Molecular identification of Hymenopteran insects collected by using Malaise traps from Hazarganji Chiltan National Park Quetta, Pakistan.

The order Hymenoptera holds great significance for humans, particularly in tropical and subtropical regions, due to its role as a pollinator of wild and cultivated flowering plants, parasites of destructive insects and honey producers. Despite this importance, limited attention has been given to the genetic diversity and molecular identification of Hymenopteran insects in most protected areas. This study provides insights into the first DNA barcode of Hymenopteran insects collected from Hazarganji Chiltan National Park (HCNP) and contributes to the global reference library of DNA barcodes. A total of 784 insect specimens were collected using Malaise traps, out of which 538 (68.62%) specimens were morphologically identified as Hymenopteran insects. The highest abundance of species of Hymenoptera (133/538, 24.72%) was observed during August and least in November (16/538, 2.97%). Genomic DNA extraction was performed individually from 90/538 (16.73%) morphologically identified specimens using the standard phenol-chloroform method, which were subjected separately to the PCR for their molecular confirmation via the amplification of cytochrome c oxidase subunit 1 (cox1) gene. The BLAST analyses of obtained sequences showed 91.64% to 100% identities with related sequences and clustered phylogenetically with their corresponding sequences that were reported from Australia, Bulgaria, Canada, Finland, Germany, India, Israel, and Pakistan. Additionally, total of 13 barcode index numbers (BINs) were assigned by Barcode of Life Data Systems (BOLD), out of which 12 were un-unique and one was unique (BOLD: AEU1239) which was assigned for Anthidium punctatum. This indicates the potential geographical variation of Hymenopteran population in HCNP. Further comprehensive studies are needed to molecularly confirm the existing insect species in HCNP and evaluate their impacts on the environment, both as beneficial (for example, pollination, honey producers and natural enemies) and detrimental (for example, venomous stings, crop damage, and pathogens transmission).

Comparing modern identification methods for wild bees: Metabarcoding and image-based morphological taxonomic assignment.

With the decline of bee populations worldwide, studies determining current wild bee distributions and diversity are increasingly important. Wild bee identification is often completed by experienced taxonomists or by genetic analysis. The current study was designed to compare two methods of identification including: (1) morphological identification by experienced taxonomists using images of field-collected wild bees and (2) genetic analysis of composite bee legs (multiple taxa) using metabarcoding. Bees were collected from conservation grasslands in eastern Iowa in summer 2019 and identified to the lowest taxonomic unit using both methods. Sanger sequencing of individual wild bee legs was used as a positive control for metabarcoding. Morphological identification of bees using images resulted in 36 unique taxa among 22 genera, and >80% of Bombus specimens were identified to species. Metabarcoding was limited to genus-level assignments among 18 genera but resolved some morphologically similar genera. Metabarcoding did not consistently detect all genera in the composite samples, including kleptoparasitic bees. Sanger sequencing showed similar presence or absence detection results as metabarcoding but provided species-level identifications for cryptic species (i.e., Lasioglossum). Genus-specific detections were more frequent with morphological identification than metabarcoding, but certain genera such as Ceratina and Halictus were identified equally well with metabarcoding and morphology. Genera with proportionately less tissue in a composite sample were less likely to be detected using metabarcoding. Image-based methods were limited by image quality and visible morphological features, while genetic methods were limited by databases, primers, and amplification at target loci. This study shows how an image-based identification method compares with genetic techniques, and how in combination, the methods provide valuable genus- and species-level information for wild bees while preserving tissue for other analyses. These methods could be improved and transferred to a field setting to advance our understanding of wild bee distributions and to expedite conservation research.

Improving the efficiency of adaptive management methods in multiple fishways using environmental DNA.

Dams and weirs impede the continuity of rivers and transit of migratory fish. To overcome this obstacle, fishways are installed worldwide; however, management after installation is important. The Miyanaka Intake Dam has three fish ladders with different flow velocities and discharges and has been under adaptive management since 2012. Fish catch surveys, conducted as an adaptive management strategy, place a heavy burden on fish. Furthermore, a large number of investigators must be mobilized during the 30-day investigation period. Thus, a monitoring method using environmental DNA that exerts no burden on fish and requires only a few surveyors (to obtain water samples) and an in-house analyst was devised; however, its implementation in a fishway away from the point of analysis and with limited flow space and its effective water sampling frequency have not been reported. Therefore, in 2019, we started a trial aiming to evaluate the methods and application conditions of environmental DNA surveys for the continuous and long-term monitoring of various fish fauna upstream and downstream of the Miyanaka Intake Dam. To evaluate the fish fauna, the results of an environmental DNA survey (metabarcoding method) for 2019 to 2022 were compared to those of a catch survey in the fishway from 2012 to 2022. The results confirmed the use of environmental DNA surveys in evaluating the contribution of fishways to biodiversity under certain conditions and introduced a novel method for sample collection.

Fecal DNA metabarcoding helps characterize the Canada jay's diet and confirms its reliance on stored food for winter survival and breeding.

Accurately determining the diet of wild animals can be challenging if food items are small, visible only briefly, or rendered visually unidentifiable in the digestive system. In some food caching species, an additional challenge is determining whether consumed diet items have been previously stored or are fresh. The Canada jay (Perisoreus canadensis) is a generalist resident of North American boreal and subalpine forests with anatomical and behavioural adaptations allowing it to make thousands of arboreal food caches in summer and fall that are presumably responsible for its high winter survival and late winter/early spring breeding. We used DNA fecal metabarcoding to obtain novel information on nestling diets and compiled a dataset of 662 published and unpublished direct observations or stomach contents identifications of natural foods consumed by Canada jays throughout the year. We then used detailed natural history information to make informed decisions on whether each item identified to species in the diets of winter adults and nestlings was best characterized as 'likely cached', 'likely fresh' (i.e., was available as a non-cached item when it appeared in a jay's feces or stomach), or 'either possible'. Of the 87 food items consumed by adults in the winter, 39% were classified as 'likely cached' and 6% were deemed to be 'likely fresh'. For nestlings, 29% of 125 food items identified to species were 'likely cached' and 38% were 'likely fresh'. Our results support both the indispensability of cached food for Canada jay winter survival and previous suggestions that cached food is important for late winter/early spring breeding. Our work highlights the value of combining metabarcoding, stomach contents analysis, and direct observations to determine the cached vs. non-cached origins of consumed food items and the identity of food caches, some of which could be especially vulnerable to degradation through climate change.

Decoding the molecular phylogenetics of ornamental catfishes (siluriformes) of North East India using DNA barcoding approach.

BACKGROUND: Catfishes (order Siluriformes) are among the most diverse and widely distributed fish groups in the world. They are not only used for human consumption but are also a major part of the ornamental fish trade. Being a Biodiversity Hotspot, the North Eastern Region of India is home to a diverse population of ornamental fishes. Catfishes contain a humongous number of species; in this study, the authors have tried to elucidate the phylogenetic relationship of some important ornamental catfishes found in North East India using DNA barcodes. METHODS AND RESULTS: In this study, we have tried to explore the phylogenetic history of 13 species (41 specimens) of ornamental catfishes spanning 12 genera and 9 families of Siluriformes using DNA barcoding. Pairwise genetic distances using Kimura 2-Parameter (K2P) were calculated at intra-specific and inter-specific levels. A Neighbor-Joining tree was constructed to understand the phylogenetic relationship among the nine different catfish families. All the specimens under this study clustered with their respective species under the same family and formed three sub-clades. However, Olyra longicaudata, belonging to the Bagridae family, did not cluster with other species from the same family. In this study, the authors have suggested a revision of the classification of O. longicaudata back to its original family, Olyridae. CONCLUSIONS: In this study, the maximum intraspecific genetic distance of 0.03 and the minimum interspecific genetic distance of 0.14 were observed among the species. Therefore, it is evident that there is a barcoding gap among the species, which helped in the correct identification of the species. Thus, DNA barcoding helped complement the phenetic approach and also revealed a different phylogenetic relationship among the catfishes belonging to the Bagridae family.

Low coverage of species constrains the use of DNA barcoding to assess mosquito biodiversity.

Mosquitoes (Culicidae) represent the main vector insects globally, and they also inhabit many of the terrestrial and aquatic habitats of the world. DNA barcoding and metabarcoding are now widely used in both research and routine practices involving mosquitoes. However, these methodologies rely on information available in databases consisting of barcode sequences representing taxonomically identified voucher specimens. In this study, we assess the availability of public data for mosquitoes in the main online databases, focusing specifically on the two most widely used DNA barcoding markers in Culicidae: COI and ITS2. In addition, we test hypotheses on possible factors affecting species coverage (i.e., the percentage of species covered in the online databases) for COI in different countries and the occurrence of the DNA barcode gap for COI. Our findings showed differences in the data publicly available in the repositories, with a taxonomic or species coverage of 28.4-30.11% for COI in BOLD + GenBank, and 12.32% for ITS2 in GenBank. Afrotropical, Australian and Oriental biogeographic regions had the lowest coverages, while Nearctic, Palearctic and Oceanian had the highest. The Neotropical region had an intermediate coverage. In general, countries with a higher diversity of mosquitoes and higher numbers of medically important species had lower coverage. Moreover, countries with a higher number of endemic species tended to have a higher coverage. Although our DNA barcode gap analyses suggested that the species boundaries need to be revised in half of the mosquito species available in the databases, additional data must be gathered to confirm these results and to allow explaining the occurrence of the DNA barcode gap. We hope this study can help guide regional species inventories of mosquitoes and the completion of a publicly available reference library of DNA barcodes for all mosquito species.

Development and validation of a DNA-based multi-species biomonitoring toolkit using a high-throughput qPCR platform: A case study of Irish shellfish species.

Biomonitoring of marine life has been enhanced in recent years by the integration of innovative DNA-based approaches, which offer advantages over more laborious techniques (e.g. microscopy). However, trade-offs between throughput, sensitivity and quantitative measurements must be made when choosing between the prevailing molecular methodologies (i.e. metabarcoding or qPCR/dPCR). Thus, the aim of the present study was to demonstrate the utility of a microfluidic-enabled high-throughput quantitative PCR platform (HTqPCR) for the rapid and cost-effective development and validation of a DNA-based multi-species biomonitoring toolkit, using larvae of 23 commercially targeted bivalve and crustacean species as a case study. The workflow was divided into three main phases: definition of (off-) target taxa and establishment of reference databases (PHASE 1); selection/development and assessment of molecular assays (PHASE 2); and protocol optimization and field validation (PHASE 3). 42 assays were eventually chosen and validated. Genetic signal not only showed good correlation with direct visual counts by microscopy but also showed the ability to provide quantitative data at the highest taxonomic resolution (species level) in a time- and cost-effective fashion. This study developed a biomonitoring toolkit, demonstrating the considerable advantages of this state-of-the-art technology in boosting the developmental testing and application of panels of molecular assays for the monitoring and management of natural resources. Once developed, this approach provides a cost and time-effective alternative compared to other multi-species approaches (e.g. metabarcoding). In addition, it is transferable to a wide range of species and will aid future monitoring programmes.

A comprehensive DNA barcoding reference database for Plecoptera of Switzerland.

DNA barcoding is an essential tool in modern biodiversity sciences. Despite considerable work to barcode the tree of life, many groups, including insects, remain partially or totally unreferenced, preventing barcoding from reaching its full potential. Aquatic insects, especially the three orders Ephemeroptera, Plecoptera, and Trichoptera (EPT), are key freshwater quality indicators worldwide. Among them, Plecoptera (stoneflies), which are among the most sensitive aquatic insects to habitat modification, play a central role in river monitoring surveys. Here, we present an update of the Plecoptera reference database for (meta)barcoding in Switzerland, now covering all 118 species known from this country. Fresh specimens, mostly from rare or localized species, were collected, and 151 new CO1 barcodes were generated. These were merged with the 422 previously published sequences, resulting in a dataset of 573 barcoded specimens. Our CO1 dataset was delimited in 115 CO1 clusters based on a priori morphological identifications, of which 17% are newly reported for Switzerland, and 4% are newly reported globally. Among the 115 CO1 clusters, 85% showed complete congruence with morphology. Distance-based analysis indicated local barcoding gaps in 97% of the CO1 clusters. This study significantly improves the Swiss reference database for stoneflies, enhancing future species identification accuracy and biodiversity monitoring. Additionally, this work reveals cryptic diversity and incongruence between morphology and barcodes, both presenting valuable opportunities for future integrative taxonomic studies. Voucher specimens, DNA extractions and reference barcodes are available for future developments, including metabarcoding and environmental DNA surveys.

Integrated DNA barcoding methods to identify species in the processed fish products from Chinese market.

DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets.

TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Species delimitation of tea plants (Camellia sect. Thea) based on super-barcodes.

BACKGROUND: The era of high throughput sequencing offers new paths to identifying species boundaries that are complementary to traditional morphology-based delimitations. De novo species delimitation using traditional or DNA super-barcodes serve as efficient approaches to recognizing putative species (molecular operational taxonomic units, MOTUs). Tea plants (Camellia sect. Thea) form a group of morphologically similar species with significant economic value, providing the raw material for tea, which is the most popular nonalcoholic caffeine-containing beverage in the world. Taxonomic challenges have arisen from vague species boundaries in this group. RESULTS: Based on the most comprehensive sampling of C. sect. Thea by far (165 individuals of 39 morphospecies), we applied three de novo species delimitation methods (ASAP, PTP, and mPTP) using plastome data to provide an independent evaluation of morphology-based species boundaries in tea plants. Comparing MOTU partitions with morphospecies, we particularly tested the congruence of MOTUs resulting from different methods. We recognized 28 consensus MOTUs within C. sect. Thea, while tentatively suggesting that 11 morphospecies be discarded. Ten of the 28 consensus MOTUs were uncovered as morphospecies complexes in need of further study integrating other evidence. Our results also showed a strong imbalance among the analyzed MOTUs in terms of the number of molecular diagnostic characters. CONCLUSION: This study serves as a solid step forward for recognizing the underlying species boundaries of tea plants, providing a needed evidence-based framework for the utilization and conservation of this economically important plant group.

New records of native and introduced fish species in a river basin of Western Ecuador, the Chocó-Darien Ecoregion, using DNA barcoding.

DNA barcoding, based on mitochondrial markers, is widely applied in species identification and biodiversity studies. The aim of this study was to establish a barcoding reference database of fishes inhabiting the Cube River from Western Ecuador in the Chocó-Darien Global Ecoregion (CGE), a threatened ecoregion with high diversity and endemism, and evaluate the applicability of using barcoding for the identification of fish species. Barcode sequences were obtained from seven orders, 17 families, 23 genera and 26 species, which were validated through phylogenetic analysis, morphological measurements, and literature review. Our results showed that 43% of fish species in this region are endemic, confirmed the presence of known species in the area, and included the addition of three new records of native (Hoplias microlepis, Rhamdia guatemalensis and Sicydium salvini) and an introduced species (Xiphophorus maculatus) to Ecuador. In addition, eight species were barcoded for the first time. Species identification based on barcoding and morphology showed discrepancy with species lists from previous studies in the CGE, suggesting that the current baseline of western fishes of Ecuador is still incomplete. Because this study analyzed fishes from a relatively small basin (165 km2), more molecular-based studies focusing on fish are needed to achieve a robust sequence reference library of species inhabiting Western Ecuador. The new sequences of this study will be useful for future comparisons and biodiversity monitoring, supporting the application of barcoding tools for studying fish diversity in genetically unexplored regions and to develop well-informed conservation programs.

A DNA barcode library of Austrian geometridae (Lepidoptera) reveals high potential for DNA-based species identification.

Situated in the Eastern section of the European Alps, Austria encompasses a great diversity of different habitat types, ranging from alpine to lowland Pannonian ecosystems, and a correspondingly high level of species diversity, some of which has been addressed in various DNA barcoding projects. Here, we report a DNA barcode library of all the 476 species of Geometridae (Lepidoptera) that have been recorded in Austria. As far as possible, species were sampled from different Austrian regions in order to capture intraspecific genetic variation. In total, 2500 DNA barcode sequences, representing 438 species, were generated in this study. For complete coverage of Austrian geometrid species in the subsequent analyses, the dataset was supplemented with DNA barcodes from specimens of non-Austrian origin. Species delimitations by ASAP, BIN and bPTP methods yielded 465, 510 and 948 molecular operational taxonomic units, respectively. Congruency of BIN and ASAP partitions with morphospecies assignments was reasonably high (85% of morphospecies in unique partitions), whereas bPTP appeared to overestimate the number of taxonomic units. The study furthermore identified taxonomically relevant cases of morphospecies splitting and sharing in the molecular partitions. We conclude that DNA barcoding and sequence analysis revealed a high potential for accurate DNA-based identification of the Austrian Geometridae species. Additionally, the study provides an updated checklist of the geometrid moths of Austria.

Enhancing amphibian biomonitoring through eDNA metabarcoding.

Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and non-invasiveness. This approach emerges as a game-changer for the elusive realm of endangered and rare species-think nocturnal, environmentally elusive amphibians. Here, we have established a framework for constructing a reliable metabarcoding pipeline for amphibians, covering primer design, performance evaluation, laboratory validation, and field validation processes. The Am250 primer, located on the mitochondrial 16S gene, was optimal for the eDNA monitoring of amphibians, which demonstrated higher taxonomic resolution, smaller species amplification bias, and more extraordinary detection ability compared to the other primers tested. Am250 primer exhibit an 83.8% species amplification rate and 75.4% accurate species identification rate for Chinese amphibians in the in silico PCR and successfully amplified all tested species of the standard samples in the in vitro assay. Furthermore, the field-based mesocosm experiment showed that DNA can still be detected by metabarcoding even days to weeks after organisms have been removed from the mesocosm. Moreover, field mesocosm findings indicate that eDNA metabarcoding primers exhibit different read abundances, which can affect the relative biomass of species. Thus, appropriate primers should be screened and evaluated by three experimental approaches: in silico PCR simulation, target DNA amplification, and mesocosm eDNA validation. The selection of a single primer set or multiple primers' combination should be based on the monitoring groups to improve the species detection rate and the credibility of results.

Finding the perfect pairs: A matchmaking of plant markers and primers for multi-marker eDNA metabarcoding.

As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi-marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior-supported primers for immediate applications. The ITS2 was the best-performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications.

Optimizing waterborne eDNA capture from waterholes in savanna systems under remote field conditions.

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA-based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 µm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor-prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

Environmental DNA metabarcoding of freshwater fish in Malaysian tropical rivers using short-read nanopore sequencing as a potential biomonitoring tool.

The approach of combining cost-effective nanopore sequencing and emerging environmental DNA (eDNA) metabarcoding could prove to be a promising tool for biodiversity documentation, especially in Malaysia. Given the substantial funding constraints in recent years, especially in relation to the country's biodiversity, many researchers have been limited to conduct restricted research without extended monitoring periods, potentially hindering comprehensive surveys and could compromise the conservation efforts. Therefore, the present study aimed to evaluate the application of eDNA metabarcoding on freshwater fish using short reads generated through nanopore sequencing. This assessment focused on species detection in three selected rivers within the Endau Rompin Landscape in Malaysia. Additionally, the study compared levels of species detection between eDNA metabarcoding and conventional sampling methods, examined the effectiveness of primer choice, and applied both metabarcoding and shotgun sequencing to the eDNA approach. We successfully identified a total of 22 and 71 species with an identification threshold of >97% and >90%, respectively, through the MinION platform. The eDNA metabarcoding approach detected over 13% more freshwater fish species than when the conventional method was used. Notably, the distinction in freshwater fish detection between eDNA primers for 12S rRNA and cytochrome oxidase I was insignificant. The cost for eDNA metabarcoding proved to be more effective compared to conventional sampling with cost reduction at 33.4%. With favourable cost-effectiveness and increased species detection, eDNA metabarcoding could complement existing methods, enhance holistic diversity documentation for targeted habitats and facilitate effective conservation planning.

Environmental DNA metabarcoding reveals the effects of seafloor litter and trawling on marine biodiversity.

Environmental DNA (eDNA) techniques are emerging as promising tools for monitoring marine communities. However, they have not been applied to study the integrated effects of anthropogenic pressures on marine biodiversity. This study examined the relationships between demersal community species composition, key environmental features, and anthropogenic impacts such as fishing effort and seafloor litter using eDNA data in the central Tyrrhenian Sea. The results indicated that both fishing effort and seafloor litter influenced species composition and diversity. The adaptive traits of marine species played a critical role in their response to debris accumulation and fishing. Mobile species appeared to use relocation strategies, while sessile species showed flexibility in the face of disturbance. Epibiotic species relied on passive transport. The use of eDNA-based methods is a valuable resource for monitoring anthropogenic impacts during scientific surveys, enhancing our ability to monitor marine ecosystems and more effectively assess the effects of pollution.

DNA and RNA Stability of Marine Microalgae in Cold-Stored Sediments and Its Implications in Metabarcoding Analyses.

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection.

Extracting, filtering and simulating cellular barcodes using CellBarcode tools.

Identifying true DNA cellular barcodes among polymerase chain reaction and sequencing errors is challenging. Current tools are restricted in the diversity of barcode types supported or the analysis strategies implemented. As such, there is a need for more versatile and efficient tools for barcode extraction, as well as for tools to investigate which factors impact barcode detection and which filtering strategies to best apply. Here we introduce the package CellBarcode and its barcode simulation kit, CellBarcodeSim, that allows efficient and versatile barcode extraction and filtering for a range of barcode types from bulk or single-cell sequencing data using a variety of filtering strategies. Using the barcode simulation kit and biological data, we explore the technical and biological factors influencing barcode identification and provide a decision tree on how to optimize barcode identification for different barcode settings. We believe that CellBarcode and CellBarcodeSim have the capability to enhance the reproducibility and interpretation of barcode results across studies.