Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.     

Detecting invertebrate species in archived collections using next‐generation sequencing

Invertebrate biodiversity measured at mostly family level is widely used in biological monitoring programmes to assess anthropogenic impacts on ecosystems. However, next‐generation sequencing (NGS) could allow development of new more sensitive biomonitoring tools by allowing rapid species identification. This could be accelerated if archived invertebrate collections and environmental information from past programmes are used to understand species distributions and their environmental responses. In this study, we take archived macroinvertebrate samples from two sites collected on multiple occasions and test whether NGS can successfully detect species. Samples had been stored in 70% ethanol at room temperature for up to 12 years. Three amplicons ranging from 197 to 274 bps within the DNA barcode region were amplified from samples and compared to DNA barcoding libraries to identify species. We were able to amplify partial DNA barcodes from most samples, and species were often detected with multiple amplicons. However, some singletons and taxa poorly covered by DNA barcoding were missed. This suggests additional DNA barcodes will be required to fill ‘gaps’ in current DNA barcode libraries for aquatic macroinvertebrates and/or that it may not be possible to detect all taxa in a sample. Furthermore, older samples often detected fewer taxa and were less reliable for amplification, suggesting NGS is best used on samples within 8 years of collection. Nevertheless, many common taxa with existing DNA barcodes were reliably identified with NGS and were often present at sites across multiple years, showing the potential of NGS for detecting common and abundant species in archived material.     

Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs.     

Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes)

Digeneans and cestodes are species‐rich taxa and can seriously impact human health, fisheries, aqua‐ and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both ‘universal’ and taxon‐specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.     

COI barcodes for identification of Merodon hoverflies (Diptera, Syrphidae) of Lesvos Island, Greece

DNA barcoding has become a useful system for linking different biological life stages, and for identification of species within a known taxonomic framework. In this study, we generated mitochondrial DNA COI barcodes using adult specimens of all 22 species of the hoverfly genus Merodon (Diptera, Syrphidae) occurring on Lesvos island (Greece). The generated COI barcodes could well discriminate between all Merodon taxa of Lesvos, except for M. loewi and M. papillus that shared the same haplotype, despite their clear morphological differences. In addition, the barcodes revealed two cases of hitherto unknown morphologically cryptic species close to M. avidus and M. nigritarsis, respectively. Because only few successful rearings of immature stages of Merodon hoverflies are available, the larval host plant remains unknown for these phytophagous taxa. The obtained COI barcode library for the Merodon spp. of Lesvos will constitute a tool to link any unknown immature stages with already known species, and thus provide important life-history information and promise for ecological studies.     

DNA barcoding of crickets,katydids and grasshoppers (Orthoptera) from Central Europe with focus on Austria,Germany and Switzerland

We present a DNA barcoding study on the insect order Orthoptera that was generated in collaboration between four barcoding projects in three countries, viz. Barcoding Fauna Bavarica (Germany), German Barcode of Life, Austrian Barcode of Life and Swiss Barcode of Life. Our data set includes 748 COI sequences from 127 of the 162 taxa (78.4%) recorded in the three countries involved. Ninety‐three of these 122 species (76.2%, including all Ensifera) can be reliably identified using DNA barcodes. The remaining 26 caeliferan species (families Acrididae and Tetrigidae) form ten clusters that share barcodes among up to five species, in three cases even across different genera, and in six cases even sharing individual barcodes. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the species concerned are phylogenetically young and hybridization has been previously observed. We also highlight the problem of nuclear mitochondrial pseudogenes (numts), a known problem in the barcoding of orthopteran species, and the possibility of Wolbachia infections. Finally, we discuss the possible taxonomic implications of our barcoding results and point out future research directions.     

Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae

Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell‐based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species‐rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin^® Tissue XS Kit, DNeasy^® Blood and Tissue kit, and QuickExtract^? DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments.     

Comprehensive DNA barcodes for species identification and discovery of cryptic diversity in mayfly larvae from South Korea: Implications for freshwater ecosystem biomonitoring

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing baseline reference for phylogenetic analysis and aquatic ecosystem biomonitoring. We obtained 112 novel sequences of the barcode region of the mitochondrial DNA cytochrome c oxidase subunit I gene representing 11 families, 25 genera, and 43 species of mayfly (Insecta: Ephemeroptera) from South Korea. No species shared barcode sequences and all can be identified with barcodes with a possible exception of some species. Minimum levels of interspecific genetic distances ranged from 6.7 to 32.9% (mean: 23.7%), whereas average levels of intraspecific divergence was 3.7%. The latter value was inflated by the presence of very high divergences within some taxa. In fact, approximately 33.3% (15/45) of the species included two or more haplotype clusters showing greater than 5.0% sequence divergence and some values were as high as 32.9%. Many of the species with high intraspecific divergences are para‐ or polyphyletic and represent the possibility of species complexes. Our study suggests that type or topotype specimens should be sequenced to identify accurate barcoding clusters with morphological species concepts and also to determine the status of currently synonymized species.     

线粒体COⅠ基因在昆虫DNA条形码中的研究与应用

自2003年DNA条形码(DNA barcodes)概念出现以来,DNA条形码技术(DNA barcoding)受到生物分类学领域普遍关注,线粒体细胞色素氧化酶亚基I(mtDNACOⅠ)被用作动物类群的主要条形码序列,基于该基因片段的昆虫条形码研究在国内外广泛开展。本文在概述DNA条形码、条形码技术及已开展的昆虫条形码研究计划的基础上,总结了昆虫mtDNACOⅠ条形码及其技术在发现和描述隐种、种类分子鉴定以及系统发育等方面的研究进展,分析了细胞核线粒体假基因(Numts)对mtDNACOⅠ条形码扩增的影响,提出检测和避免Numts的方法,并对DNA条形码技术的进一步研究和应用进行了讨论和展望。     

An evaluation of candidate plant DNA barcodes and assignment methods in diagnosing 29 species in the genus Agalinis (Orobanchaceae)

? Premise of the study: DNA barcoding has been proposed as a useful technique within many disciplines (e.g., conservation biology and forensics) for determining the taxonomic identity of a sample based on nucleotide similarity to samples of known taxonomy. Application of DNA barcoding to plants has primarily focused on evaluating the success of candidate barcodes across a broad spectrum of evolutionary divergence. Less attention has been paid to evaluating performance when distinguishing congeners or to differential success of analytical techniques despite the fact that the practical application and utility of barcoding hinges on the ability to distinguish closely related species. ? Methods: We tested the ability to distinguish among 92 samples representing 29 putative species in the genus Agalinis (Orobanchaceae) using 13 candidate barcodes and three analytical methods (i.e., threshold genetic distances, hierarchical tree-based, and diagnostic character differences). Due to questions regarding evolutionary distinctiveness of some taxa, we evaluated success under two taxonomic hypotheses. ? Key results: The psbA-trnH and trnT-trnL barcodes in conjunction with the "best close match" distance-based method best met the objectives of DNA barcoding. Success was also a function of the taxonomy used. ? Conclusions: In addition to accurately identifying query sequences, our results showed that DNA barcoding is useful for detecting taxonomic uncertainty; determining whether erroneous taxonomy or incomplete lineage sorting is the cause requires additional information provided by traditional taxonomic approaches. The magnitude of differentiation within and among the Agalinis species sampled suggests that our results inform how DNA barcoding will perform among closely related species in other genera.     

Limitations of mitochondrial gene barcoding in Octocorallia

The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub-class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral-specific mitochondrial protein-coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra- and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character-based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2× greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north‐eastern part of the Congo basin

This study evaluates the utility of DNA barcoding to traditional morphology‐based species identifications for the fish fauna of the north‐eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio ‘nearest‐neighbour distance/maximum intraspecific divergence’ was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative.     

DNA Barcoding and Species Boundary Delimitation of Selected Species of Chinese Acridoidea (Orthoptera: Caelifera)

We tested the performance of DNA barcoding in Acridoidea and attempted to solve species boundary delimitation problems in selected groups using COI barcodes. Three analysis methods were applied to reconstruct the phylogeny. K2P distances were used to assess the overlap range between intraspecific variation and interspecific divergence. “Best match (BM)”, “best close match (BCM)”, “all species barcodes (ASB)” and “back-propagation neural networks (BP-based method)” were utilized to test the success rate of species identification. Phylogenetic species concept and network analysis were employed to delimitate the species boundary in eight selected species groups. The results demonstrated that the COI barcode region performed better in phylogenetic reconstruction at genus and species levels than at higher-levels, but showed a little improvement in resolving the higher-level relationships when the third base data or both first and third base data were excluded. Most overlaps and incorrect identifications may be due to imperfect taxonomy, indicating the critical role of taxonomic revision in DNA barcoding study. Species boundary delimitation confirmed the presence of oversplitting in six species groups and suggested that each group should be treated as a single species.     

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae)

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007–0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known.     

Efficacy of using DNA barcoding to identify parasitoid wasps of the melon-cotton aphid (<Emphasis Type="Italic">Aphis gossypii</Emphasis>) in watermelon cropping system

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.     

Botany without borders: barcoding in focus

This recent meeting, held on the campus of the University of British Columbia, attracted 1200 delegates and a vast array of talks, but was notable for a remarkable showing of talks and posters on DNA barcoding in plants, spread through many sessions. The Canadian Centre for DNA Barcoding defines barcoding as ‘species identification and discovery through the analysis of short, standardized gene regions known as DNA barcodes’. This approach is somewhat controversial in animals ( Rubinoff et al., 2006 ), although it has been shown to be useful and reliable in many metazoan taxa ( Meyer & Paulay 2005 ; Hajibabaei et al., 2007 ), in which the mitochondrial cytochrome oxidase I (COI) gene is used. However, in land plants, COI evolves far too slowly to be useful, and there is no obvious single universal alternative ( Fazekas et al., 2008 ). Genes that work well in one taxon may perform poorly in other taxa. Additionally, some perfectly good plant species, reproductively isolated and morphologically and ecologically distinct, are too young to show much sequence divergence at most loci. Nevertheless, as we saw at this conference, progress has been made towards identifying genes that serve many of the functions of DNA barcodes, at least in some plant taxa.     

关于植物DNA条形码研究技术规范

DNA条形码是利用标准的基因片段对物种进行快速鉴定的技术,已经成功用于生物物种分类和鉴定、生态学调查和生物多样性评估等研究领域。尽管生命条形码数据（BOLD）系统提供了主要针对动物类群DNA条形码研究的技术规范,但由于植物本身的生物学特性与所使用的条形码不同,因此已有技术规范并不完全适用于植物DNA条形码的研究。本文根据植物DNA条形码研究的特点与我国的实际情况,编写了植物DNA条形码研究技术标准和规范指南,具体包括十个方面的内容,即植物DNA条形码研究的样品采集策略;植物标本和野外数据的采集规范;植物标本图像信息的采集规范;植物DNA材料的采集规范;植物DNA材料的干燥与保存规范;植物总DNA的质量标准及保存规范;植物标准DNA条形码的选择与通用引物;DNA条形码的扩增与测序;DNA条形码数据的命名、编辑和提交规范;以及DNA条形码数据分析。我们期望通过这些标准规范的实施和在实践中的不断修订和完善,能为我国学者开展植物DNA条形码和iFlora研究提供参考和借鉴。
关键词：植物DNA条形码;技术规范;物种鉴定;标准;新一代植物志     

‘Direct PCR’ optimization yields a rapid,cost‐effective,nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.