Comprehensive DNA Barcode Research Articles

Toward a global DNA barcode reference library of the intolerant nonbiting midge genus Rheocricotopus Brundin, 1956.

Environmental DNA metabarcoding is becoming a predominant tool in biodiversity assessment, as this time‐ and cost‐efficient tactics have the ability to increase monitoring accuracy. As a worldwide distributed genus, Rheocricotopus Brundin, 1956 still does not possess a complete and comprehensive global DNA barcode reference library for biodiversity monitoring. In the present study, we compiled a cytochrome c oxidase subunit 1 (COI) DNA barcode library of Rheocricotopus with 434 barcodes around the world, including 121 newly generated DNA barcodes of 32 morphospecies and 313 public barcodes. Automatic Barcode Gap Discovery (ABGD) was applied on the 434 COI barcodes to provide a comparison between the operational taxonomic units (OTU) number calculated from the Barcode Index Number (BIN) with the “Barcode Gap Analysis” and neighbor‐joining (NJ) tree analysis. Consequently, these 434 COI barcodes were clustered into 78 BINs, including 42 new BINs. ABGD yielded 51 OTUs with a prior intraspecific divergence of Pmax = 7.17%, while NJ tree revealed 52 well‐separated clades. Conservatively, 14 unknown species and one potential synonym were uncovered with reference to COI DNA barcodes. Besides, based on our ecological analysis, we discovered that annual mean temperature and annual precipitation could be considered as key factors associated with distribution of certain members from this genus. Our global DNA barcode reference library of Rheocricotopus provides one fundamental database for accurate species delimitation in Chironomidae taxonomy and facilitates the biodiversity monitoring of aquatic biota.

Coverage and quality of DNA barcode references for Central and Northern European Odonata.

BackgroundDragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches.MethodsThis study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD.ResultsOver 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

High resolution DNA barcode library for European butterflies reveals continental patterns of mitochondrial genetic diversity

The study of global biodiversity will greatly benefit from access to comprehensive DNA barcode libraries at continental scale, but such datasets are still very rare. Here, we assemble the first high-resolution reference library for European butterflies that provides 97% taxon coverage (459 species) and 22,306 COI sequences. We estimate that we captured 62% of the total haplotype diversity and show that most species possess a few very common haplotypes and many rare ones. Specimens in the dataset have an average 95.3% probability of being correctly identified. Mitochondrial diversity displayed elevated haplotype richness in southern European refugia, establishing the generality of this key biogeographic pattern for an entire taxonomic group. Fifteen percent of the species are involved in barcode sharing, but two thirds of these cases may reflect the need for further taxonomic research. This dataset provides a unique resource for conservation and for studying evolutionary processes, cryptic species, phylogeography, and ecology.

The first step towards a DNA barcode reference database for mayflies (Ephemeroptera) of Slovakia

Mayflies (Ephemeroptera) represent a small but diverse order of amphibiotic insects, whose larvae contribute to several essential processes in freshwater habitats, such as bioturbation and bioirrigation, decomposition, nutrient cycling, and also serve as a primary source of nutrients for numerous organisms. Due to their cosmopolitan distribution and high-quality water requirements, they are also important indicators of ecosystem health and an integral part of biomonitoring protocols. Although the Slovak mayfly fauna is well researched, studies on genetic diversity, including DNA barcoding, are still lacking. The absence of the comprehensive DNA barcode reference libraries from various biogeographical regions and the presence of so-called cryptic lineages may prevent further efficient use and application of new approaches to aquatic ecosystem biomonitoring (Biomonitoring 2.0) based on eDNA analyses. Therefore, in the initial stage of our research, we bring the first insight into the genetic diversity of mayflies (based on mtDNA COI-5P barcoding fragment) from 47 localities of Slovakia mostly situated in the biogeographically significant Western Carpathians' territory. A total of 403 sequences of 42 morphologically determined species were added to the BOLD (Barcode of Life Data System) database, representing more than 1/3 of the mayfly fauna of Slovakia and covering 10 of 16 families. Sequences of these species were finally assigned to 62 BINs (Barcode Index Numbers) in BOLD (Fig. 1), whereby sequences of 12 species were divided into more than one BIN, indicating the presence of cryptic lineages. The largest number of BINs was represented by widely distributed species such as Baetis rhodani Pictet, 1843-1845 (6 BINs), Habroleptoides confusa Sartori & Jacob, 1986 (4 BINs) and Ecdyonurus venosus (Fabricius, 1775) (3 BINs). The sequences of the remaining nine species were split into two BINs. Maximum intraspecific variability (calculated by K2P) of some representatives was surprisingly high [e.g., E. venosus – 27.1 %; Baetis muticus (Linneaus, 1758) – 23.6 %; Caenis luctuosa (Burmeister, 1839) – 23.34 %, Baetis rhodani – 18.66 % and B. vernus Curtis, 1834 – 15.25 %] and far exceeded the level of intraspecific variability of the COI fragment based on the BOLD standards. The sequences of 23 individuals determined as Habroleptoides confusa, Baetis rhodani, B. buceratus Eaton, 1870, Caenis beskidensis Sowa, 1973 and Torleya major (Klapálek, 1905) created seven unique BINs, which represent the distant phylogenetic lineages of already existing BINs, that are currently unique to Slovakia. The coexistence of Baetis rhodani individuals of two different BINs was confirmed at five localities. Our study indicates clear importance of more detailed sampling and DNA barcoding due to the presence of unexpected intraspecific genetic diversity of mayflies captured in a relatively small area of the Western Carpathians.

DNA barcoding provides new insights on the distribution, systematics and conservation of the freshwater genus Pelasgus (Cypriniformes: Cyprinidae) in Greece

The freshwater ichthyofauna of Greece is characterized by high diversity and endemism and faces various conservation challenges. Despite significant efforts, mainly with traditional assessment methods, the precise taxonomic status and geographic range of some of the currently acknowledged fish species remain insufficiently explored. Here, the results of the first comprehensive DNA barcoding survey of the genus Pelasgus are presented, covering its entire known range in Greece. Two hundred and forty COI sequences (retrieved from collected samples, museum specimens and the GenBank) were analysed to determine the number of putative species and delineate their current distribution. Data support the existence of seven haplogroups, corresponding to the currently valid Pelasgus species. Analysis of museum samples of the critically endangered species P. epiroticus from its type locality (now extinct there) uncovers its presence in contemporary samples from a neighbouring aquatic system (Zaravina Lake) and from aquatic systems well outside its native range (possibly as a result of past translocations) for the first time. A subgroup of P. epiroticus haplotypes was also identified in North-western Greece in regions previously believed to host P. thesproticus or P. stymphalicus. Based on the above, hotspots for further Pelasgus taxonomic investigations are identified and conservation actions are discussed.

The first comprehensive DNA barcode reference library of Chinese Tanytarsus (Diptera: Chironomidae) for environmental DNA metabarcoding

AbstractAimReliable DNA barcode reference libraries are critical for biodiversity monitoring based on Environmental DNA (eDNA). DNA barcoding has proven successful in species delimitation and association of life stages in many groups of animals, such as non‐biting midges (Chironomidae). We use the genus Tanytarsus van der Wulp, 1874, second largest genus of the family Chironomidae, to test whether DNA barcodes can be used to identify unknown and cryptic species. A comprehensive DNA barcode library will be built to facilitate further eDNA studies for biodiversity monitoring and conservation in freshwater ecosystems.LocationMainland China.MethodsIn this study, the cytochrome c oxidase subunit 1 (COI) DNA barcodes from 298 individuals, representing 56 morphospecies from China, were analysed, including 136 sequences from previous studies and 162 newly generated DNA barcodes. 298 COI barcodes were applied into the Automatic Barcode Gap Discovery (ABGD) to compare the operational taxonomic units (OTU) number resulting from the Barcode Index Number (BIN) based “Barcode Gap Analysis” and neighbour joining (NJ) tree.ResultsTotal 298 barcode sequences were applied to 66 BINs, including 20 singleton BINs and 46 concordant BINs. ABGD yielded 55 OTUs with a prior intraspecific divergence of Pmax = 3.59%. By Integrating DNA barcodes with morphology, we revealed 21 putative species new to science and two unidentified species from the 56 well‐separated morphospecies. Besides, several cryptic species are uncovered using the NJ tree and TCS haplotype network based on COI DNA barcodes.Main conclusionsOur results demonstrate that DNA barcodes enable the discovery and identification of Tanytarsus non‐biting midges. A comprehensive DNA barcode reference library could speed up accurate species delimitation and discovery by providing a reliable resource for taxonomic studies, and improve the application of eDNA metabarcoding in biomonitoring of freshwater ecosystems.

BAGS: An automated Barcode, Audit & Grade System for DNA barcode reference libraries.

Biodiversity studies greatly benefit from molecular tools, such as DNA metabarcoding, which provides an effective identification tool in biomonitoring and conservation programmes. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries. The role of these libraries is to support species identification, but accidental errors in the generation of the barcodes may compromise their accuracy. Here, we present an R-based application, Barcode, Audit & Grade System (BAGS) (https://github.com/tadeu95/BAGS), that performs automated auditing and annotation of cytochrome c oxidase subunit I (COI) sequences libraries, for a given taxonomic group of animals, available in the Barcode of Life Data System (BOLD). This is followed by implementing a qualitative ranking system that assigns one of five grades (A to E) to each species in the reference library, according to the attributes of the data and congruency of species names with sequences clustered in barcode index numbers (BINs). Our goal is to allow researchers to obtain the most useful and reliable data, highlighting and segregating records according to their congruency. Different tests were performed to perceive its usefulness and limitations. BAGS fulfils a significant gap in the current landscape of DNA barcoding research tools by quickly screening reference libraries to gauge the congruence status of data and facilitate the triage of ambiguous data for posterior review. Thereby, BAGS has the potential to become a valuable addition in forthcoming DNA metabarcoding studies, in the long term contributing to globally improve the quality and reliability of the public reference libraries.

A DNA barcode library for ground beetles of Germany: the genus Pterostichus Bonelli, 1810 and allied taxa (Insecta, Coleoptera, Carabidae).

Species of the ground beetle genus Pterostichus Bonelli, 1810 are some of the most common carabids in Europe. This publication provides a first comprehensive DNA barcode library for this genus and allied taxa including Abax Bonelli, 1810, Molops Bonelli, 1810, Poecilus Bonelli, 1810, and Stomis Clairville, 1806 for Germany and Central Europe in general. DNA barcodes were analyzed from 609 individuals that represent 51 species, including sequences from previous studies as well as more than 198 newly generated sequences. The results showed a 1:1 correspondence between BIN and traditionally recognized species for 44 species (86%), whereas two (4%) species were characterized by two BINs. Three BINs were found for one species (2%), while one BIN for two species was revealed for two species pairs (8%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for four species pairs. Haplotype sharing was found for two closely related species pairs: Pterostichus adstrictus Eschscholtz, 1823/Pterostichus oblongopunctatus (Fabricius, 1787) and Pterostichus nigrita Paykull, 1790/Pterostichus rhaeticus Heer, 1837. In contrast to this, high intraspecific sequence divergences with values above 2.2% were shown for three species (Molops piceus (Panzer, 1793), Pterostichus panzeri (Panzer, 1805), Pterostichus strenuus (Panzer, 1793)). Summarizing the results, the present DNA barcode library does not only allow the identification of most of the analyzed species, but also provides valuable information for alpha-taxonomy as well as for ecological and evolutionary research. This library represents another step in building a comprehensive DNA barcode library of ground beetles as part of modern biodiversity research.

A DNA barcode library for ground beetles of Germany: the genus Agonum Bonelli, 1810 (Insecta, Coleoptera, Carabidae)

The ground beetle genusAgonumBonelli, 1810 is a large genus of the tribe Platynini with many species that show high amounts of intraspecific variations, making a correct identification challenging. As part of the German Barcode of Life initiative, this publication provides a comprehensive DNA barcode library for species ofAgonumthat are reported for Germany. In total, DNA barcodes from 258 beetles and 23 species were analysed using the Barcode of Life Data System (BOLD) workbench, including sequences from former studies and 68 newly-generated sequences. The neighbour-joining analyses, based on K2P distances, revealed distinct clustering for all studied species, with unique Barcode Index Numbers (BINs) for 15 species (65%). BIN sharing but distinct clustering was found for three species pairs:Agonum micans/Agonum scitulum,Agonum impressum/Agonum sexpunctatumandAgonum duftschmidi/Agonum emarginatum. The given dataset and its analysis represent another important step in generating a comprehensive DNA barcode library for the ground beetles of Germany and Central Europe in terms of modern biodiversity research.

Italian odonates in the Pandora's box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale.

The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA-based monitoring relies on validated reference data sets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658-bp 5' end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the data set to 1,294 DNA barcodes. A multi-approach species delimitation analysis involving two distance (OT and ABGD) and four tree-based (PTP, MPTP, GMYC and bGMYC) methods was used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct molecular operational units, whereas the remaining ones were classified as 'warnings' (i.e. showing a mismatch between morphospecies assignment and DNA-based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped into three categories depending on if they showed low, high or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA-based monitoring studies.

DNA barcoding to characterize biodiversity of freshwater fishes of Egypt.

The current study represents the first molecular characterization of freshwater fish species in Egypt from two major fish resources; the River Nile and Lake Nasser. A total of 160 DNA barcodes using a 655-bp-long fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene were generated from 37 species belonging to 32 genera that represent 15 families from nine orders. The studied species were identified using different molecular-based identification approaches, in addition to the morphological identification, including neighbor-joining (NJ) trees, Barcode Index Number, and Automatic Barcode Gap Discovery (ABGD). The average genetic divergence based on the Kimura two-parameter model (K2P) within orders, families, genera, and species were 0.175, 0.067, 0.02, and 0.008, respectively. The minimum and maximum K2P distance-based genetic divergences were 0.0 and 0.154, respectively. Nucleotide diversity (π) varied among families and ranged between 0.0% for families Malapteruridae, Auchenoglanididae, Schilbeidae, Anguillidae, Centropomidae and Tetraodontidae and 17% for family Cyprinidae. The current study cautions against the lack of species coverage at public databases which limits the accurate identification of newly surveyed species and recommends that multiple methods are encouraged for accurate species identification. The findings of the current study also support that COI barcode enabled effective fish species identification in River Nile and Lake Nasser. Moreover, the results of the current study will establish a comprehensive DNA barcode library for freshwater fishes along the River Nile in Egypt. Egyptian freshwater fish DNA barcodes will contribute substantially to future efforts in monitoring, conservation, and management of fisheries in Egypt.

Gap-analysis and annotated reference library for supporting macroinvertebrate metabarcoding in Atlantic Iberia

DNA metabarcoding provides a rapid and effective identification tool of macroinvertebrate species. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries, which, due to incompleteness, are currently a recognized limitation for metabarcoding applications. In this study, we assembled a comprehensive reference library of DNA barcodes for Atlantic Iberia marine macroinvertebrate species, assessed gaps in species coverage and examined data ambiguities. Initially, an Iberian species checklist for the three dominant groups of marine macroinvertebrates was compiled, comprising 2827 species (926 Annelida, 638 Crustacea and 1263 Mollusca). A total of 18162 DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) matching the species checklist were compiled in a BOLD dataset, where taxonomic discordances were evaluated and cases of deep intraspecific divergence flagged. Gap-analysis showed that 63% of the Iberian macroinvertebrate species still lack a DNA barcode. Coverage gaps varied considerably across taxonomic groups with Mollusca displaying the highest sequence representation in the dataset (427 species, 49% of the total number of sequences), and Crustacea the highest species coverage with 338 species barcoded (53% of the checklist). In contrast, Polychaeta displayed the lower levels of completion (288 species, 16% of the total number of sequences). In total, 1545 Barcode Index Numbers (BINs) were assigned to 1053 barcoded species, of which 66% were taxonomically concordant, 26% displayed multiple BINs and 8% were discordant. Overall, results show that there is still a large portion of marine invertebrate taxa in this region of Europe pending barcode coverage, even considering only the dominant groups. However, the most notable finding was the relevant proportion of species flagged for significant intraspecific divergence and possible hidden diversity. The annotated reference library and gap-analysis here provided can therefore contribute to prioritize marine macroinvertebrate taxa for future research efforts and barcode coverage.

A reference DNA barcode library for Austrian amphibians and reptiles.

In the last few years, DNA barcoding became an established method for species identification in biodiversity inventories and monitoring studies. Such studies depend on the access to a comprehensive reference data base, covering all relevant taxa. Here we present a comprehensive DNA barcode inventory of all amphibian and reptile species native to Austria, except for the putatively extinct Vipera ursinii rakosiensis and Lissotriton helveticus, which has been only recently reported for the very western edge of Austria. A total of 194 DNA barcodes were generated in the framework of the Austrian Barcode of Life (ABOL) initiative. Species identification via DNA barcodes was successful for most species, except for the hybridogenetic species complex of water frogs (Pelophylax spp.) and the crested newts (Triturus spp.), in areas of sympatry. However, DNA barcoding also proved powerful in detecting deep conspecific lineages, e.g. within Natrix natrix or the wall lizard (Podarcis muralis), resulting in more than one Barcode Index Number (BIN) per species. Moreover, DNA barcodes revealed the presence of Natrix helvetica, which has been elevated to species level only recently, and genetic signatures of the Italian water frog Pelophylax bergeri in Western Austria for the first time. Comparison to previously published DNA barcoding data of European amphibians and reptiles corroborated the results of the Austrian data but also revealed certain peculiarities, underlining the particular strengths and in the case of the genus Pelophylax also the limitations of DNA barcoding. Consequently, DNA barcoding is not only powerful for species identification of all life stages of most Austrian amphibian and reptile species, but also for the detection of new species, the monitoring of gene flow or the presence of alien populations and/or species. Thus, DNA barcoding and the data generated in this study may serve both scientific and national or even transnational conservation purposes.

From DNA barcoding to nanoparticle-based colorimetric testing: a new frontier in cephalopod authentication

Food-item authentication and traceability is an issue of primary concern, due to both socio-economical and health implications. DNA-based methods are increasingly being recognised as powerful tools to assess the reliability of supplier labels for any type of food. This is especially true for products characterised by a short shelf life and high-processing supply chain, such as seafood. In this work, a DNA barcoding approach was applied to assess the accuracy of species labelling in 150 cephalopod seafood products sold in the Italian market. Overall, high levels of mislabelling in squid, cuttlefish, and octopus items were identified, and in some cases, even species not included in the current food Regulations. Additionally, an application of the recently developed naked-eye detection tool ‘NanoTracer’, consisting in the combination of DNA barcoding with gold nanoparticle-based, was demonstrated to authenticate common cuttlefish (Sepia officinalis) seafood. The primer pairs used to set the fast detection system for S. officinalis were designed based on the most comprehensive DNA barcoding (COI and 16s rRNA) datasets ever assembled for cephalopods, assuring the specificity of the method. ‘NanoTracer’ allowed a simple, rapid, accurate and cost-effective authentication, revealing its potential adaptability to any type of seafood and other food categories.

DNA Barcoding Subtropical Aphids and Implications for Population Differentiation.

DNA barcoding has proven its worth in species identification, discovering cryptic diversity, and inferring genetic divergence. However, reliable DNA barcode reference libraries that these applications depend on are not available for many taxonomic groups and geographical regions. Aphids are a group of plant sap sucking insects, including many notorious pests in agriculture and forestry. The aphid fauna of the subtropical region has been understudied. In this study, based on extensive sampling effort across main subtropical areas, we sequenced 1581 aphid specimens of 143 morphospecies, representing 75 genera, and 13 subfamilies, to build the first comprehensive DNA barcode library for subtropical aphids. We examined the utility of DNA barcodes in identifying aphid species and population differentiation and evaluated the ability of different species delimitation methods (automatic barcode gap discovery (ABGD), generalized mixed Yule-coalescent (GMYC), and Bayesian Poisson tree processes (bPTP)). We found that most aphid species demonstrated barcode gaps and that a threshold value of 2% genetic distance is suitable for distinguishing most species. Our results indicated that ten morphospecies may have species divergence related to factors such as host plant or geography. By using two pest species Aphis spiraecola and A. gossypii as examples, we also discussed the effect of the sampling scale of host plants on the results and reliability of DNA barcoding of phytophagous insects. This DNA barcode library will be valuable for future studies and applications.

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

Abstract Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub‐sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.

A DNA barcode reference library of French Polynesian shore fishes

The emergence of DNA barcoding and metabarcoding opened new ways to study biological diversity, however, the completion of DNA barcode libraries is fundamental for such approaches to succeed. This dataset is a DNA barcode reference library (fragment of Cytochrome Oxydase I gene) for 2,190 specimens representing at least 540 species of shore fishes collected over 10 years at 154 sites across the four volcanic archipelagos of French Polynesia; the Austral, Gambier, Marquesas and Society Islands, a 5,000,000 km2 area. At present, 65% of the known shore fish species of these archipelagoes possess a DNA barcode associated with preserved, photographed, tissue sampled and cataloged specimens, and extensive collection locality data. This dataset represents one of the most comprehensive DNA barcoding efforts for a vertebrate fauna to date. Considering the challenges associated with the conservation of coral reef fishes and the difficulties of accurately identifying species using morphological characters, this publicly available library is expected to be helpful for both authorities and academics in various fields.

Assessing the effectiveness of molecular data for species identification and diversity studies of Nigerian herpetofauna

We evaluated the effectiveness of molecular data (mitochondrial cytochrome c oxidase subunit I – COI and 16S ribosomal RNA – 16S) for species identification and diversity studies of Nigerian herpetofauna. We collected 189 specimens consisting of 91 amphibians and 98 reptiles from the largest and under-studied national park in Nigeria – Gashaka Gumti National Park (GGNP), eastern Nigeria. The resulting COI and 16S data allowed us to compare our sequences with others in global databases for species identification. During query searches, we observed poor representation of COI sequences for African herpetofauna in the GenBank and BOLD databases. Neighbour-joining (NJ) trees constructed based on COI and 16S datasets for all specimens sequenced for this study clustered most individuals of the same species. On the other hand, morphologically unidentified amphibian (Leptopelis sp and Ptychadena sp) and reptile (Hemidactylus sp and Lycophidion sp) species formed strongly supported lineages in the NJ trees, and were clearly separated from their sister species. Further, the NJ tree-based analyses recovered at least two well supported divergent lineages within Agama agama and Trachylepis maculilabris. Our study confirms the efficiency of integrating morphological and DNA barcoding approaches in identification and diversity studies of Nigerian herpetofauna. Finally, we recommend development of comprehensive and reliable DNA barcode reference libraries for global amphibians and reptiles.

DNA Barcoding in Forensic Entomology - Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species.

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High-quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1-5P' DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high-quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.

Integrating a comprehensive DNA barcode reference library with a global map of yews (Taxus L.) for forensic identification.

Rapid and accurate identification of endangered species is a critical component of biosurveillance and conservation management, and potentially policing illegal trades. However, this is often not possible using traditional taxonomy, especially where only small or preprocessed parts of plants are available. Reliable identification can be achieved via a comprehensive DNA barcode reference library, accompanied by precise distribution data. However, these require extensive sampling at spatial and taxonomic scales, which has rarely been achieved for cosmopolitan taxa. Here, we construct a comprehensive DNA barcode reference library and generate distribution maps using species distribution modelling (SDM), for all 15 Taxus species worldwide. We find that trnL-trnF is the ideal barcode for Taxus: It can distinguish all Taxus species and in combination with ITS identify hybrids. Among five analysis methods tested, NJ was the most effective. Among 4,151 individuals screened for trnL-trnF, 73 haplotypes were detected, all species-specific and some population private. Taxonomical, geographical and genetic dimensions of sampling strategy were all found to affect the comprehensiveness of the resulting DNA barcode library. Maps from SDM showed that most species had allopatric distributions, except T. mairei in the Sino-Himalayan region. Using the barcode library and distribution map data, two unknown forensic samples were identified to species (and in one case, population) level and another was determined as a putative interspecific hybrid. This integrated species identification system for Taxus can be used for biosurveillance, conservation management and to monitor and prosecute illegal trade. Similar identification systems are recommended for other IUCN- and CITES-listed taxa.