights into how gene expression changes over the life-cycle of an organism. Our comparison of several RNAseq datasets shows the power of transcriptomic data to accurately characterize genetic polymorphism and for comparing different populations or sources of sequencing material.