Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.     

Detecting invertebrate ecosystem service providers in orchards: traditional methods versus barcoding of environmental DNA in soil

1. The objective of this study was to assess barcoding of environmental DNA as a method for monitoring invertebrate ecosystem service providers in soil samples.
2. We selected 26 invertebrate ecosystem service providers that occur in New Zealand kiwifruit or apple orchards and produced mitochondrial cytochrome c oxidase gene subunit I (cytochrome oxidase I) and/or 28S ribosomal DNA sequences for each. Specific barcode primers were designed for each invertebrate ecosystem service provider and tested, along with generic barcoding cytochrome oxidase I primers, for their ability to detect DNA from invertebrate ecosystem service providers that had been added to sterilized and unsterilized soil samples.
3. Although the specific primers accurately detected the invertebrate ecosystem service providers in more than 96% of the samples, the generic cytochrome oxidase I primers detected only 37% of the invertebrate ecosystem service providers added to the sterilized samples and 2.5% in the unsterilized samples.
4. In a field test, we compared metabarcoding with traditional invertebrate trapping methods to detect the invertebrate ecosystem service providers in 10 kiwifruit and 10 apple orchards. All invertebrate ecosystem service providers were collected in traps in at least one orchard, but very few were identified by metabarcoding of soil environmental DNA.
5. Although the specific primers can be used as a tool for monitoring invertebrate ecosystem service providers in soil samples, methodological improvements are needed before metabarcoding of soil environmental DNA can be used to monitor these taxa.

     

Biosurveillance for invasive insect pest species using an environmental DNA metabarcoding approach and a high salt trap collection fluid

1. With the increase in global trade and warming patterns, the movement, introduction, and establishment of non‐native insect species has increased. A rapid and effective early detection biosurveillance program to identify species of concern is needed to reduce future impacts and costs associated with introduced non‐native species. One of the challenges facing insect surveillance trapping methods is the sheer volume of individual specimens in the collections. Although molecular identification methods are improving, they currently have limitations (e.g., destructive processing of specimens) and a protocol addressing these limitations can support regulatory applications that need morphological evidence to corroborate molecular data.
2. The novel protocol presented here uses a metabarcoding approach to amplify environmental DNA from a saturated salt solution trap fluid, which retains trap specimens for downstream morphological identifications. The use of a saturated salt solution to preserve specimens in traps addresses issues with the high evaporation rate of ethanol in traps, and public safety concerns with other fluid preservation options with unattended traps in public settings.
3. Using a metabarcoding approach, a 407‐nucleotide segment of the cytochrome c oxidase subunit 1 (COI) animal barcode region was successfully amplified from Lindgren funnel trap collection fluids. These traps were placed in forested areas to survey for wood‐boring beetles of regulatory concern. Our results displayed successful amplification of target taxa, including the molecular identification of the Japanese Beetle Popillia japonica, a species regulated in Canada. A second species, Anisandrus maiche, recently introduced to North America, was identified in every trap. The genus Lymantria, which contains numerous species of concern to North American woodlands, was also detected. Also, there were six other species identified of interest due to their potential impacts on native and crop flora and fauna.
4. Our results show how this protocol can be used as an efficient method for the surveillance of insects using a trap with a saturated salt solution and eDNA metabarcoding to detect species of regulatory concern.

     

Targeted and passive environmental DNA approaches outperform established methods for detection of quagga mussels,Dreissena rostriformis bugensis in flowing water

1. The early detection of invasive non‐native species (INNS) is important for informing management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS—particularly during the early stages of an invasion.
2. Here, we compared the use of traditional kick‐net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR and passive detection via metabarcoding with conserved primers) for detection of quagga mussel, Dreissena rostriformis bugensis, a high priority INNS, along a density gradient on the River Wraysbury, UK.
3. All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and relative read count (metabarcoding) were significantly influenced by both mussel density and distance from source population, with distance being the most significant predictor.
4. Synthesis and application. All three molecular approaches were more sensitive than traditional kick‐net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community and consequently the impacts of INNS.

     

Detecting invertebrate species in archived collections using next‐generation sequencing

Invertebrate biodiversity measured at mostly family level is widely used in biological monitoring programmes to assess anthropogenic impacts on ecosystems. However, next‐generation sequencing (NGS) could allow development of new more sensitive biomonitoring tools by allowing rapid species identification. This could be accelerated if archived invertebrate collections and environmental information from past programmes are used to understand species distributions and their environmental responses. In this study, we take archived macroinvertebrate samples from two sites collected on multiple occasions and test whether NGS can successfully detect species. Samples had been stored in 70% ethanol at room temperature for up to 12 years. Three amplicons ranging from 197 to 274 bps within the DNA barcode region were amplified from samples and compared to DNA barcoding libraries to identify species. We were able to amplify partial DNA barcodes from most samples, and species were often detected with multiple amplicons. However, some singletons and taxa poorly covered by DNA barcoding were missed. This suggests additional DNA barcodes will be required to fill ‘gaps’ in current DNA barcode libraries for aquatic macroinvertebrates and/or that it may not be possible to detect all taxa in a sample. Furthermore, older samples often detected fewer taxa and were less reliable for amplification, suggesting NGS is best used on samples within 8 years of collection. Nevertheless, many common taxa with existing DNA barcodes were reliably identified with NGS and were often present at sites across multiple years, showing the potential of NGS for detecting common and abundant species in archived material.     

Increased accuracy of species lists developed for alpine lakes using morphology and cytochrome oxidase I for identification of specimens

The first step in many community ecology studies is to produce a species list from a sample of individuals. Community ecologists now have two viable ways of producing a species list: morphological and barcode identification. In this study, we compared the taxonomic resolution gained by a combined use of both methods and tested whether a change in taxonomic resolution significantly impacted richness estimates for benthic macroinvertebrates sampled from ten lakes in Sequoia National Park, USA. Across all lakes, 77 unique taxa were identified and 42% (32) were reliably identified to species using both barcode and morphological identification. Of the 32 identified to species, 63% (20) were identified solely by comparing the barcode sequence from cytochrome oxidase I to the Barcode of Life reference library. The increased resolution using a combined identification approach compared to identifications based solely on morphology resulted in a significant increase in estimated richness within a lake at the order, family, genus and species levels of taxonomy (P < 0.05). Additionally, young or damaged individuals that could not be identified using morphology were identified using their COI sequences to the genus or species level on average 75% of the time. Our results demonstrate that a combined identification approach improves accuracy of benthic macroinvertebrate species lists in alpine lakes and subsequent estimates of richness. We encourage the use of barcodes for identification purposes and specifically when morphology is insufficient, as in the case of damaged and early life stage specimens of benthic macroinvertebrates.     

Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding

Environmental bulk samples often contain many different taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high‐throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here, we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate bulk samples, collected from a low‐mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5 × 5, 5 × 10, and up to 10 × 20 mm). Tissue powder from each size category was extracted individually and pooled based on tissue weight to simulate samples that were not sorted by biomass (“Unsorted”). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass (“Sorted”). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately fivefold when sorting the samples into three size classes and pooling by specimen abundance. Even coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high‐throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass or size is a simple yet efficient method to reduce current sequencing costs.     

Beavers alter stream macroinvertebrate communities in north-eastern Utah

1. Understanding changes in macroinvertebrate communities is important because they play a large role in stream ecosystem functioning, and they are an important food resource for fish. Beaver-induced changes to stream morphology could alter macroinvertebrate communities, which in turn could affect food webs and ecosystem function. However, studies investigating the effects of North American beaver activities on macroinvertebrates are rare in the inter-mountain west, an area with high potential for beaver-assisted restoration.
2. The aim of this study was to quantify differences in the macroinvertebrate community between unaltered segments of streams and within beaver ponds in north-eastern Utah, U.S.A. We assessed macroinvertebrate species richness, biomass, density, functional feeding group composition, mobility group composition, and macroinvertebrate habitat characteristics to test the hypothesis that macroinvertebrate communities will differ among habitat types (undammed stream segments and beaver ponds) in beaver-occupied streams.
3. Beaver pond communities significantly differed from lotic reach communities in many ways. Beaver ponds were less diverse with 25% fewer species. Although there was variability among streams, in general, beaver ponds had 75% fewer individuals and 90% lower total macroinvertebrate biomass compared to lotic reaches.
4. Regarding functional feeding groups, beaver ponds contained more engulfers, while lotic reaches contained more scrapers, filterers, and gatherers. For mobility groups, beaver ponds had more sprawlers, while lotic reaches had more clingers. Swimmers were also more prevalent in lotic reaches, although this is probably due to the abundance of Baetis within lotic reaches. More beaver pond taxa were classified as lentic-dwelling insects, while more lotic reach taxa were categorised as preferring lotic habitats.
5. The creation of ponds by beavers fundamentally altered the macroinvertebrate community in north-eastern Utah streams. Such changes to stream macroinvertebrate communities suggest that recolonisation of beavers across North America may be altering stream functioning and food webs. Our study highlights the need to further investigate the effects of beaver recolonisation on stream communities.

     

Different surrogacy approaches for stream macroinvertebrates in discriminating human disturbances in Central China

Due to the difficulty of identifying many taxa of freshwater invertebrates to species, many researchers have assessed the utility of surrogates for species-level identifications (e.g. higher taxa) in bioassessment programs. Here, we examined the efficiency of two different approaches to species surrogacy, one using coarser taxonomic resolution and a second approach based on random aggregation (“Best practicable aggregation of species”, BestAgg), in portraying patterns of stream macroinvertebrates in Central China. The main objectives were: (1) to compare the discriminatory power of biodiversity indices and assemblage structure for different levels of human disturbances based on different taxonomic resolution and on BestAgg; (2) to identify the congruence of assemblage-environment and biodiversity-indices-environment relationships for datasets at the species level versus those at surrogate levels. We found that genus-level and BestAgg datasets accurately reproduced the pattern of species-level communities, whereas family- and order-level datasets did not. Specifically, both genus-level and BestAgg approaches performed almost as well as species-level data in distinguishing sites subjected to different disturbance levels. Most of the environmental variables that were important for species-level assemblages, also emerged as significant when analyzing genera and BestAgg surrogates, as shown by both analyses of indices and assemblage composition according to distance-based ordination models. Our results suggest that genus-level taxonomy, which resulted in the least loss of ecological information relative to species-level identification, is sufficient in studies of community ecology and bioassessment of stream macroinvertebrates in Central China. In addition, the BestAgg approach, which required identification of fewer taxa than genus-level analysis, has a similar ability to depict multivariate patterns of macroinvertebrate assemblages and differentiate different disturbance levels. Applying our results could enhance speed and cost-effectiveness of freshwater biomonitoring and bioassessment programs; however, independent determination of best taxonomic level and BestAgg will be required whenever a new geographic area or habitat type is assessed.     

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.     

Considerations for metabarcoding‐based port biological baseline surveys aimed at marine nonindigenous species monitoring and risk assessments

1. Monitoring introduction and spread of nonindigenous species via maritime transport and performing risk assessments require port biological baseline surveys. Yet, the comprehensiveness of these surveys is often compromised by the large number of habitats present in a port, the seasonal variability, and the time‐consuming morphological approach used for taxonomic identification. Metabarcoding represents a promising alternative for rapid comprehensive port biological baseline surveys, but its application in this context requires further assessments.
2. We applied metabarcoding (based on barcodes of the cytochrome c oxidase subunit I and of the 18S ribosomal RNA gene) to 192 port samples collected (a) from diverse habitats (water column—including environmental DNA and zooplankton, sediment, and fouling structures), (b) at different sites (from inner to outer estuary), and iii) during the four seasons of the year.
3. By comparing the biodiversity metrics derived from each sample group, we show that each sampling method resulted in a distinct community profile and that environmental DNA alone cannot substitute for organismal sampling, and that, although sampling at different seasons and locations resulted in higher observed biodiversity, operational results can be obtained by sampling selected locations and seasons.
4. By assessing the taxonomic composition of the samples, we show that metabarcoding data allowed the detection of previously recorded nonindigenous species as well as to reveal presence of new ones, even if in low abundance.
5. Synthesis and application. Our comprehensive assessment of metabarcoding for port biological baseline surveys sets the basics for cost‐effective, standardized, and comprehensive monitoring of nonindigenous species and for performing risk assessments in ports. This development will contribute to the implementation of the recently entered into force International Convention for the Control and Management of Ships' Ballast Water and Sediments.

     

Toward accurate species‐level metabarcoding of arthropod communities from the tropical forest canopy

相似文献   

Relating macroinvertebrate community structure to environmental characteristics and sediment contamination at the scale of the St. Lawrence River

There is still no assessment of the impact of sediment chemicals and environmental conditions on macroinvertebrates at the scale of the St. Lawrence River. In order to assess these impacts in the fluvial section of the St. Lawrence River including the Montreal harbour, the community structure of macroinvertebrates using different taxonomic aggregations (genus and family) and taxa attributes (abundance, presence–absence, indicator taxa) was assessed. The goal of the study was to determine the indicator taxa of macroinvertebrates along the fluvial continuum and relate changes in macroinvertebrate community to sediment chemical conditions and environmental characteristics of habitats using variance partitioning. This study also evaluated which taxonomic level and taxa attributes of macroinvertebrates were the most suitable for bioassessment of quality of sediments and habitat environment in the St. Lawrence River. Four different macroinvertebrate assemblages were found distributed along the fluvial continuum using either abundance or presence–absence data and genus or family levels. Indicator taxa characteristic of the different macroinvertebrate communities were associated with the sediment contamination gradient. However, habitat environmental characteristics (water masses, sulphur and DOC in sediments) had more influence on macroinvertebrate assemblages than sediment contamination. Our study confirms that family level analysis can give information comparable to the genus level analysis using presence–absence or abundance of macroinvertebrates, yet a higher number of indicator taxa were detected at the genus level.     

Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs.     

Environmental DNA metabarcoding primers for freshwater fish detection and quantification: In silico and in tanks

Environmental DNA (eDNA) techniques refer to utilizing the organisms’ DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high‐throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein‐coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs—AcMDB07, MiFish‐U, Ve16S1, and Ve16S3—designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish‐U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.     

Salamander diversity alters stream macroinvertebrate community structure

相似文献   

Minimizing polymerase biases in metabarcoding

DNA metabarcoding is an increasingly popular method to characterize and quantify biodiversity in environmental samples. Metabarcoding approaches simultaneously amplify a short, variable genomic region, or “barcode,” from a broad taxonomic group via the polymerase chain reaction (PCR), using universal primers that anneal to flanking conserved regions. Results of these experiments are reported as occurrence data, which provide a list of taxa amplified from the sample, or relative abundance data, which measure the relative contribution of each taxon to the overall composition of amplified product. The accuracy of both occurrence and relative abundance estimates can be affected by a variety of biological and technical biases. For example, taxa with larger biomass may be better represented in environmental samples than those with smaller biomass. Here, we explore how polymerase choice, a potential source of technical bias, might influence results in metabarcoding experiments. We compared potential biases of six commercially available polymerases using a combination of mixtures of amplifiable synthetic sequences and real sedimentary DNA extracts. We find that polymerase choice can affect both occurrence and relative abundance estimates and that the main source of this bias appears to be polymerase preference for sequences with specific GC contents. We further recommend an experimental approach for metabarcoding based on results of our synthetic experiments.     

The efficacy of DNA barcoding in the classification,genetic differentiation,and biodiversity assessment of benthic macroinvertebrates

Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.     

Effects of consumer surface sterilization on diet DNA metabarcoding data of terrestrial invertebrates in natural environments and feeding trials

1. DNA metabarcoding is an emerging tool used to quantify diet in environments and consumer groups where traditional approaches are unviable, including small‐bodied invertebrate taxa. However, metabarcoding of small taxa often requires DNA extraction from full body parts (without dissection), and it is unclear whether surface contamination from body parts alters presumed diet presence or diversity.
2. We examined four different measures of diet (presence, rarefied read abundance, richness, and species composition) for a terrestrial invertebrate consumer (the spider Heteropoda venatoria) both collected in its natural environment and fed an offered diet item in contained feeding trials using DNA metabarcoding of full body parts (opisthosomas). We compared diet from consumer individuals surface sterilized to remove contaminants in 10% commercial bleach solution followed by deionized water with a set of unsterilized individuals.
3. We found that surface sterilization did not significantly alter any measure of diet for consumers in either a natural environment or feeding trials. The best‐fitting model predicting diet detection in feeding trial consumers included surface sterilization, but this term was not statistically significant (β = −2.3, p‐value = .07).
4. Our results suggest that surface contamination does not seem to be a significant concern in this DNA diet metabarcoding study for consumers in either a natural terrestrial environment or feeding trials. As the field of diet DNA metabarcoding continues to progress into new environmental contexts with various molecular approaches, we suggest ongoing context‐specific consideration of the possibility of surface contamination.

     

Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.