A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding‐based biomonitoring

This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species‐level assignment, so called “dark taxa.” Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the “taxonomic impediment”; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species‐rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.     

A from‐benchtop‐to‐desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

The main objective of this work was to develop and validate a robust and reliable “from‐benchtop‐to‐desktop” metabarcoding workflow to investigate the diet of invertebrate‐eaters. We applied our workflow to faecal DNA samples of an invertebrate‐eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross‐contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.     

Quantitative and qualitative assessment of pollen DNA metabarcoding using constructed species mixtures

Pollen DNA metabarcoding—marker‐based genetic identification of potentially mixed‐species pollen samples—has applications across a variety of fields. While basic species‐level pollen identification using standard DNA barcode markers is established, the extent to which metabarcoding (a) correctly assigns species identities to mixes (qualitative matching) and (b) generates sequence reads proportionally to their relative abundance in a sample (quantitative matching) is unclear, as these have not been assessed relative to known standards. We tested the quantitative and qualitative robustness of metabarcoding in constructed pollen mixtures varying in species richness (1–9 species), taxonomic relatedness (within genera to across class) and rarity (5%–100% of grains), using Illumina MiSeq with the markers rbcL and ITS2. Qualitatively, species composition determinations were largely correct, but false positives and negatives occurred. False negatives were typically driven by lack of a barcode gap or rarity in a sample. Species richness and taxonomic relatedness, however, did not strongly impact correct determinations. False positives were likely driven by contamination, chimeric sequences and/or misidentification by the bioinformatics pipeline. Quantitatively, the proportion of reads for each species was only weakly correlated with its relative abundance, in contrast to suggestions from some other studies. Quantitative mismatches are not correctable by consistent scaling factors, but instead are context‐dependent on the other species present in a sample. Together, our results show that metabarcoding is largely robust for determining pollen presence/absence but that sequence reads should not be used to infer relative abundance of pollen grains.     

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.     

Sorting specimen‐rich invertebrate samples with cost‐effective NGS barcodes: Validating a reverse workflow for specimen processing

Biologists frequently sort specimen‐rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a “reverse workflow” is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next‐generation sequencing (NGS) barcoding pipeline that allows for cost‐effective high‐throughput generation of short specimen‐specific barcodes (313 bp of COI; laboratory cost <$0.50 per specimen) through next‐generation sequencing of tagged amplicons. We applied our approach to a large sample of tropical ants, obtaining barcodes for 3,290 of 4,032 specimens (82%). NGS barcodes and their corresponding specimens were then sorted into molecular operational taxonomic units (mOTUs) based on objective clustering and Automated Barcode Gap Discovery (ABGD). High diversity of 88–90 mOTUs (4% clustering) was found and morphologically validated based on preserved vouchers. The mOTUs were overwhelmingly in agreement with morphospecies (match ratio 0.95 at 4% clustering). Because of lack of coverage in existing barcode databases, only 18 could be accurately identified to named species, but our study yielded new barcodes for 48 species, including 28 that are potentially new to science. With its low cost and technical simplicity, the NGS barcoding pipeline can be implemented by a large range of laboratories. It accelerates invertebrate species discovery, facilitates downstream taxonomic work, helps with building comprehensive barcode databases and yields precise abundance information.     

Site‐specific distribution of oak rhizosphere‐associated oomycetes revealed by cytochrome c oxidase subunit II metabarcoding

The phylum Oomycota comprises important tree pathogens like Phytophthora quercina, involved in central European oak decline, and Phytophthora cinnamomi shown to affect holm oaks among many other hosts. Despite the importance to study the distribution, dispersal and niche partitioning of this phylum, metabarcoding surveys, and studies considering environmental factors that could explain oomycete community patterns are still rare. We investigated oomycetes in the rhizosphere of evergreen oaks in a Spanish oak woodland using metabarcoding based on Illumina sequencing of the taxonomic marker cytochrome c oxidase subunit II (cox2). We developed an approach amplifying a 333 bp long fragment using the forward primer Hud‐F (Mycologia, 2000) and a reverse primer found using DegePrime (Applied and Environmental Microbiology, 2014). Factors reflecting topo‐edaphic conditions and tree health were linked to oomycete community patterns. The majority of detected OTUs belonged to the Peronosporales. Most taxa were relatives of the Pythiaceae, but relatives of the Peronosporaceae and members of the Saprolegniales were also found. The most abundant OTUs were related to Globisporangium irregulare and P. cinnamomi, both displaying strong site‐specific patterns. Oomycete communities were strongly correlated with the environmental factors: altitude, crown foliation, slope and soil skeleton and soil nitrogen. Our findings illustrate the significance of small scale variation in habitat conditions for the distribution of oomycetes and highlight the importance to study oomycete communities in relation to such ecological patterns.     

DNA barcoding the ichthyofauna of the Yangtze River: Insights from the molecular inventory of a mega‐diverse temperate fauna

Intensification of inland fisheries and aquatic landscape conversion led to a drastic decline of fish populations in the Yangtze River (YR) during the last decades. This situation urges for the development of a large‐scale molecular assessment of YR ichthyofauna to further develop standardized methods of molecular identification for conservation and fisheries management purposes. We present here the results of a large‐scale campaign to DNA barcode YR freshwater fishes that succeeded in producing 1,424 new DNA barcodes for 123 species. Together with 1,406 sequences mined from BOLD and GenBank, a reference library including 2,830 DNA barcodes for 238 species was compiled. By using four DNA‐based species delimitation methods, RESL, ABGD, mPTP and mGMYC, 230 operational taxonomic units (OTUs) were identified and 195 species displayed OTUs that tightly match species boundaries. No barcoding gap was observed; however, and conflicting cases of species and OTU delimitation were identified. A total of 23 species with maximum intraspecific distances above 2% were detected and null genetic distances to the nearest phylogenetic relatives were detected in 11 species. Among those 23 species, 16 were represented by multiple OTUs amounting to 40 OTUs delineated. Several cases of multiple OTUs confined to species boundaries were detected suggesting the presence of overlooked species. A total of 18 OTUs, however, were shared by several species and particularly so for the Qinghai‐Tibet plateau endemic species. These results are discussed with reference to previous large‐scale DNA barcoding campaign and compared to previous phylogeographic studies in the YR.     

Metabarcoding for stomach‐content analyses of Pygmy devil ray (Mobula kuhlii cf. eregoodootenkee): Comparing tissue and ethanol preservative‐derived DNA

The application of high‐throughput sequencing to retrieve multi‐taxon DNA from different substrates such as water, soil, and stomach contents has enabled species identification without prior knowledge of taxon compositions. Here we used three minibarcodes designed to target mitochondrial COI in plankton, 16S in fish, and 16S in crustaceans, to compare ethanol‐ and tissue‐derived DNA extraction methodologies for metabarcoding. The stomach contents of pygmy devilrays (Mobula kuhlii cf. eregoodootenkee) were used to test whether ethanol‐derived DNA would provide a suitable substrate for metabarcoding. The DNA barcoding assays indicated that tissue‐derived operational taxonomic units (OTUs) were greater compared to those from extractions performed directly on the ethanol preservative. Tissue‐derived DNA extraction is therefore recommended for broader taxonomic coverage. Metabarcoding applications should consider including the following: (i) multiple barcodes, both taxon specific (e.g., 12S or 16S) and more universal (e.g., COI or 18S) to overcome bias and taxon misidentification and (ii) PCR inhibitor removal steps that will likely enhance amplification yields. However, where tissue is limited or no longer available, but the ethanol‐preservative medium is still available, metabarcoding directly from ethanol does recover the majority of common OTUs, suggesting the ethanol‐retrieval method could be applicable for dietary studies. Metabarcoding directly from preservative ethanol may also be useful where tissue samples are limited or highly valued; bulk samples are collected, such as for rapid species inventories; or mixed‐voucher sampling is conducted (e.g., for plankton, insects, and crustaceans).     

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High‐throughput sequencing (“HTS”) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed “environmental DNA” or “eDNA”). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called “eDNA metabarcoding” and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

Evaluating the utility of cox1 for cetacean species identification

The mitochondrial cytochrome c oxidase I (cox1) gene has been promoted as a universal reference gene, or barcode, to identify organisms to the species level. We evaluated whether cox1 would be appropriate to diagnose cetacean species. The 5′ end of cox1 (686 base pairs, bp) was sequenced for 46 of 86 recognized species of cetaceans. In addition, we included 105 sequences from GenBank, increasing our taxonomic coverage to 61 species. Particular focus was placed on sampling two subfamilies that contain closely related taxa: the Delphininae and the Globicephalinae. Species‐specific sequences were observed for all but three taxa (Delphinus delphis, D. capensis, and Stenella coeruleoalba). Although correct assignment was seen for most species, significant overlap between intra‐ and interspecific variation makes cox1 an imperfect barcode for cetaceans. The efficacy of cox1 was compared to the 5′ end of the cytochrome b (cytb) gene, a mitochondrial region routinely used for cetacean species identification. Although cytb performed better than cox1 for some species, this marker could not differentiate other closely related taxa (Eubalaena spp.). Species identification for taxa not reliably identified using cox1 or cytb might be best addressed through use of multiple mitochondrial DNA fragments or other newly developed markers.     

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

Studies of insect assemblages are suited to the simultaneous DNA‐based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR‐amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR‐amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR‐amplified the blend using five primer sets, targeting either COI or 16S, with high‐throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.     

Integrative taxonomy reveals hidden diversity in the southern African darters genus Nannocharax Günther 1867 (Characiformes: Distichodontidae)

The present study explored the diversity of Nannocharax within southern Africa by implementing three species delimitation methods for a data set consisting of 37 mitochondrial cytochrome oxidase subunit I sequences. Two unilocus coalescent methods, the General Mixed Yule Coalescent (GMYC) and the Bayesian implementation of the Poisson Tree Processes (bPTP), and a genetic distance method, the Automatic Barcode Gap Discovery (ABGD), were applied. Both GMYC and bPTP delimited the same operational taxonomic units (OTUs), revealing a higher diversity for the genus in the region than previously recognised, whereas the ABGD failed to delimit the same candidate species. All methods delimited two species groups, and these are supported based on colouration patterning and morphology; the Nannocharax multifasciatus and the Nannocharax macropterus species groups and the delimited OTUs were assigned to each. Two putative new species were identified, Nannocharax cf. lineostriatus “Okavango” from the Okavango River in Angola and N. cf. lineostriatus “Kwanza” from the Kwanza River system in Angola. The distribution of Nannocharax dageti was confirmed for the Upper Zambezi and extended to the Okavango system, and an identification key for the southern Africa Nannocharax species is provided.     

Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries

The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next‐generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom‐designed group‐specific primer set and next‐generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture‐based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co‐occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture‐based methods, for improved mapping of species distribution patterns.     

Plant DNA‐barcode library and community phylogeny for a semi‐arid East African savanna

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA‐barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL‐F, trnH–psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra‐ and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack “barcode gaps”, defined as disparities between the maximum intra‐ and minimum interspecific genetic distances. We found barcode gaps for 72%–89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode‐gap presence or absence for all of the markers in combination and for matK, trnL‐F, and trnH–psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations.     

Comparison of morphological,DNA barcoding,and metabarcoding characterizations of freshwater nematode communities

Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single‐specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.     

Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island,France)

Diatoms are excellent ecological indicators of water quality because they are broadly distributed, they show high species diversity and they respond rapidly to human pressures. In Europe, the Water Framework Directive (WFD) gives the legal basis for the use of this indicator for water quality assessment and its management. Several quality indices, like the Specific Polluosensitivity Index (SPI), were developed to assess the ecological quality status of rivers based on diatom communities. It is based on morphological identifications and count of diatom species present in natural biofilms using a microscope. This methodology requires high taxonomic skills and several hours of analysis per sample as 400 individuals must be identified to species level. Since several years, a molecular approach based on DNA metabarcoding combined to High-Throughput Sequencing (HTS) is developed to characterize species assemblages in environmental samples which is potentially faster and cheaper. The ability of this approach to provide reliable diatom inventories has been demonstrated and its application to water quality assessment is currently being improved. Despite optimization of the DNA metabarcoding process with diatoms, few studies had yet extended it at the scale of a freshwater monitoring network and evaluated the reliability of its quality assessment compared to the classical morphological approach.In the present study we applied DNA metabarcoding to the river monitoring network of the tropical Island Mayotte. This island is a French département since 2011 and the WFD has to be applied. This offered the opportunity to scale up the comparison of molecular and morphological approaches and their ability to produce comparable community inventories and water quality assessments. Benthic diatoms were sampled following WFD standards in 45 river sites in 2014 and 2015 (80 samples). All samples were submitted in parallel to the molecular and the morphological approaches. DNA metabarcoding was carried out using Genelute DNA extraction method, rbcL DNA barcode and PGM sequencing, while microscopic counts were carried out for the classical methodology. Diatom community structures in terms of molecular (OTUs) and of morphological (species) were significantly correlated. However, only 13% of the species was shared by both approaches, with qualitative and quantitative variation due to i) the incompleteness of the reference library (82% of morphological species are not represented in the database), ii) limits in taxonomic knowledge and iii) biases in the estimation of relative abundances linked to diatom cell biovolume. However, ecological quality status assessed with the molecular and morphological SPI values were congruent, and little affected by sequencing depth. DNA metabarcoding of diatom communities allowed a reliable estimation of the quality status for most of the rivers at the scale of the full biomonitoring network of Mayotte Island.     

Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

18S rDNA gene metabarcoding of microeukaryotes and epi-endophytes in the holobiome of seven species of large brown algae

Brown algae (Phaeophyceae) are habitat-forming species in coastal ecosystems and include kelp forests and seaweed beds that support a wide diversity of marine life. Host-associated microbial communities are an integral part of phaeophyte biology, and whereas the bacterial microbial partners have received considerable attention, the microbial eukaryotes associated with brown algae have hardly been studied. Here, we used broadly targeted “pan-eukaryotic” primers (metabarcoding) to investigate brown algal-associated eukaryotes (the eukaryome). Using this approach, we aimed to investigate the eukaryome of seven large brown algae that are important and common species in coastal ecosystems. We also aimed to assess whether these macroalgae harbor novel eukaryotic diversity and to ascribe putative functional roles to the host-associated eukaryome based on taxonomic affiliation and phylogenetic placement. We detected a significant diversity of microeukaryotic and algal lineages associated with the brown algal species investigated. The operational taxonomic units (OTUs) were taxonomically assigned to 10 of the eukaryotic major supergroups, including taxonomic groups known to be associated with seaweeds as epibionts, endobionts, parasites, and commensals. Additionally, we revealed previously unrecorded sequence types, including novel phaeophyte OTUs, particularly in the Fucus spp. samples, that may represent fucoid genomic variants, sequencing artifacts, or undescribed epi-/endophytes. Our results provide baseline data and technical insights that will be useful for more comprehensive seaweed eukaryome studies investigating the evidently lineage-rich and functionally diverse symbionts of brown algae.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.