Barcode Database Research Articles

DNA Barcoding and New Records of Water Mites (Acari, Hydrachnidia) from Bosnia and Herzegovina

During the last decade, DNA barcoding has become a widely applicated approach in the identification of water mite species. Still, our knowledge about the molecular diversity and number of DNA barcodes of water mite species that inhabit Bosnia and Herzegovina is limited. In this study a total of 50 mitochondrial barcode sequences were obtained from 18 water mite species collected in Bosnia and Herzegovina. Seven species, i.e. Sperchon violaceus Walter, 1944, S. milisai Pešić, 2022, Lebertia glabra Thor, 1897, L. reticulata (Koenike, 1919), Torrenticola lukai Pešić, Valdecasas & Garcia-Jimenez, 2012, Hygrobates turcicus Pešić, Esen & Dabert, 2017, and Arrenurus cylindratus Piersig, 1896 are added as new for the water mite fauna of Bosnia and Herzegovina. Our results revealed that all recorded water mite species in our study could be identified when we consider global and regional DNA barcode database.

Comparative analysis of the whole mitochondrial genomes of four species in sect. Chrysantha (Camellia L.), endemic taxa in China

BackgroundThe sect. Chrysantha Chang of plants with yellow flowers of Camellia species as the “Queen of the Tea Family”, most of these species are narrowly distributed endemics of China and are currently listed Grde-II in National Key Protected Wild Plant of China. They are commercially important plants with horticultural medicinal and scientific research value. However, the study of the sect. Chrysantha species genetics are still in its infancy, to date, the mitochondrial genome in sect. Chrysantha has been still unexplored.ResultsIn this study, we provide a comprehensive assembly and annotation of the mitochondrial genomes for four species within the sect. Chrysantha. The results showed that the mitochondrial genomes were composed of closed-loop DNA molecules with sizes ranging from 850,836 bp (C. nitidissima) to 1,098,121 bp (C. tianeensis) with GC content of 45.71–45.78% and contained 48–58 genes, including 28–37 protein-coding genes, 17–20 tRNA genes and 2 rRNA genes. We also examined codon usage, sequence repeats, RNA editing and selective pressure in the four species. Then, we performed a comprehensive comparison of their basic structures, GC contents, codon preferences, repetitive sequences, RNA editing sites, Ka/Ks ratios, haplotypes, and RNA editing sites. The results showed that these plants differ little in gene type and number. C. nitidissima has the greatest variety of genes, while C. tianeensis has the greatest loss of genes. The Ka/Ks values of the atp6 gene in all four plants were greater than 1, indicating positive selection. And the codons ending in A and T were highly used. In addition, the RNA editing sites differed greatly in number, type, location, and efficiency. Twelve, six, five, and twelve horizontal gene transfer (HGT) fragments were found in C. tianeensis, Camellia huana, Camellia liberofilamenta, and C. nitidissima, respectively. The phylogenetic tree clusters the four species of sect. Chrysantha plants into one group, and C. huana and C. liberofilamenta have closer affinities.ConclusionsIn this study, the mitochondrial genomes of four sect. Chrysantha plants were assembled and annotated, and these results contribute to the development of new genetic markers, DNA barcode databases, genetic improvement and breeding, and provide important references for scientific research, population genetics, and kinship identification of sect. Chrysantha plants.

Ghost species form an important component of the epiphytic lichens in temperate forests

Sequencing of environmental samples has great potential for biodiversity research, but its application is limited by the lack of reliable DNA barcode databases for species identifications. Such a database has been created for epiphytic lichens of Europe, allowing us to compare the results of environmental sequencing with standard taxonomic surveys. The species undetected by taxonomic surveys (what we term the ghost component) amount to about half of the species actually present in hectare plots of Central European forests. Some of these, which currently occur only as diaspores or weakly developed thalli, are likely to be favoured in the course of global change. The ghost component usually represents a larger fraction in managed forests than in old-growth unmanaged forests. The total species composition of different plots is much more similar than suggested by taxonomic surveys alone. On a regional scale, this supports the well-known statement that “everything is everywhere, but, the environment selects”.

Eyeing DNA barcoding for species identification of fish larvae.

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.

Role of forest fuelbreaks for browsers: Implications from dietary pattern and food resources survey for sika deer (Cervus nippon)

Fuelbreaks are used to manage wildfires worldwide. Regular mowing within fuelbreaks can alter species composition and transform them into grassland habitats. These changes may provide more herbaceous plants to large herbivores, subsequently influencing their distribution. To clarify the impact of fuelbreaks on herbivores' food resources, we conducted molecular dietary analysis to quantify the dietary composition of sika deer (Cervus nippon) in both the Sino-Russian border fuelbreak and forest interior, with an average distance of 1.6 km away from the fuelbreak. We constructed a local barcode database to enhance the resolution of the molecular dietary analysis. In addition, we surveyed the vegetation in these two habitats to determine the availability of food resources. We also used camera traps to monitor the visitation intensity of sika deer in both habitats. The results indicated that sika deer increased their visitation intensity at the fuelbreak during the snow-free season. The biomass of graminoids and herbaceous plants was high in the fuelbreak. However, the results of the molecular dietary analysis showed that during the snow-free season, the sika deer that visited the fuelbreak consumed fewer herbaceous plants. We used the Jacobs index to determine the dietary preferences of the sika deer and found that they were consistent browsers with a stable preference for woody plants. By assessing habitat quality, specifically the proportion of preferred plants, we found that the habitat quality of the fuelbreak was relatively low. In conclusion, fuelbreaks may attract herbivores. However, for browsers, such as sika deer, the major function of fuelbreaks is not to provide food resources.

Identification of earthworms (Clitellata, Oligochaeta) from Kottayam district in Southwestern India: an integrated traditional and barcoding approach

Earthworms from various land uses in the Kottayam district of Kerala, part of the Western Ghats hotspot in India, were studied using an integrative approach that involved morphoanatomical methods and cytochrome c oxidase I (COI) barcoding techniques for the first time here. The study unveiled the presence of nine earthworm species: <i>Drawida ghatensis</i> Michaelsen, 1910; <i>Moniligaster julkai</i> Narayanan and Paliwal, 2022; <i>Glyphidrilus annandalei</i> Michaelsen, 1910; <i>Pontoscolex corethrurus</i> (Müller, 1857); <i>Eukerria kuekenthali</i> (Michaelsen, 1908); <i>Dichogaster bolaui</i> (Michaelsen, 1891); <i>Megascolex cochinensis</i> Stephenson, 1915; <i>M. konkanensis</i> Fedarb, 1898; and <i>Lampito mauritii</i> Kinberg, 1867, belonging to eight genera. The COI sequences of all nine species were deposited in GenBank. Notably, <i>M. julkai</i> and <i>E. kuekenthali</i> are novel additions to the GenBank barcode database. The Bayesian phylogram and maximum-likelihood indicated that the species <i>M. julkai</i> in this study exhibited a closer relationship to <i>D. ghatensis</i> than to other <i>Moniligaster</i> species. This study provides detailed descriptions and illustrations of all nine species, supplemented by their DNA barcodes, to facilitate future species identification.

Applications and challenges of DNA barcoding in rapid radiation groups: Rhodiola (Crassulaceae) as a case study

ObjectiveRhodiolae Crenulatae Radix et Rhizoma (Hongjingtian in Chinese, RCRR), the roots and rhizomes of Rhodiola crenulata and its application in the medicinal market is very chaotic. In this study, DNA barcoding database and identification engine of Rhodiola species were established, decoction pieces from the medicinal market were identified, and the application and challenges of DNA barcoding in the rapid radiation of Rhodiola species were analyzed. This study provides reference for the protection, rational development, and utilization of endangered resources within Rhodiola species. MethodsA total of 50 original plant samples from 20 species of the genus Rhodiola from Hebei, Xinjiang, Tibet, Jilin, and other major production areas were collected. Theses samples cover the typical distribution area (Qinghai-Tibetan Platea) of Rhodiola species and other scattered alpine regions (Changbai Mountain, Taibai Mountain, Lushan Mountain, etc.), it encompasses all Rhodiola species with thick rhizomes in China. ITS2 and psbA-trnH barcode of Rhodiola database (BORD) were established and an identification engine named Rhodiola-IDE was developed. The stability and accuracy of the standard DNA barcoding database were evaluated using two datasets. Rhodiola-IDE identified 31 decoction pieces of RCRR from the medicinal material market. ResultsThe BORD containing 1 532 sequences of 88 Rhodiola species has been established, and the identification efficiency results showed good accuracy and stability. According to the Chinese Pharmacopoeia (2020 edition), 23 samples (74.2%) were identified as authentic R. crenulata, while the rest of the marketed varieties were R. kirilowii, R. dumulosa, and R. fastigiata. The product label “Larger flower, Hongjingtian” was identified as R. crenulata. Samples labeled as “Smaller flower, Hongjingtian” were identified as R. crenulata, R. kirilowii, and R. fastigiata. ConclusionITS2 and psbA-trnH barcodes can identify monophyletic groups represented by R. crenulata. However, for non-monophyletic species, it is necessary to collect as many samples as possible and combine them with multiple markers for joint identification. This study discussed the application and challenges of DNA barcodes in Rhodiola under rapid radiation conditions, providing a scientific basis for the rational development and utilization of Rhodiola varieties.

In-silico approaches for discrimination of Curcuma species and their closely related family using the novel technique of DNA Barcoding

In this study, we have discriminated and identified the Genus of Curcuma and related species Zingiberaceae using rbcL and trnL DNA barcode primers. Curcuma genus related to the family Zingiberaceae comprises a significant number of medicinal plants renowned for their use in ethnomedicine, playing a pivotal role in the medical, health, and pharmaceutical sectors. Traditionally, morphological methods alone have proven insufficient for accurately identifying species within this family. However, DNA barcoding technology provides a contemporary solution by utilizing plant DNA sequences for species identification, thus enabling effective conservation efforts. We used DNA barcoding techniques and for analysis used the Maximum Parsimony tree in MEGA 11 with the Kimura 2-parameter (KP model) to analyse the genetic relationships between species. Out of the 13 accessions that were studied, 12 accessions belonged to Curcuma caesia and 1 accession belonged to Curcuma aeruginosa. The genetic relationships observed were correlated with the geographical distributions of these species. It was determined that C. aeruginosa is a mutated species of C. caesia. Additionally, 1 specimen of Alpinia galanga, a plant species related to the Zingiberaceae. Barcode primer trnL primer demonstrated a 92% efficiency during the investigation. The rbcL and trnL loci are recommended as potential barcode markers for discriminating between different plant species. This study developed a comprehensive DNA barcoding database that can confidently differentiate between species by combining morphological and molecular data. This database has the potential to identify adulteration in herbal products, combat illegal trade and adulteration of plant species, and assist in germplasm conservation efforts.

Authentication of sushi and sashimi products from northeastern Pará, coastal Amazon, by DNA barcoding reveals commercial frauds and potential threats to human health and environment

The Japanese cuisine has become a huge success in Brazil over the last years, but little is known about the fisheries resources commercialized to supply this specific market. In the present study, we carried out the first molecular characterization of sushi and sashimi products available in northeastern Pará, coastal Amazon, in order to infer what species have been actually exploited in this trade. We sequenced two mitochondrial DNA regions from 172 samples (138 of fish and 34 of shrimp-derived products) that were divided into 28 haplotypes. The comparative analyses with public DNA barcode databases revealed eight species of fish and three of shrimps among the samples, including cases of commercial frauds. For instance, Ruvettus pretiosus and Lepidocybium flavobrunneum were traded as “anchova” (bluefish), a valid and specific commercial name of Pomatomus saltatrix. Both species can cause gastrointestinal issues, which has led to restrictions on their sale in several countries. All commercialized shrimp-derived products were labeled as “camarão” (shrimp), as generic trade name that could not be applied to identify each species. Furthermore, potentially threatened species have also been recorded in the commercial products, such as Salmo salar and Cynoscion acoupa, classified as “nearly threatened” and “vulnerable”, respectively. The later, popularly known as “pescada-amarela” (acoupa weakfish) has been commercialized as “peixe-branco” (white fish), a designation that is not recognized by the Brazilian regulatory agencies. Both DNA markers (COI and 16S rRNA) were highly efficient in identifying the species from all samples, providing the first list of exploited species for Japanese cuisine along the coastal Amazon. Besides revealing issues in the commercialization of Japanese food items, we recorded a largely overlooked diversity of traded species. These data represent a potential risk to public health and to the environmental conservation such as the presence of exotic species and the exploitation of threatened species that should be considered to support effective policies for the regional regulation of this market.

DNA Barcoding Analysis of Betok Fish (Anabas testudineus) from Kampar, Riau Based on Cytochrome Oxidase Subunit I (COI)

Ikan betok (Anabas testudineus, Bloch 1792) is one of fishes which is a member of family Anabantidae. Betok is an endemic fish in Riau Province. The scientific study about DNA barcoding of betok fish is still rarely. This research aims to analyze the DNA barcode sequence of cytochrome c oxidase I (COI) on betok fish. Methods were used in this study such as sampling, total DNA extraction, PCR, electrophoresis, sequencing and data analysis. The COI sequence studied had size of 694 bp. The BLASTn analysis showed that the betok fish had the highest similarity of 99.7% with A. testudineus and the lowest 93.00% with A. cobojinus. There was one nucleotide that characterize the betok fish based on the COI sequence namely nucleotide number 309. This study may enrich the DNA barcode database of betok fish in GeneBank.

CoSFISH: a comprehensive reference database of COI and 18S rRNA barcodes for fish.

Fish, being a crucial component of aquatic ecosystems, holds significant importance from both economic and ecological perspectives. However, the identification of fish at the species level remains challenging, and there is a lack of a taxonomically complete and comprehensive reference sequence database for fish. Therefore, we developed CoSFISH, an online fish database. Currently, the database contains 21 535 cytochrome oxidase I sequences and 1074 18S rRNA sequences of 21 589 species, belonging to 8 classes and 90 orders. We additionally incorporate online analysis tools to aid users in comparing, aligning and analyzing sequences, as well as designing primers. Users can upload their own data for analysis, in addition to using the data stored in the database directly. CoSFISH offers an extensive fish database and incorporates online analysis tools, making it a valuable resource for the study of fish diversity, phylogenetics and biological evolution. Database URL: http://210.22.121.250:8888/CoSFISH/home/indexPage.

The geographic and phylogenetic structure of public DNA barcode databases: an assessment using Chrysomelidae (leaf beetles)

IntroductionDNA barcoding in insects has progressed rapidly, with the ultimate goal of a complete inventory of the world’s species. However, the barcoding effort to date has been driven by a few national campaigns and leaves much of the world unsampled. This study investigates to what degree the current barcode data cover the species diversity across the globe, using the leaf beetle family Chrysomelidae as an example.MethodsA recent version (June 2023) of the Barcode-of-Life database was subjected to test of sampling completeness using the barcode-to-BIN ratio and sampling coverage (SC) metric. All barcodes were placed in a phylogenetic tree of ~600 mitochondrial genomes, applying phylogenetic diversity (PD) and metrics of community phylogenetics to national barcode sets to test for sampling completeness at clade level and reveal the global structure of species diversity.ResultsThe database included 73342 barcodes, grouped into 5310 BINs (species proxies) from 101 countries. Costa Rica contributed nearly half of all barcode sequences, while nearly 50 countries were represented by less than ten barcodes. Only five countries, Costa Rica, Canada, South Africa, Germany, and Spain, had a high sampling completeness, although collectively the barcode database covers most major taxonomic and biogeographically confined lineages. PD showed moderate saturation as more species diversity is added in a country, and community phylogenetics indicated clustering of national faunas. However, at the species level the inventory remained incomplete even in the most intensely sampled countries, and the sampling was insufficient for assessment of global species richness patterns.DiscussionThe sequence-based inventory in Chrysomelidae needs to be greatly expanded to include more areas and deeper local sampling before reaching a knowledge base similar to the existing Linnaean taxonomy. However, placing the barcodes into a backbone phylogenetic tree from mitochondrial genomes, a taxonomically and biogeographically highly structured pattern of global diversity emerges into which all species can be integrated via their barcodes.

DNA Barcoding for Species Identification of Moss-Dwelling Invertebrates: Performance of Nanopore Sequencing and Coverage in Reference Database

In view of the current biodiversity crisis and our need to preserve and improve ecosystem functioning, efficient means for characterizing and monitoring biodiversity are required. DNA barcoding, especially when coupled with new sequencing technologies, is a promising method that can, in principle, also be employed by taxonomic lay people. In this study we compare the performance of DNA barcoding by means of a third-generation sequencing technology, nanopore sequencing with classical Sanger sequencing, based on a sample of invertebrates collected from moss pads in a bog in Austria. We find that our nanopore sequencing pipeline generates DNA barcodes that are at least as good as barcodes generated with Sanger sequencing, with the MinION producing better results than the Flongle flowcell. We further find that while many arthropod taxa are well covered in the international reference DNA barcode database BOLD, this clearly is not the case for important taxa like mites and springtails, which hampers large-scale biodiversity assessments. Based on examples from our study we further highlight which factors might be responsible for ambiguous species identification based on BOLD and how this can, at least partly, be solved.

DNA Barcoding of Invasive Terrestrial Plant Species in India.

Invasive plants are known to cause biodiversity loss and pose a major risk to human health and environment. Identification of invasive plants and distinguishing them from native species has been relied on morphological examination. Stringent requirement of floral characters and decreasing number of expert taxonomists are making conventional morphology-based identification system tedious and resource-intensive. DNA barcoding may help in quick identification of invasive species if distinct sequence divergence pattern at various taxonomic levels is observed. The present work evaluates the utility of four molecular markers; rbcL, matK, their combination (rbcL + matK), and psbA-trnH for identification of 37 invasive plant species from India and also in distinguishing them from 97 native species. A psbA-trnH locus was found to be of restricted utility in this work as it was represented by the members of a single family. A hierarchical increase in K2P mean divergence across different taxonomic levels was found to be the maximum for matK alone followed by rbcL + matK and rbcL alone, respectively. NJ clustering analysis, however, confirmed the suitability of combined locus (rbcL + matK) over individual rbcL and matK as the DNA barcode. RbcL showed the lowest resolution power among the three markers studied. MatK exhibited much better performance compared to rbcL alone in identifying most of the species accurately although it failed to show monophyly of genus Dinebra. Two families; Asteraceae and Poaceae, remained polyphyletic in the trees constructed by all three markers. Combined locus (rbcL + matK) was found to be the most suitable marker as it raised the resolution power of both the markers and could identify more than 90% of genera correctly. Phylogenetic tree constructed by Maximum-Parsimony method using combined locus as a molecular marker exhibited the best resolution, thus, supporting the significance of two-locus combination of rbcL + matK for barcoding invasive plant species from India. Present study contributes to the global barcode data of invasive plant species by adding fifty-one new sequences to it. Effective barcoding of additional number of native as well as invasive plant species from India is possible using this dual locus if it is combined with one or more new molecular plastid markers. Expansion of barcode database with a focus on barcode performance optimisation to improve discrimination ability at species level can be undertaken in future.

Conservation Strategies for Aquilaria sinensis: Insights from DNA Barcoding and ISSR Markers.

The evergreen tree species Aquilaria sinensis holds significant economic importance due to its specific medicinal values and increasingmarket demand. However, the unrestricted illegal exploitation of its wild population poses a threat to its survival. This study aims to contribute to the conservation efforts of A. sinensis by constructing a library database of DNA barcodes, including two chloroplast genes (psbA-trnH and matK) and two nuclear genes (ITS and ITS2). Additionally, the genetic diversity and structure were estimated using inter-simple sequence repeats (ISSR) markers. Four barcodes of 57 collections gained 194 sequences, and 1371 polymorphic bands (98.63%) were observed using DNA ISSR fingerprinting. The Nei's gene diversity (H) of A. sinensis at the species level is 0.2132, while the Shannon information index (I) is 0.3128. The analysis of molecular variance revealed a large significant proportion of total genetic variations and differentiation among populations (Gst = 0.4219), despite a relatively gene flow (Nm = 0.6853) among populations, which were divided into two groups by cluster analysis. There was a close genetic relationship among populations with distances of 0.0845 to 0.5555. This study provides evidence of the efficacy and dependability of establishing a DNA barcode database and using ISSR markers to assess the extent of genetic diversity A. sinensis. Preserving the genetic resources through the conservation of existing populations offers a valuable proposition. The effective utilization of these resources will be further deliberated in subsequent breeding endeavors, with the potential to breed agarwood commercial lines.

Towards a Canary Islands barcode database for soil biodiversity: revealing cryptic and unrecorded mite species diversity within insular soils.

Soil arthropod diversity contributes to a high proportion of the total biodiversity on Earth. However, most soil arthropods are still undescribed, hindering our understanding of soil functioning and global biodiversity estimations. Inventorying soil arthropods using conventional taxonomical approaches is particularly difficult and costly due to the great species richness, abundance and local-scale heterogeneity of mesofauna communities and the poor taxonomic background knowledge of most lineages. To alleviate this situation, we have designed and implemented a molecular barcoding framework adapted to soil fauna. This pipeline includes different steps, starting with a morphology-based selection of specimens which are imaged. Then, DNA is extracted non-destructively. Both images and voucher specimens are used to assign a taxonomic identification, based on morphology that is further checked for consistency with molecular information. Using this procedure, we studied 239 specimens of mites from the Canary Islands including representatives of Mesostigmata, Sarcoptiformes and Trombidiformes, of which we recovered barcode sequences for 168 specimens that were morphologically identified to 49 species, with nine specimens that could only be identified at the genus or family levels. Multiple species delimitation analyses were run to compare molecular delimitations with morphological identifications, including ASAP, mlPTP, BINs and 3% and 8% genetic distance thresholds. Additionally, a species-level search was carried out at the Biodiversity Databank of the Canary Islands (BIOTA) to evaluate the number of species in our dataset that were not previously recorded in the archipelago. In parallel, a sequence-level search of our sequences was performed against BOLD Systems. Our results reveal that multiple morphologically identified species correspond to different molecular lineages, which points to significant levels of unknown cryptic diversity within the archipelago. In addition, we evidenced that multiple species in our dataset constituted new records for the Canary Islands fauna and that the information for these lineages within online genetic repositories is very incomplete. Our study represents the first systematic effort to catalogue the soil arthropod mesofauna of the Canary Islands and establishes the basis for the Canary Islands Soil Biodiversity barcode database. This resource will constitute a step forward in the knowledge of these arthropods in a region of special interest.

Scalable, Cost-Effective, and Decentralized DNA Barcoding with Oxford Nanopore Sequencing.

DNA barcodes are useful in biodiversity research, but sequencing barcodes with dye termination methods ("Sanger sequencing") has been so time-consuming and expensive that DNA barcodes are not as widely used as they should be. Fortunately, MinION sequencers from Oxford Nanopore Technologies have recently emerged as a cost-effective and efficient alternative for barcoding. MinION barcodes are now suitable for large-scale species discovery and enable specimen identification when the target species are represented in barcode databases. With a MinION, it is possible to obtain 10,000 barcodes from a single flow cell at a cost of less than 0.10 USD per specimen. Additionally, a Flongle flow cell can be used for small projects requiring up to 300 barcodes (0.50 USD per specimen). We here describe a cost-effective laboratory workflow for obtaining tagged amplicons, preparing ONT libraries, sequencing amplicon pools, and analyzing the MinION reads with the software ONTbarcoder. This workflow has been shown to yield highly accurate barcodes that are 99.99% identical to Sanger barcodes. Overall, we propose that the use of MinION for DNA barcoding is an attractive option for all researchers in need of a cost-effective and efficient solution for large-scale species discovery and specimen identification.

Environmental DNA in a Biofilm Can Be Used to Assess Diatom Ecological Health in Stream Water Ecology

In urban and agricultural streams, assessing aquatic ecosystem health is critical due to widespread pollution. Traditional methods for evaluating attached diatoms crucial for ecosystem monitoring face limitations such as species misidentification and sample damage. This study was conducted in the Miho River within the Geum River system and highlights the effectiveness of environmental DNA (eDNA) techniques for more accurate and efficient genetic-based analysis than conventional microscopic analysis methods. When eDNA-based assessments were compared with traditional microscopic methods, this study found that eDNA analysis often revealed poorer ecosystem health. Notably, eDNA assessments showed a stronger correlation with phosphorus concentrations, underlining their precision and importance in ecological studies. These findings suggest that eDNA has potential as a valuable tool for comprehensive biomonitoring. However, the use of international genetic barcode databases in eDNA analysis could lead to the identification of unrecorded species in Korea. Therefore, this study recommends developing a localized genetic barcode database and constructing eDNA information through meta-barcoding, focusing on native species. This approach is crucial for enhancing the accuracy and applicability of eDNA-based health assessments in national biomonitoring efforts.

Revisiting the species list of freshwater fish in Israel based on DNA barcoding.

Israel's region forms a continental bridge; hence, the freshwater fish fauna in Israel consists of unique populations of species that originated from Africa, Asia, or Europe and are often endemic or at the edge of their distribution range. Worldwide, fish biodiversity suffers significantly from pressures and disturbances of freshwater habitats, especially in arid regions, such as in parts of Israel. Biodiversity conservation requires efficient tools for monitoring changes in populations. DNA barcoding, by complementing and enhancing species identification, provides such monitoring tools. In this study, over 200 specimens representing over 28 species were DNA barcoded and together with previously available records, a DNA barcoding database for freshwater fish of Israel was established. Of the 71 distinct barcodes generated, 37% were new, attesting to the uniqueness of fish populations in Israel. For most species, morphological and molecular species identifications agreed. However, discrepancies were found for five genera. Based on DNA barcoding, we propose Acanthobrama telavivensis as a junior synonym for Acanthobrama lissneri. In Garra spp., we propose splitting Garra nana into two species and assigning Garra rufa in the region to Garra jordanica, or possibly to two species. Israeli Pseudophoxinus kervillei is not the same species as in Syria and Lebanon. However, Pseudophoxinus syriacus might not be endangered since it is genetically very similar to Pseudophoxinus drusensis. In Israel, instead of five reported Oxynoemacheilus species, combining DNA barcoding with morphology suggests only three. Genetic and geographic separation suggested that Aphanius mento is likely a species complex. The study provides a thorough barcoding database, suggests significant species reconsiderations in the region, and highlights the Sea of Galilee and the Beit She'an valley streams as biodiversity "hotspots." This study will therefore promote further studying of the fish species in the region and their ecology, as well as the monitoring and conservation of freshwater fish biodiversity in Israel and the region.

RCRUX: A Rapid and Versatile Tool for Generating Metabarcoding Reference libraries in R.

The sequencing revolution requires accurate taxonomic classification of DNA sequences. Key to making accurate taxonomic assignments are curated, comprehensive reference barcode databases. However, the generation and curation of such databases has remained challenging given the large and continuously growing volumes of both DNA sequence data and novel reference barcode targets. Monitoring and research applications require a greater diversity of specialized gene regions and targeted taxa then are currently curated by professional staff. Thus there is a growing need for an easy to implement computational tool that can generate comprehensive metabarcoding reference libraries for any bespoke locus. We address this need by reimagining CRUX from the Anacapa Toolkit and present the rCRUX package in R which, like it's predecessor, relies on sequence homology and PCR primer compatibility instead of keyword-searches to avoid limitations of user-defined metadata. The typical workflow involves searching for plausible seed amplicons (get_seeds_local() or get_seeds_remote()) by simulating in silico PCR to acquire a set of sequences analogous to PCR products containing a user-defined set of primer sequences. Next, these seeds are used to iteratively blast search seed sequences against a local copy of the National Center for Biotechnology Information (NCBI) formatted nt database using a taxonomic-rank based stratified random sampling approach ( blast_seeds() ). This results in a comprehensive set of sequence matches. This database is dereplicated and cleaned (derep_and_clean_db()) by identifying identical reference sequences and collapsing the taxonomic path to the lowest taxonomic agreement across all matching reads. This results in a curated, comprehensive database of primer-specific reference barcode sequences from NCBI. Databases can then be compared (compare_db()) to determine read and taxonomic overlap. We demonstrate that rCRUX provides more comprehensive reference databases for the MiFish Universal Teleost 12S, Taberlet trnl, fungal ITS, and Leray CO1 loci than CRABS, MetaCurator, RESCRIPt, and ecoPCR reference databases. We then further demonstrate the utility of rCRUX by generating 24 reference databases for 20 metabarcoding loci, many of which lack dedicated reference database curation efforts. The rCRUX package provides a simple to use tool for the generation of curated, comprehensive reference databases for user-defined loci, facilitating accurate and effective taxonomic classification of metabarcoding and DNA sequence efforts broadly.