Comprehensive DNA Barcode Research Articles

A DNA barcode reference library for the Tipulidae (Insecta, Diptera) of Germany.

Tipulidae, commonly known as true crane flies, represent one of the most species-rich dipteran families, boasting approximately 4,500 known species globally. Their larvae serve as vital decomposers across diverse ecosystems, prompting their frequent and close observation in biomonitoring programs. However, traditional morphological identification methods are laborious and time-consuming, underscoring the need for a comprehensive DNA barcode reference library to speed up species determination. In this study, we present the outcomes of the German Barcode of Life initiative focused on Tipulidae. Our DNA barcode library comprises 824 high-quality cytochrome c oxidase I (COI) barcodes encompassing 76 crane fly species, counting for ca. 54% of the German tipulid fauna. Our results significantly increased the number of European tipulid species available in the Barcode of Life Data System (BOLD) by 14%. Additionally, the number of barcodes from European tipulid specimens more than doubled, with an increase of 118%, bolstering the DNA resource for future identification inquiries. Employing diverse species delimitation algorithms - including the multi-rate Poisson tree processes model (mPTP), Barcode Index Number assignments (BIN), Assemble Species by Automatic Partitioning (ASAP), and the TaxCI R-script - we successfully match 76-86% of the morphologically identified species. Further validation through neighbor-joining tree topology analysis and comparison with 712 additional European tipulid barcodes yield a remarkable 89% success rate for the species identification of German tipulids based on COI barcodes. This comprehensive DNA barcode dataset not only enhances species identification accuracy but also serves as a pivotal resource for ecological and biomonitoring studies, fostering a deeper understanding of crane fly diversity and distribution across terrestrial landscapes.

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.

Delimiting species, revealing cryptic diversity, and population divergence in Qinghai-Tibet Plateau weevils through DNA barcoding.

The Leptomias group represents one of the most diverse taxonomic group of weevils in the Qinghai-Tibet Plateau and its adjacent areas. Despite the potential of hidden diversity, relatively few comprehensive studies have been conducted on species diversity in this taxonomic group. In this study, we performed DNA barcoding analysis for species of the Leptomias group using a comprehensive DNA barcode dataset that included 476 sequences representing 54 morphospecies. Within the dataset, our laboratory contributed 474 sequences, and 390 sequences were newly generated for this study. The average Kimura 2-parameter distances among morphospecies and genera were 0.76% and 19.15%, respectively. In 94.4% of the species, the minimum interspecific distances exceeded the maximum intraspecific distances, indicating the presence of barcode gaps in most species of Leptomias group. The application of Automatic Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Bayesian Poisson tree processes, jMOTU, and Neighbor-joining tree methods revealed 45, 45, 63, 54, and 55 distinct clusters representing single species, respectively. Additionally, a total of four morphospecies, Leptomias kangmarensis, L. midlineatus, L. siahus, and L. sp.9RL, were found to be assigned to multiple subclade each, indicating the geographical divergences and the presence of cryptic diversity. Our findings of this study demonstrate that Qinghai-Tibet Plateau exhibits a higher species diversity of the Leptomias group, and it is imperative to investigate cryptic species within certain morphospecies using integrative taxonomic approaches in future studies. Moreover, the construction of a DNA barcode reference library presented herein establishes a robust foundational dataset to support forthcoming research on weevil taxonomy, phylogenetics, ecology, and evolution.

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.

Next‐generation species delimitation and taxonomy: Implications for biogeography

AbstractAn accurate species‐level taxonomy is paramount for biogeographical research, and conversely, biogeographical data are of importance for species delimitation. We here review recent developments and future perspectives of taxonomy of direct relevance for biogeographers. The understanding that species are independently evolving segments of population‐level lineages, the rise of integrative approaches to delimit such lineages, and the advent of high‐throughput sequencing have considerably renewed the discipline of taxonomy. Using genome‐scale molecular datasets, the extent of admixture across hybrid zones can now be effectively assessed and the evolutionary independence of lineages inferred, leading to more reliable and comparable species delimitation criteria. Substantially divergent but admixing phylogeographical lineages can conveniently be named as subspecies, thus avoiding taxonomic oversplitting and taxonomic inflation. At the same time, comprehensive DNA barcoding and DNA metabarcoding efforts are uncovering an enormous proportion of undiscovered biotic diversity, and we encourage the development of bioinformatic pipelines that combine high‐throughput species discovery and delimitation with diagnosis and scientific naming, to approach a biotic inventory of the globe without abandoning the established Linnaean system.

A comprehensive DNA barcoding of Indian freshwater fishes of the Indus River system, Beas

The Beas River is one of the important rivers of the Indus River system located in Himachal Pradesh, India, that harbors a diverse range of freshwater fish species. The present study employed COI gene to investigate the ichthyofaunal diversity of river Beas. Through the sequencing of 203 specimens from Beas River, we identified 43 species, belonging to 31 genera, 16 families, and 10 orders. To analyze the genetic divergence and phylogeny of identified species, 485 sequences of Indian origin were retrieved from BOLD, resulting in a dataset of 688 sequences. Our findings consistently revealed a hierarchical increase in the mean K2P genetic divergence within species (0.80%), genus (9.06%), and families (15.35%). Automated Barcode Gap discovery, Neighbour Joining, and Bayesian inference consensus tree methodologies were employed to determine the putative species and their phylogeny, successfully delimiting most of the species with only a few exceptions. The results unveiled six species exhibiting high intra-species divergence (> 2%), suggesting the presence of sibling species and falsely identified sequences on online databases. The present study established the first DNA barcoding-based inventory of freshwater fish species in the Beas River providing comprehensive insights into economically exploited endangered and vulnerable species. In order to ensure the sustainable use of aquatic resources in the Beas River, we recommend the implementation of species measures to protect biodiversity and genetic resources.

Cuticular lipid profiles of selected species of cyclocephaline beetles (Melolonthidae, Cyclocephalini).

Neotropical cyclocephaline beetles, a diverse group of flower-loving insects, significantly impact natural and agricultural ecosystems. In particular, the genus Cyclocephala, with over 350 species, displays polymorphism and cryptic complexes. Lacking a comprehensive DNA barcoding framework, accessible tools for species differentiation are needed for research in taxonomy, ecology, and crop management. Moreover, cuticular hydrocarbons are believed to be involved in sexual recognition mechanisms in these beetles. In the present study we examined the cuticular chemical profiles of six species from the genus Cyclocephala and two populations of Erioscelis emarginata and assessed their efficiency in population, species, and sex differentiation. Overall we identified 74 compounds in cuticular extracts of the selected taxa. Linear alkanes and unsaturated hydrocarbons were prominent, with ten compounds between them explaining 85.6% of species dissimilarity. Although the cuticular chemical profiles efficiently differentiated all investigated taxa, only C. ohausiana showed significant cuticular profile differences between sexes. Our analysis also revealed two E. emarginata clades within a larger group of 'Cyclocephala' species, but they were not aligned with the two studied populations. Our research underscores the significance of cuticular lipid profiles in distinguishing selected cyclocephaline beetle species and contemplates their potential impact as contact pheromones on sexual segregation and speciation.

Most soil and litter arthropods are unidentifiable based on current DNA barcode reference libraries.

We are far from knowing all species living on the planet. Understanding biodiversity is demanding and requires time and expertise. Most groups are understudied given problems of identifying and delimiting species. DNA barcoding emerged to overcome some of the difficulties in identifying species. Its limitations derive from incomplete taxonomic knowledge and the lack of comprehensive DNA barcode libraries for so many taxonomic groups. Here, we evaluate how useful barcoding is for identifying arthropods from highly diverse leaf litter communities in the southern Appalachian Mountains (USA). We used 3 reference databases and several automated classification methods on a data set including several arthropod groups. Acari, Araneae, Collembola, Coleoptera, Diptera, and Hymenoptera were well represented, showing different performances across methods and databases. Spiders performed the best, with correct identification rates to species and genus levels of ~50% across databases. Springtails performed poorly, no barcodes were identified to species or genus. Other groups showed poor to mediocre performance, from around 3% (mites) to 20% (beetles) correctly identified barcodes to species, but also with some false identifications. In general, BOLD-based identification offered the best identification results but, in all cases except spiders, performance is poor, with less than a fifth of specimens correctly identified to genus or species. Our results indicate that the soil arthropod fauna is still insufficiently documented, with many species unrepresented in DNA barcode libraries. More effort toward integrative taxonomic characterization is needed to complete our reference libraries before we can rely on DNA barcoding as a universally applicable identification method.

DNA barcoding of marine teleost fishes (Teleostei) in Cebu, the Philippines, a biodiversity hotspot of the coral triangle

A morphology-based barcoding library of market teleost fishes (Teleostei) in Cebu is built based on cytochrome c oxidase subunit I (COI) sequences and voucher specimens which aimed to establish a reliable reference of frequently traded fishes in the province, a biodiversity hotspot at the center of the Philippine archipelago. A total of 1721 specimens were collected from 18 fish markets and landing sites around the province, in which 538 specimens were sequenced belonging to 393 species from 229 genera, 86 families, and 37 orders. Most speciose families are coral reef or reef-related shallow-water species. Twelve species from 11 families are newly recorded in the Philippine waters, among which 7 species are deep-sea inhabitants, while 3 species have expanded their distribution range. Only 20 taxa could not be identified to the species level due to the difficulty in morphological examinations, absence of matched reference sequences in online databases, and/or problematic species awaiting further studies. This first comprehensive DNA barcoding survey of Cebu fishes can facilitate further taxonomic research as well as the conservation and management of fisheries in the Philippines.

DNA barcode-based survey documents underestimated diversity and intricate phylogeographic patterns of aquatic Heteroptera in an endangered Balkan biodiversity hotspot: ancient Lake Skadar basin

Lake Skadar with its surrounding springs, wetlands and larger affluents is among the most diverse freshwater ecosystems in the Mediterranean region and a key biodiversity/endemism hotspot in Europe. It is also highly endangered due to climate change and rapid tourism development in the area. Being abundant, diverse and mostly predatory, true aquatic bugs play an important role in the functioning of freshwater ecosystems and are used as indicators of aquatic habitat quality. Nevertheless, this taxonomic group has been scarcely studied in the area. Our survey provides the first comprehensive DNA barcode library for 24 out of 25 species of aquatic Heteroptera collected in the Skadar Lake basin and adjacent regions. By this, we extend the list of species known from the area by 60%. In the case of three species, Notonecta maculata, Hydrometra stagnorum and Nepa cinerea, we detected multiple highly divergent, and also new BINs indicating possible taxonomic inconsistencies, the potential for (pseudo)cryptic diversity and intricate phylogeographic patterns. We show that presumably well-known hotspots, such as Lake Skadar region, are heavily understudied regarding even the prominent insect taxa and, thus, particularly vulnerable to undocumented biodiversity loss. Finally, we underline the value of simple DNA-barcoding-based surveys for providing reference barcode libraries for effective biomonitoring and signalling taxonomic and biogeographic issues.

DNA barcoding of black flies (Diptera: Simuliidae) in Indonesia

BackgroundDNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences.MethodsGenomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested.ResultsThe DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32–13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa.ConclusionsThe findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.Graphical abstract

Revealing cryptic diversity and population divergence in subtropical aphids throughDNAbarcoding

AbstractAphids are worldwide distributed pests, which feed on plant sap and cause serious losses to agricultural and forestry industries. Relatively few comprehensive studies have been conducted on aphid diversity in subtropics, although these regions may harbour hidden aphid diversity due to high plant diversity. In this study, we conducted DNA barcoding analysis for aphids in subtropical regions based on a comprehensive DNA barcode reference library including 5821 sequences and related ecological information (e.g. geography, host plant) of 379 morphospecies. In the reference library, 2140 sequences and related data of 151 morphospecies belonging to 77 genera and 11 subfamilies were newly produced by the current study. The average Kimura 2‐parameter distances within species, genera and subfamilies were 0.92%, 3.55% and 8.3%, respectively. The minimum interspecific distances were greater than the maximum intraspecific distances in 79.8% species, suggesting that barcode gaps existed in most aphid species. Automatic barcode gap discovery, Barcode Index Number, Bayesian Poisson tree processes and generalized mixed Yule‐coalescent revealed 419, 401, 462 and 455 single species‐representing clusters, respectively. A total of 32 morphospecies were assigned to more than one subclade respectively, suggesting geography or host plant‐mediated divergences and the existence of cryptic diversity. This study demonstrates that subtropical regions should have a higher species diversity of aphids, and cryptic species in certain morphospecies indicated in this study need to be investigated based on integrative taxonomic practices in the future. The DNA barcode reference library constructed herein provides a robust baseline data set to support future research in taxonomy, phylogenetics, ecology and evolution of aphids.

Molecular identification and mapping of mixed root samples to species and individuals.

This article is a Commentary on Osborne et al. (2023), 238: 1305–1317.

A DNA barcode reference library for the native woody seed plants of Japan.

DNA barcode databases are increasingly available for a range of organisms, facilitating the wide application of DNA barcode-based studies. Here we announce the development of a comprehensive DNA barcode reference library of Japanese native woody seed plants representing 43 orders, 99 families, 303 genera and 834 species, and comprising 77.3% of the genera and 72.2% of the species of native woody seed plants in Japan. A total of 6216 plant specimens were collected from 223 sites across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This reference library utilized three chloroplast DNA regions (rbcL, trnH-psbA and matK) and consists of 14,403 barcode sequences. Individual regions varied in their identification rates, with species-level and genus-level rates for rbcL, trnH-psbA and matK based on blast being 57.4%/96.2%, 78.5%/99.1% and 67.8%/98.1%, respectively. Identification rates were higher using region combinations, with total species-level rates for two region combinations (rbcL & trnH-psbA, rbcL & matK and trnH-psbA & matK) ranging between 90.6% and 95.8%, and for all three regions being equal to 98.6%. Genus-level identification rates were even higher, ranging between 99.7% and 100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode reference library is an effective resource for investigations of native woody seed plants in Japan using DNA barcodes and provides a useful template for the development of libraries for other components of the Japanese flora.

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes.

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.

Gap analysis for DNA-based biomonitoring of aquatic ecosystems in China

DNA-based taxon identification is improving the assessment and management of biodiversity in rivers. However, the lack of comprehensive DNA barcode reference libraries and globally highly unequal coverage are still hindering the application prospects of this method worldwide. Here, we analyzed the COI barcode gap in two reference libraries, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on three aquatic animal groups (freshwater fish, aquatic insects and molluscs) in Chinese rivers. Our data show gaps in barcode coverage (e.g., organisms without barcodes) of ca. 40–70% of taxa in these groups in the BOLD or NCBI GenBank database, respectively. These gaps can rise even further if the barcode thresholds are set to contain at least five reference sequences per taxon. Furthermore, most barcodes are from non-local samples, and only 14.4% (BOLD) and 28.8% (NCBI GenBank) of reference sequences were from organisms sampled in China, respectively. The pairwise genetic distance of local barcodes is 3 to 5 times lower than non-local barcodes, indicating that the latter may not be a good substitute. When looking at individual catchments, ca. 60% of the potentially occurring aquatic species have one or more barcodes, yet the barcode coverage varies slightly across ten major river catchments, ranging from 54.3% (Liao River basin) to 68.2% (Huai River basin). The taxa Salmoniformes and Perciformes in freshwater fish, Odonata and Diptera in aquatic insects, and Bivalvia in molluscs have the best barcode coverage in most catchments (mean coverage >70%). This study gives the first overview and current status of barcode reference libraries of three major aquatic animal groups in Chinese rivers. Our results will help to better interpret current metabarcoding studies from China, and also provide a basis to develop a strategy of filling the gaps in the reference libraries of aquatic species in China.

A DNA barcode library for the water mites of Montenegro

Water mites (Acari, Hydrachnidia) are a significant component of freshwater ecosystems inhabiting a wide range of aquatic habitats. This study provides a first comprehensive DNA barcode library for the water mites of Montenegro. DNA barcodes were analysed from 233 specimens of water mites morphologically assigned to 86 species from 28 genera and 15 families. In the course of the study, four species, i.e. Lebertiareticulata (Koenike, 1919), Atractidesinflatipalpis K.Viets, 1950, A.latipes (Szalay, 1935) and Parabrachypodamontii (Maglio, 1924) were molecularly confirmed as new for Montenegro and three species, i.e. Protziaoctopora Lundblad, 1954, Pionalaminata (Thor, 1901) and Unionicolaypsilophora (Bonz, 1783) are new for the Balkan Peninsula. Results are analysed using the Barcode Index Number system (BIN) and the Refined Single Linkage (RESL) of BOLD. The BIN assigned sequences to 98 clusters, while the RESL reveal 103 operational taxonomic units (OTUs). Unique BINs were revealed for 72 species (83.7%), whereas twelve species (14%) were characterised by two BINs and two species (2.3%) with three BINs. Amongst the studied taxa, 14 species were found with a high intraspecific sequence divergences (˃ 2.2%), emphasising the need for additional comprehensive morphological and molecu­lar analysis of these species.

Recent advances in environmental DNA‐based biodiversity assessment and conservation

Recent advances in environmental DNA‐based biodiversity assessment and conservation

The dark side of pseudoscorpion diversity: The German Barcode of Life campaign reveals high levels of undocumented diversity in European false scorpions.

DNA barcoding is particularly useful for identification and species delimitation in taxa with conserved morphology. Pseudoscorpions are arachnids with high prevalence of morphological crypsis. Here, we present the first comprehensive DNA barcode library for Central European Pseudoscorpiones, covering 70% of the German pseudoscorpion fauna (35 out of 50 species). For 21 species, we provide the first publicly available COI barcodes, including the rare Anthrenochernes stellae Lohmander, a species protected by the FFH Habitats Directive. The pattern of intraspecific COI variation and interspecific COI variation (i.e., presence of a barcode gap) generally allows application of the DNA barcoding approach, but revision of current taxonomic designations is indicated in several taxa. Sequences of 36 morphospecies were assigned to 74 BINs (barcode index numbers). This unusually high number of intraspecific BINs can be explained by the presence of overlooked cryptic species and by the accelerated substitution rate in the mitochondrial genome of pseudoscorpions, as known from previous studies. Therefore, BINs may not be an appropriate proxy for species numbers in pseudoscorpions, while partitions built with the ASAP algorithm (Assemble Species by Automatic Partitioning) correspond well with putative species. ASAP delineated 51 taxonomic units from our data, an increase of 42% compared with the present taxonomy. The Neobisium carcionoides complex, currently considered a polymorphic species, represents an outstanding example of cryptic diversity: 154 sequences from our dataset were allocated to 23 BINs and 12 ASAP units.

Unraveling the plant diversity of the Amazonian canga through DNA barcoding.

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.