DNA Barcoding Methods Research Articles

Sixteen Years of DNA Barcoding in China: What Has Been Done? What Can Be Done?

Over the past 16 years, more than half (59.68%) of research papers in China on DNA barcoding have been published in Chinese rather than English. Using the records in BOLD (Barcode of Life Data) system, we found Chinese scientists have contributed nearly 120 thousand DNA barcodes for more than 16 thousand species as of September, 2019, with barcoded species distributed throughout China. Based on 2,624 articles and 494 dissertations published during the last 16 years, we reviewed the basic statistics of these studies, including the type of articles contributed by Chinese scientists, the preference of taxonomic groups, the characteristic of barcoding studies in China, the current limitations, and potential future directions as well. We found that most barcode data pertain primarily to plants and animals. Most work in China has focused on verification of the authenticity of species used in traditional Chinese medicine, while other applications paid more attention to food safety, inspection and quarantine, and control of pests and invasive species. In methodology and technology, a number of new DNA barcoding methods were developed by Chinese scientists. However, there are several severe limitations of research on DNA barcoding in China in general, such as the lack of leadership in pioneering international projects, the absence of an open bioinformatics infrastructure, and the fact that some Chinese journals do not clearly require data transparency and availability for DNA barcodes, impeding the further development of barcode libraries and research in China. In the future, Chinese scientists should build authoritative online libraries, while aiming for theoretical innovations for both concepts and methodology of DNA barcoding.

Genetic diversity of genus Vespa including an invaded species of V. velutina (Hymenoptera: Vespidae) in Korea inferred from DNA barcoding data

Abstract With about 5000 known species, the Vespidae is a large family belongs to order Hymenoptera. The genus Vespa with 22 species is one of the four genera of the subfamily Vespinae. In Korea, 10 species and subspecies are recognized. Because of their social behavior, their treat to human health and their impact in apiculture, the reliable and sometimes automated identification of these insects to species level are important. To test the efficacy of DNA barcoding method for identification of species of the genus Vespa in Korea, 30 samples of eight Korean species of genus Vespa were collected and mitochondrial DNAs of 658 bp fragment cytochrome oxidase subunit 1 (CO1) region were sequenced. A Bayesian Inference based on COI gene of the Korean Vespa species was constructed. The phylogenetic tree shoed that identification of all specimens is possible based on COI gene and we found strong relation between the sequences of the collected species from different localities in South Korea which clustered together with 100% support with sequences of the same species in GenBank. The results demonstrate that DNA barcoding is a useful technique for rapid and accurate species recognition in Korean Vespa species. The DNA barcode part of COI for V. binghami is provided for the first time that can help for identification of this species through DNA barcoding. Also, the genetic diversity among Korean Vespa velutina was zero suggests that the invasion might have occurred in a single event with small number of founders.

Preliminary multi analytical approach to address geographic traceability at the intraspecific level in Scombridae family

ABSTRACT Globalization of seafood product marketing caused the increase of request of an effective fish traceability that enhances the consumer confidence in food safety. In this study, an integrated multi analytical approach based on two different and independent analytical techniques (carbon and nitrogen stable isotopes and fatty acids analysis) was applied in order to identify different fish species and trace their geographical provenience. The investigation was focused on four species (Thunnus thynnus, Thunnus alalunga, Auxis rochei and Scomber scombrus) belonging to the Scombridae family. The DNA barcoding method confirmed genus and species for S. scombrus and A. rochei, but only genus for T. alalunga and T. thynnus. Carbon and nitrogen stable isotopes results evidenced different fish diets and trophic positions, whereas fatty acids analysis displayed that the unsaturated prevailed (∼60 %) over the saturated compounds with a variation among the species and the geographical area in particular for docosahexaenoic and eicosapentaenoic acids percentage. The principal component analysis applied to stable isotopes and fatty acids evidenced a good discrimination among species and their geographical catching area. This multi-disciplinary analytical approach could represent a promising tool to identify the commercial fish and trace their origin in order to guarantee the health of consumers.

DNA Barcoding for Delimitation of Putative Mexican Marine Nematodes Species

Nematode biodiversity is mostly unknown; while about 20,000 nematode species have been described, estimates for species diversity range from 0.1 to 100 million. The study of nematode diversity, like that of meiofaunal organisms in general, has been mostly based on morphology-based taxonomy, a time-consuming and costly task that requires well-trained specialists. This work represents the first study on the taxonomy of Mexican nematodes that integrates morphological and molecular data. We added eleven new records to the Mexican Caribbean nematode species list: Anticomidae sp.1, Catanema sp.1, Enoploides gryphus, Eurystomina sp.1, Haliplectus bickneri, Metachromadora sp.1, Odontophora bermudensis, Oncholaimus sp.1, Onyx litorale, Proplatycoma fleurdelis, and Pontonema cf. simile. We improved the COI database with 57 new sequences from 20 morphotypes. All COI sequences obtained in this work are new entries for the international genetic databases GenBank and BOLD. Among the studied sites, we report the most extensive species record (12 species) at Cozumel. DNA barcoding and species delineation methods supported the occurrence of 20 evolutionary independent entities and confirmed the high taxonomic resolution of the COI gene. Different approaches provided consistent results: ABGD and mPTP methods disentangled 20 entities, whereas Barcode Index Numbers (BINs) recovered 22 genetic species. Results support DNA barcoding being an efficient, fast, and low-cost method to integrate into morphological observations in order to address taxonomical shortfalls in meiofaunal organisms.

Comparative analysis of different DNA barcodes for applications in the identification and production of Pyropia

To evaluate the potential application of the DNA barcoding method for Pyropia identification and to select the most appropriate barcode or barcode combination, five barcodes (UPA, V9, Cox1, 5.8S rDNA-ITS, and rbcL) were used to analyze twenty-two different Pyropia samples (including naturally distributed, aquaculture, and processed samples). A total of 99 sequences were obtained from amplification and sequencing of the five barcodes for Bayesian analysis. Except for Cox1, the other four barcodes could accurately identify five different species, i.e., Py. dentata, Py. sp., Py. yamadae, Py. haitanensis and Py. yezoensis. The 5.8S rDNA-ITS region could distinguish closely related species, with interspecific genetic distance values ranging from 40% to 70% and high resolution for intraspecific samples (0.7%). UPA had the shortest sequence length among all five barcodes, with mean length of 410 bp and could also correctly distinguish interspecific samples. Despite some limitations of the candidate markers, the data strongly suggest that DNA barcodes can be used to identify Pyropia. Further, the ability to successfully extract, PCR amplify, and sequence DNA from processed Pyropia samples indicates that processed samples can be analyzed using DNA barcodes. We also found that barcode combination cannot increase genetic distances compared with a single barcode. Our results indicate that 5.8S rDNA-ITS can be used for Pyropia traceability, while UPA is the most viable for quality inspection of Pyropia.

Towards conserving natural diversity: A biotic inventory by observations, specimens, DNA barcoding and high-throughput sequencing methods.

The Kenai National Wildlife Refuge has been given a broad conservation mandate to conserve natural diversity. A prerequisite for fulfilling this purpose is to be able to identify the species and communities that make up that biodiversity.We tested a set of varied methods for inventory and monitoring of plants, birds and terrestrial invertebrates on a grid of 40 sites in a 938 ha study area in the Slikok Creek watershed, Kenai Peninsula, Alaska. We sampled plants and lichens through observation and specimen-based methods. We surveyed birds using bird call surveys on variable circular plots. We sampled terrestrial arthropods by sweep net sampling, processing samples with High Throughput Sequencing methods. We surveyed for earthworms, using the hot mustard extraction method and identified worm specimens by morphology and DNA barcoding. We examined community membership using clustering methods and Nonmetric Multidimensional Scaling.We documented a total of 4,764 occurrences of 984 species and molecular operational taxonomic units: 87 vascular plants, 51 mosses, 12 liverworts, 111 lichens, 43 vertebrates, 663 arthropods, 9 molluscs and 8 annelid worms. Amongst these records, 102 of the arthropod species appeared to be new records for Alaska. We found three non-native species: Derocerasagreste (Linnaeus, 1758) (Stylommatophora: Agriolimacidae), Dendrobaenaoctaedra (Savigny, 1826) (Crassiclitellata: Lumbricidae) and Heterarthrusnemoratus (Fallén, 1808) (Hymenoptera: Tenthredinidae). Both D.octaedra and H.nemoratus were found at sites distant from obvious human disturbance. The 40 sites were grouped into five community groups: upland mixed forest, black spruce forest, open deciduous forest, shrub-sedge bog and willow.We demonstrated that, at least for a subset of species that could be detected using these methods, we were able to document current species distributions and assemblages in a way that could be efficiently repeated for the purposes of biomonitoring. While our methods could be improved and additional methods and groups could be added, our combination of techniques yielded a substantial portion of the data necessary for fulfilling Kenai National Wildlife Refuge's broad conservation purposes.

Preliminary application of DNA barcoding toward the detection of viable plant propagules at an initial, international point-of-entry in Georgia, USA

Over 90% of global commercial trade occurs between seaports, which are initial points-of-entry for nonnative, potentially invasive propagules. As such, there is a need to develop means to both rapidly intercept and identify propagules as they arrive. Here, we focus on plant propagules that are assumed to be non-native, in seed form. Because standard morphological techniques alone are laborious and require taxonomic expertise, we sought to address if identification through barcoding of the plastid DNA (rbcL + matK genes) of plant seeds could improve current processes in the early detection and rapid response to prevent entry/establishment of nonnative plant species. This research conducted a preliminary foray to evaluate the utility of widely accepted plant plastid DNA barcodes to identify plant propagules (seeds, hereafter) collected from the air-intake grilles of refrigerated shipping containers of a single agricultural commodity arriving at the Port of Savannah, Georgia, USA. We ask four questions: (1) Can DNA barcoding be used to detect seeds collected from shipping containers at the port? (2) What is the genetic composition of propagules entering the port? (3) How do morphological identifications compare to those based on genetic analysis? (4) Are nonnative invasive plant species present on shipping containers entering the Port of Savannah? This research collected 11,044 seeds from 628 refrigerated shipping containers between 2015 and 2017. Seeds were then morphologically sorted into Seed Types. Barcoding of the matK and rbcL gene regions of the plastid genomes directly isolated from seeds resulted in poor amplification. This is likely due to a host of potential confounding factors. Therefore, we germinated seeds and utilized leaf-tissues for sequencing of these two gene regions. From BLASTn analyses, results returned top hits for a variety of species, with up to 22 possible nonnative plant species and one definite Federal Noxious Weed. This work investigates the interception application of DNA barcoding to improve agro- and bio-security issues posed by nonnative and invasive species. Though this study required the germination of the seeds to obtain leaf-tissue suitable for our DNA barcoding method, we effectively demonstrated seed viability. Our seed identification process was lengthy and understandably not feasible for real-time application. Therefore, we seek to improve our methods for future applications by testing other approaches that may better complement morphological identification. Next reasonable steps include improved extraction protocols, metabarcoding to generate DNA barcode sequences directly from groups of seeds harvested from shipping containers and implementing other next-generation sequencing techniques.

Feeding intensity and molecular prey identification of the common long-armed octopus, Octopus minor (Mollusca: Octopodidae) in the wild.

The common long-armed octopus, Octopus minor, is an important component of systems and supports the local fisheries in the coastal areas of northern China. For the fishery management and artificial breeding, especially for the management of exclusive conservation reserves, its role in the ecosystem requires assessment. Therefore, the feeding intensity of O. minor was studied from April to July 2014 when females reaching ovary maturation, and prey composition was identified from stomach contents using a DNA barcoding method. Of the 172 sampled octopuses, 66 had stomach contents that were nearly digested into pulp. On the whole, the feeding intensity of octopus remained more or less the same during the first three months and significantly decreased in July. The changes of feeding intensity were different between females and males; in females, the intensity of feeding decreased from April to July; in case of males, however, the feeding activity increased from April to June and decreased thereafter. The feeding intensity of the females was extremely greater than that of the males. O. minor was a generalist predator and based on homology searches and phylogenetic analysis, a total of 10 different taxa were identified in the stomach contents. In terms of percent composition by frequency of occurrences (%N), fishes accounted for the most of the octopuses diet (50%), followed by cephalopod (25%), crustaceans (21.7%), annelid (1.7%) and nematode (1.7%). The families of Gobiidae and Octopodidae appeared in all months and Protunidae appeared in three months. The results confirmed that Gobiidae family (45.8%, by frequency of occurrences) was an important source of food during the time when females reaching ovarian maturation. From April to July, the observed cannibalism showed an increasing trend. Controlling and reducing fishing production of Gobiidae fishes in conservation area are recommended from April to June when female octopuses are actively feeding.

First report of the lace bug Neoplerochila paliatseasi (Rodrigues, 1981) (Hemiptera: Tingidae) infesting cultivated olive trees in South Africa, and its complete mitochondrial sequence.

Olive lace bugs are small phytophagous Hemipteran insects known to cause agricultural losses in olive production in South Africa. Plerochila australis (Distant, 1904) has been reported as the species responsible for damage to olive trees; however, the diversity of olive lace bug species in the region has lacked attention. Adult olive lace bugs were collected incidentally from wild and cultivated olive trees in the Western Cape Province, and identified as P. australis and Neoplerochila paliatseasi (Rodrigues, 1981). The complete mitochondrial genome of a representative specimen of N. paliatseasi was sequenced, and used for comparative mitogenomics and phylogenetic reconstruction within the family. Furthermore, the value of DNA barcodes for species identification in Tingidae was assessed using genetic clustering and estimates of genetic divergence. The patterns of genetic clustering and genetic divergence of COI sequences supported the morphological identification of N. paliatseasi, and the utility of DNA barcoding methods in Tingidae. The complete mitogenome sequence had the typical Metazoan gene content and order, including 13 PCGs, 22 tRNAs, two rRNAs, and an AT-rich non-coding region. A+T content was high, as commonly found in Tingidae. The phylogenetic reconstruction recovered Agramma hupehanum (Drake Maa 1954) as basal to Tingini, and as a sister species to N. paliatseasi. Stephanitis Stål 1873 and Corythucha Stål 1873 were monophyletic, but Metasalis populi (Takeya 1932) was not recovered as sister to Tingis cardui (Linnaeus 1746), as expected. The mitochondrial phylogeny of the family Tingidae has been recovered inconsistently across different studies, possibly due to sequence heterogeneity and high mutation rates. Species diversity of olive lace bugs in South Africa was previously underestimated. The presence of P. australis was confirmed in both wild and cultivated olives, and N. paliatseasi is reported in cultivated olives for the first time. These results warrant further investigation on the diversity and distribution of olive lace bugs in the Western Cape to inform pest control strategies.

Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding

Ethnopharmacological relevanceMantidis Oötheca (mantis egg case; sangpiaoxiao) is a medicine from an insect source, which has been widely used in Asian countries. However, misidentification due to a lack of information given variations in the medicinal portion of the ootheca and morphological similarities of the ootheca as an egg chamber. Aim of the studyThus, this study aims to provide the first comprehensive data for discriminating authentic of Mantidis Oötheca. Here, we provide detailed ootheca morphology and their molecular information to accurately identify Mantidis Oötheca. Materials and methodsOothecae of Tenodera angustipennis (Saussure, 1869), Tenodera sinensis (Saussure, 1871), Hierodula patellifera Serville, 1839, and Hierodula sp. were used in the comparative morphological, principal component analysis, and DNA barcoding. ResultsThe morphological analyses revealed that the emergence area, outline, angle of distal end, width of air-filled layer, and weight are useful diagnostic characters. Using these quantitative and qualitative characteristics, we developed the effective identification key. Furthermore, our CO1 sequences from all individuals were monophyletic with high bootstrap values at genus and species levels. Moreover, morphological identification using our developed key among all studied individuals agreed with molecular identification results using CO1 barcoding data. ConclusionsThese multilateral approaches, including morphological, statistical, and DNA barcoding methods are highly reliable identification tools. Moreover, our diagnostic key characteristics and molecular barcoding should aid in the accurate identification, authentication, and quality control of Mantidis Oötheca medicinal materials.

Integrative taxonomy reveals a new species of pacu (Characiformes: Serrasalmidae: Myloplus) from the Brazilian Amazon

ABSTRACT Pacus of the genus Myloplus represent a formidable taxonomic challenge, and particularly so for the case of M. asterias and M. rubripinnis, two widespread and common species that harbor considerable morphological diversity. Here we apply DNA barcoding and multiple species discovery methods to find candidate species in this complex group. We report on one well-supported lineage that is also morphologically and ecologically distinct. This lineage represents a new species that can be distinguished from congeners by the presence of dark chromatophores on lateral-line scales, which gives the appearance of a black lateral line. It can be further diagnosed by having 25-29 branched dorsal-fin rays (vs. 18-24), 89-114 perforated scales from the supracleithrum to the end of hypural plate (vs. 56-89), and 98-120 total lateral line scales (vs. 59-97). The new species is widely distributed in the Amazon basin, but seems to have a preference for black- and clearwater habitats. This ecological preference and black lateral line color pattern bears a striking similarity to the recently described silver dollar Metynnis melanogrammus.

Wild Pacific oyster Magallana gigas (Thunberg, 1793) populations in Romanian Black Sea waters – friend or foe?

A relatively small population of Magallana gigas was discovered near the Agigea harbor (Constanța, Romania) in 2017. The DNA barcoding method was used to confirm the morphological identification of the species. We consider this colony to be the first instance of a possibly stable Pacific oyster population in the Black Sea, outside of farming activities. The possible impact on native ecosystems is briefly discussed.

Species identification and genetic diversity of Aedes in Penang (Malaysia) based on Cytochrome Oxidase Subunit I

Abstract. Kamal MZM, Dangnga MS, Irmayani, Md. Naim D. 2020. Species identification and genetic diversity of Aedes in Penang (Malaysia) based on Cytochrome Oxidase Subunit I. Nusantara Bioscience 12: 6-12. Cases of dengue fever are expanding globally and recently it was identified as being notable in remote/rural regions in South-East Asia in spite of the previous belief that it is outbreak to urban areas, especially in Malaysia. In an effort to constraint, the extent of the virus, the most important thing is to know the distribution of the vector that causes the disease. This research aims to recognize all species of mosquitoes distributed in Penang utilizing DNA barcode method in which the mtDNA cytochrome oxidase subunit 1 (COI) as a gene marker. Approximately 497 larvae of mosquitoes were obtained from four regions in Penang, Malaysia namely Central Seberang Perai, North Seberang Perai, North East, and South West of Penang. All samples were extracted and PCR amplified. Sequences were employed in BLAST of GenBank and aligned with MUSCLE. The ABGD analysis was performed to partition all the samples and substitution saturation analysis was employed in DAMBE. Phylogenetic relationships among nucleotides were reconstructed utilizing Maximum Likelihood (ML) and Neighbor-Joining (NJ) calculated following Kimura 2-parameter. Intra and interspecific genetic variation among population were also conducted based on Tamura-Nei parameter and all analyses were employed in MEGA version 6.0. As a result, COI gene has successfully recognized all larvae obtained in this study. We found a total of six species (including outgroup sequence) as revealed by BLASTn, ABGD and phylogenetic analysis. Genetic distance test has further supported the results. This study revealed that there is no visible population structure and all species have a uniform distribution throughout all sampling locations in Penang. We also found that Ae. albopictus has occupied most areas in Penang, and this includes urban locations, hence, suggest that Ae. albopictus probably succeed Ae. aegypti as the main vector for dengue virus in Penang.

DNA barcoding as a method for species identification: case study in Ahaetulla snake

Species identification using molecular analyses has recently been developed, one of which is DNA barcoding method. The purpose of this study was to apply the DNA barcoding method to identify Ahaetulla snake which previously had not been identified molecularly. The specimens used came from Sumatra, Java, Bali, Lombok and additional samples obtained from Genbank. Cytochrome B gene was extracted and amplified to obtain DNA barcode. Phylogenetic analysis using ML method was used to determine the kinship relationship of the Ahaetulla snake. Morphological analyses were added to confirm the results of molecular identification. The results of molecular analysis using DNA barcoding method showed the presence of two Ahaetulla species in this study, there are A. prasina and A. mycterizans. This difference is indicated by the formation of two separate main clades, clade I contains A. prasina and clade II contains A. mycterizans. The results of the morphological analyses also showed differences in the character of the two different clade species. Thus, molecular analyses result agrees with morphological analyses. It can be concluded that the DNA barcoding method can be used as a valid method to identify Ahaetulla snake species.

DNA Barcoding: Methods and Approaches

The DNA barcoding method proposed 15 years ago has gained considerable popularity. This work provides a review of methodical approaches in this field and their progress over the past years. Direct and reverse approaches to DNA barcoding and their prospects due to the development of sequencing methods are discussed.

Ciliate Environmental Diversity Can Be Underestimated by the V4 Region of SSU rDNA: Insights from Species Delimitation and Multilocus Phylogeny of Pseudokeronopsis (Protist, Ciliophora).

Metabarcoding and high-throughput sequencing methods have greatly improved our understanding of protist diversity. Although the V4 region of small subunit ribosomal DNA (SSU-V4 rDNA) is the most widely used marker in DNA metabarcoding of eukaryotic microorganisms, doubts have recently been raised about its suitability. Here, using the widely distributed ciliate genus Pseudokeronopsis as an example, we assessed the potential of SSU-V4 rDNA and four other nuclear and mitochondrial markers for species delimitation and phylogenetic reconstruction. Our studies revealed that SSU-V4 rDNA is too conservative to distinguish species, and a threshold of 97% and 99% sequence similarity detected only one and three OTUs, respectively, from seven species. On the basis of the comparative analysis of the present and previously published data, we proposed the multilocus marker including the nuclear 5.8S rDNA combining the internal transcribed spacer regions (ITS1-5.8S-ITS2) and the hypervariable D2 region of large subunit rDNA (LSU-D2) as an ideal barcode rather than the mitochondrial cytochrome c oxidase subunit 1 gene, and the ITS1-5.8S-ITS2 as a candidate metabarcoding marker for ciliates. Furthermore, the compensating base change and tree-based criteria of ITS2 and LSU-D2 were useful in complementing the DNA barcoding and metabarcoding methods by giving second structure and phylogenetic evidence.

Using DNA Barcodes to Aid the Identification of Larval Fishes in Tropical Estuarine Waters (Malacca Straits, Malaysia).

Larval descriptions of tropical marine and coastal fishes are very few, and this taxonomic problem is further exacerbated by the high diversity of fish species in these waters. Nonetheless, accurate larval identification in ecological and early life history studies of larval fishes is crucial for fishery management and habitat protection. The present study aimed to evaluate the usefulness of DNA barcodes to support larval fish identification since conventional dichotomous keys based on morphological traits are not efficient due to the lack of larval traits and the rapid morphological changes during ontogeny. Our molecular analysis uncovered a total of 48 taxa (21 families) from the larval samples collected from the Klang Strait waters encompassing both spawning and nursery grounds of marine and estuarine fishes. Thirty-two (67%) of the larval taxa were identified at the species level, two taxa (4%) at the genus level, and 14 taxa (29%) at family level. The relatively low rate of species-level identification is not necessarily due to the DNA barcoding method per se, but a general lack of reference sequences for speciose and non- commercial fish families such as Gobiidae, Blenniidae, and Callionymidae. Larval morphology remains important in species diagnoses when molecular matches are ambiguous. A lower ethanol percentage (50%) for larva preservation is also useful to keep the body of larvae intact for morphological identification, and to preserve DNA for subsequent molecular analyses. The 10% Chelex resin used to extract DNA is also cost- effective for long term monitoring of larval fishes. Hence, the DNA barcoding method is an effective and easy way to aid the identification of estuarine larval fishes at the species level.

Identification and evaluation of Citrus grandis based on DNA barcode,UPLC and chromaticity method

In order to identify the source of Citrus grandis and evaluate its quality originate from two areas comprehensively,DNA barcode was used to identify 26 samples of C. grandis. The content of naringin,rhoifolin,naringenin and apigenin was determined by UPLC method,and the color difference was numerically studied by color difference analyzer,which was related to the effective components of C. grandis. The results showed that samples was the source of C. grandis in both regions. The ITS2 sequence length was about400-500 bp,and the sequence similarity reached 99. 82%. There was only one base deletion in the two groups. There was one base A in some medicinal materials of Guangdong at 330 bp,but no base in Chongqing. The contents of naringin and rhoifolin in Chongqing samples were higher than those in Guangdong samples,and there were statistical differences between naringenin and apigenin. The chroma value showed that L*value of Guangdong was larger,a*value was smaller,L*value of Chongqing was smaller,and a*value was larger,while the b*value of both was not significantly different; The results of correlation analysis showed that naringin,rhoifolin,naringenin were positively correlated with L*,b*value,negatively correlated with a*value,and apigenin had no correlation with L*,a*,b*value. In this study,the scientific identification and evaluation of C. grandis was carried out to provide a new idea for the further study of the rapid identification and evaluation of C. grandis.

DNA-barcoding clarifies species definitions of Finnish <i>Apatania</i> (Trichoptera: Apataniidae)

All the known 8 Finnish Apatania Kolenati species were analysed using the DNA barcoding method. The Finnish species were grouped in four clades; A. zonella (Zetterstedt, 1840) group sensu lato, A. stigmatella (Zetterstedt, 1840), A. wallengreni (McLachlan, 1871) and A. muliebris McLachlan, 1866. The A. zonella group sensu lato consists of A. zonella, A. dalecarlica (Forsslund, 1942), A. forsslundi Tobias, 1981, A. auricula (Forsslund, 1930) and A. hispida (Forsslund, 1930). The DNA barcoding results support keeping all these 8 taxa as separate, good species. The main distribution and larval environment of A. zonella and A. dalecarlica in Finland were clarified. In addition to the 8 species, a morphologically distinct taxon close to A. hispida was included in the analysis and is discussed. Photographs of the genitalia of all taxa are provided as well as a key for identifying the taxa.

Unexpected discovery of Diadema clarki in the Coral Triangle

Sea urchins of the genus Diadema, key herbivores in coral reef ecosystems, also provide habitat for other organisms. Our research extended Diadema biogeography in seaways east and west of Sulawesi and identified Diadema species associated with the endemic Banggai cardinalfish (Pterapogon kauderni) using field surveys and molecular DNA barcoding methods. Field observations (20 sites, n = 11,223) found urchins with morphological phenotypes typical of Diadema setosum (≈ 74%, all sites), D. savignyi (≈ 24%, 19 sites) and atypical or mixed traits (≈ 2%, 19 sites). Distribution of these phenotype groups across the three main habitat types (i.e. coral reef, reef flat and seagrass beds) differed significantly (χ2 = 533.03, p < 2.2e−16), indicating overlapping but non-equivalent ecological niches. Pterapogon kauderni associated with all urchin morphological phenotypes present. Diadema mtDNA CO1 sequences were obtained from tissue samples collected (4 sites, n = 62) from specimens with typical D. savignyi and D. setosum phenotypes. Phylogenetic tree analysis resolved the sequences into four clades. Three clades from our analysis were identified as D. savignyi, D. setosum and D. clarki based on additional sequences obtained from GenBank. This unexpected first record of D. clarki mtDNA in the Coral Triangle implies a substantial extension of the known range of this recently resurrected species. Our findings indicate the occurrence and/or introgression of D. clarki may be widespread, and misidentification of Diadema urchins based on external morphology may be relatively common. Further research is required to determine the distribution and functional roles of Indo-Pacific Diadema species, contributing to our understanding of processes underpinning biodiversity.