Untangling taxonomy: a DNA barcode reference library for Canadian spiders

Approximately 1460 species of spiders have been reported from Canada, 3% of the global fauna. This study provides a DNA barcode reference library for 1018 of these species based upon the analysis of more than 30 000 specimens. The sequence results show a clear barcode gap in most cases with a mean intraspecific divergence of 0.78% vs. a minimum nearest‐neighbour (NN) distance averaging 7.85%. The sequences were assigned to 1359 Barcode index numbers (BINs) with 1344 of these BINs composed of specimens belonging to a single currently recognized species. There was a perfect correspondence between BIN membership and a known species in 795 cases, while another 197 species were assigned to two or more BINs (556 in total). A few other species (26) were involved in BIN merges or in a combination of merges and splits. There was only a weak relationship between the number of specimens analysed for a species and its BIN count. However, three species were clear outliers with their specimens being placed in 11–22 BINs. Although all BIN splits need further study to clarify the taxonomic status of the entities involved, DNA barcodes discriminated 98% of the 1018 species. The present survey conservatively revealed 16 species new to science, 52 species new to Canada and major range extensions for 426 species. However, if most BIN splits detected in this study reflect cryptic taxa, the true species count for Canadian spiders could be 30–50% higher than currently recognized.     

药用植物DNA条形码鉴定研究进展

DNA条形码是利用相对较短的标准DNA片段对物种进行快速准确鉴定的一门技术。DNA条形码技术可以从分子水平弥补传统鉴定方法的一些不足。该技术具有良好的通用性,使得物种鉴定过程更加快速,已经广泛应用于动物物种的鉴定研究中。近年来,随着药用植物DNA条形码鉴定研究的快速发展,逐渐形成了药用植物和植物源中药材鉴定的完善体系。本文综述了DNA条形码技术鉴定药用植物的原理,介绍了中草药传统鉴定方法及其缺陷、使用DNA条形码技术鉴定植物源药材的意义以及DNA条形码在药用植物鉴定中的应用,对其应用前景进行了展望。     

DNA条形码与动植物分类学的研究

DNA条形码在动植物分类学中的研究最近几年非常火热。这项技术在国外研究的比较多,国内的许多方面的研究还没达到国际水平。我国的动植物种类比较繁多,地区差异也较大,怎样能将这些物种准确,快速的进行分类,是众多动植物学家一直在研究的难题。针对DNA条形码研究的现状和他在动植物学中应用以及它存在的争议来进一步认识DNA条形码。     

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.     

DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用

为了探讨DNA条形码技术在夜蛾物种鉴定中的可行性, 本研究利用条形码通用引物扩增了北京百花山地区43种夜蛾75个样本的线粒体细胞色素C氧化酶亚基I （mitochondrial cytochrome c oxidase subunit I, COI）基因序列, 以Kimura双参数模型进行种内种间遗传距离分析、 使用邻接法（neighbor-joining, NJ）和最大简约法（maximum parsimony, MP）分别构建系统发育树, 并利用分子序列差异阈值对样本进行分子可操作分类单元（molecular defined operational taxonomic units, MOTU）划分。结果表明： 所有夜蛾种类通过系统发育树可以成功区分; 种内平均遗传距离(0.03%)远远小于种间平均遗传距离(11.29%); 采用较为保守的1%的序列差异阈值将75个夜蛾样本分为42个MOTU, 正确率为95%, 除了MOTU04包含2个物种外, 剩余41个MOTU与形态种呈现一一对应的关系。结果显示, 基于COI基因的DNA条形码对于本研究中所涉及的夜蛾具有较好的区分, 可以作为一种有效的工具在夜蛾科昆虫物种鉴定中进行应用。     

Molecular operational taxonomic units as approximations of species in the light of evolutionary models and empirical data from Fungi

During the last couple of decades, an increasing number of studies use sequence clusters as units for taxonomic diversity. It is well known that such molecular operational taxonomic units (MOTUs) do not necessarily correspond to species, but they are treated as such when measuring diversity and testing theories. Here, I show that data from studies of molecular evolution and species diversification of fungi indicate that commonly used cut‐offs are likely to lump species in many cases. At the same time, empirical studies show that the mean within‐species variation is close to these cut‐offs. That the within‐species variation estimates are plausible is supported by coalescence modelling under a range of parameter settings. In addition, studies using crossing tests to delimit species show that there often is an overlap in within‐ and between‐species distances. The available data therefore indicate that sequence clusters are likely to misrepresent species. However, to keep a biological relevance, MOTUs should be kept in close agreement with species. Studies using them should therefore asses how sensitive the results are to differences between MOTUs and species – something that is rarely done. An even better solution is to directly include the uncertainty in species delimitation in the analyses, but in many cases, we need to increase our knowledge of taxonomy and evolution to do this accurately. Even if the empirical data referred to here pertain to the “barcoding” region of rDNA in fungi, there is nothing indicating that the situation is substantially better for other taxa or genes.     

DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Multiple species delimitation approaches with COI barcodes poorly fit each other and morphospecies – An integrative taxonomy case of Sri Lankan Sericini chafers (Coleoptera: Scarabaeidae)

DNA taxonomy including barcoding and metabarcoding is widely used to explore the diversity in biodiversity hotspots. In most of these hotspot areas, chafers are represented by a multitude of species, which are well defined by the complex shape of male genitalia. Here, we explore how well COI barcode data reflect morphological species entities and thus their usability for accelerated species inventorization. We conducted dedicated field surveys in Sri Lanka to collect the species‐rich and highly endemic Sericini chafers (Coleoptera: Scarabaeidae). Congruence among results of a series of protocols for de novo species delimitation and with morphology‐based species identifications was investigated. Different delimitation methods, such as the Poisson tree processes (PTP) model, Statistical Parsimony Analysis (TCS), Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Barcode Index Number (BIN) assignments, resulted in different numbers of molecular operational taxonomic units (MOTUs). All methods showed both over‐splitting and lumping of morphologically identified species. Only 18 of the observed 45 morphospecies perfectly matched MOTUs from all methods. The congruence of delimitation between MOTUs and morphospecies expressed by the match ratio was low, ranging from 0.57 to 0.67. TCS and multirate PTP (mPTP) showed the highest match ratio, while (BIN) assignment resulted in the lowest match ratio and most splitting events. mPTP lumped more species than any other method. Principal coordinate analysis (PCoA) on a match ratio‐based distance matrix revealed incongruent outcomes of multiple DNA delimitation methods, although applied to the same data. Our results confirm that COI barcode data alone are unlikely to correctly delimit all species, in particular, when using only a single delimitation approach. We encourage the integration of various approaches and data, particularly morphology, to validate species boundaries.     

Identifying fern gametophytes using DNA sequences

Identification of fern gametophytes is generally hampered by low morphological complexity. Here we explore an alternative: DNA‐based identification. We obtained a plastid rbcL sequence from a sterile gametophyte of unknown origin (cultivated for more than 30 years) and employed blast to determine its affinities. Using this approach, we identified the gametophyte as Osmunda regalis. To evaluate the robustness of this determination, and the usefulness of rbcL in differentiating among species, we conducted a phylogenetic analysis of osmundaceous fern sequences. Based on our results, it is evident that DNA‐based identification has considerable potential in exploring the ecology of fern gametophytes.     

Deep genetic divergences in Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi): a widespread 'mite harvestman' defies DNA taxonomy

Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi, Pettalidae), a widespread 'mite harvestman' endemic to the South Island of New Zealand, is found in leaf littler habitats throughout Nelson and Marlborough, and as far south as Arthur's Pass. We investigated the phylogeography and demographic history of A. denticulata in the first genetic population-level study within Opiliones. A total of 119 individuals from 17 localities were sequenced for 785 bp of the gene cytochrome c oxidase subunit I; 102 of these individuals were from the Aoraki subspecies A. denticulata denticulata and the remaining 17 were from the subspecies A. denticulata major. An extraordinarily high degree of genetic diversity was discovered in A. denticulata denticulata, with average uncorrected p-distances between populations as high as 19.2%. AMOVA, average numbers of pairwise differences, and pairwise F(ST) values demonstrated a significant amount of genetic diversity both within and between populations of this subspecies. Phylogenetic analysis of the data set revealed many well-supported groups within A. denticulata denticulata, generally corresponding to clusters of specimens from single populations with short internal branches, but separated by long branches from individuals from other populations. No haplotypes were shared between populations of the widespread small subspecies, A. denticulata denticulata. These results indicate a subspecies within which very little genetic exchange occurs between populations, a result consistent with the idea that Cyphophthalmi are poor dispersers. The highly structured populations and deep genetic divergences observed in A. denticulata denticulata may indicate the presence of cryptic species. However, we find a highly conserved morphology across sampling localities and large genetic divergences within populations from certain localities, equivalent to those typically found between populations from different localities. Past geological events may have contributed to the deep genetic divergences observed between sampling localities; additionally, the high divergence within populations of A. denticulata denticulata suggests that the rate of COI evolution may be accelerated in this taxon. In contrast, the larger subspecies A. denticulata major shows much less differentiation between and within sampling localities, suggesting that it may disperse more easily than its smaller counterpart. The fact that the remarkable genetic divergences within populations of A. denticulata denticulata from certain localities are equivalent to divergences between localities poses a challenge to the rapidly spreading practice of DNA taxonomy.     

Confirmation of the species status of the blackfly Simulium galeratum in Britain using molecular taxonomy

Since 1920 Simulium reptans (Linnaeus) (Diptera: Simuliidae) has been reported as exhibiting two different larval morphotypes, a typical S. reptans and an atypical S. reptans var. galeratum, which differ in the markings of the larval head capsule. Inconsistent variation in adults and no apparent variation in the pupae have led taxonomists to conclude that these types in Britain are a single species. We investigated populations in Britain where either the typical form or var. galeratum is found, and one population where the two exist sympatrically. A phylogenetic study based upon a region of the mitochondrial cytochrome c oxidase 1 gene (DNA barcoding) produced a tree that delineated the morphotypes into two distinct monophyletic clades. The average Kimura-2-parameter distances within each clade (i.e. within each morphotype) were very low (0.67% and 0.78%), with the distances between morphotypes being 9-10-fold greater (mean 7.06%). This is concordant with differences within and between species in other taxa; based upon the strict correlation between the molecular variation and the morphotypes, we propose the re-instatement of S. galeratum to species status.     

DNA barcoding largely supports 250 years of classical taxonomy: identifications for Central European bees (Hymenoptera,Apoidea partim)

This study presents DNA barcode records for 4118 specimens representing 561 species of bees belonging to the six families of Apoidea (Andrenidae, Apidae, Colletidae, Halictidae, Megachilidae and Melittidae) found in Central Europe. These records provide fully compliant barcode sequences for 503 of the 571 bee species in the German fauna and partial sequences for 43 more. The barcode results are largely congruent with traditional taxonomy as only five closely allied pairs of species could not be discriminated by barcodes. As well, 90% of the species possessed sufficiently deep sequence divergence to be assigned to a different Barcode Index Number (BIN). In fact, 56 species (11%) were assigned to two or more BINs reflecting the high levels of intraspecific divergence among their component specimens. Fifty other species (9.7%) shared the same Barcode Index Number with one or more species, but most of these species belonged to a distinct barcode cluster within a particular BIN. The barcode data contributed to clarifying the status of nearly half the examined taxonomically problematic species of bees in the German fauna. Based on these results, the role of DNA barcoding as a tool for current and future taxonomic work is discussed.     

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.     

DNA-based species delimitation in algae

Given the problems of species delimitation in algae using morphology or sexual compatibility, molecular data are becoming the standard for delimiting species and testing their traditional boundaries. The idea that species are separately evolving metapopulation lineages, along with theoretical progress in phylogenetic and population genetic analyses, has led to the development of new methods of species delimitation. We review these recent developments in DNA-based species delimitation methods, and discuss how they have changed and continue to change our understanding of algal species boundaries. Although single-locus approaches have proven effective for a first rapid and large-scale assessment of species diversity, species delimitation based on single gene trees falls short due to gene tree–species tree incongruence, caused by confounding processes like incomplete lineage sorting, trans-species polymorphism, hybridization and introgression. Data from unlinked loci and multi-species coalescent methods, which combine principles from phylogenetics and population genetics, may now be able to account for these complicating factors. Several of these methods also provide statistical support regarding species boundaries, which is important because speciation is a process and therefore uncertainty about precise species boundaries is inevitable in recently diverged lineages.     

Detecting signatures of competition from observational data: a combined approach using DNA barcoding,diversity partitioning and checkerboards at small spatial scales

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Integrative taxonomy does not support the occurrence of two species of the Squalius squalus complex (Actinopterygii,Cypriniformes, Cyprinidae) in Italy

The systematic is still unresolved for the genus Squalius (Cyprinidae, Leuciscinae), a rich group of small to large fishes widely distributed throughout Europe. The distinction of one of the Italian narrowly endemic species, Squalius albus (Bonaparte, 1838), described for the area surrounding lake Trasimeno, from the more common and widespread Squalius squalus (Bonaparte, 1837) is doubtful. The application of integrative taxonomy, with DNA taxonomy and quantitative morphometric, using both living and preserved individuals collected from lake Trasimeno before Squalius sp. restocking, allowed us to explicitly test for the identity of the two species in the complex. COI barcoding data, used for phylogenetic reconstructions, underlined that two clades may exist in the complex; nevertheless, DNA taxonomy (ABGD and GMYC) and morphometrics show no statistical support for their identity as separate species. Moreover, during our survey of the genetic diversity of the Italian Squalius, we provided further support for the species status of Squalius lucumonis, and found evidence of the occurrence in Southern Italy of another chub species, Squalius vardarensis (Karaman, 1928), previously known only from the Southern part of the Balkan Peninsula.     

Molecular taxonomy and species delimitation in Andean Schistocerca (Orthoptera: Acrididae)

The acridian genus Schistocerca comprises about 50 species which are endemic to the New World, except the Old World locust S. gregaria. Their morphological identification is rendered difficult by phase polyphenism, geographical overlap due to migrations or swarming, the difficulty to easily differentiate genitalia and the occurrence of interspecific hybrids. The three species reported from Peru include the swarming species S. interrita, a pest that can be recognized only by taxonomists. We show that it can be unambiguously identified using a mitochondrial DNA fragment known to have barcoding properties in this genus. We used several methods to delimitate Peruvian species. While S. interrita and S. pallens were well characterized, S. piceifrons peruviana was split into several taxa by a phylogeny-based method, whereas a combination of population genetics methods led one to identify only the three nominal species. A tentative reconstruction of the species history shows that several populations of S. piceifrons peruviana have recently increased in number, while exchanging some migrants, whereas an isolated population at the northern margin of the species range is substantially differentiated while exchanging no migrants with the others. This complex history has resulted in an atypical lineage pattern that appears to have confounded the standard assumptions underlying available species delimitation methods. Because of its behavioral property which tends to keep it panmictic, the identification of the swarming S. interrita remained unaffected.     

中国鸟类的DNA分类及系统发育研究概述

鸟类分类是鸟类学其他研究领域的基础,近年来分子技术的发展,以及计算机技术的应用为鸟类分类学和鸟类系统演化研究提供了新的研究手段,给传统的系统分类研究带来了新的机遇.Tautz等于2002年首先提出运用DNA序列作为生物分类系统的主要平台,即DNA分类学(DNA Taxonomy).而Hebert等于2003年则首次提出了DNA条形码(DNA Barcoding)的概念,并对其物种分类和鉴定意义予以肯定,建议利用线粒体细胞色素C氧化酶亚单位Ⅰ(COI)的特定区段来做DNA条形编码的基础.在鸟类DNA分类方面,国内学者应用线粒体基因Cut b,COI,c-mos,c-myc,12s rRNA,16s rRNA,ND2,ND3,CR,RAG-1以及核基因myoglobin introⅡ等不同片段对很多类群进行了分类探讨和系统发育研究.但是主要集中在鸡形目及雀形目鸟类.中国是鸟类多样性极其丰富的国家,近年来很多亚种、种及以上分类阶元依然存在问题,因此,中国鸟类物种的分类地位、系统发育与演化关系等依然有很多问题等待深入研究.目前国内基于COI的鸟类分类及系统发育研究有了一些报道,但是真正的DNA条形码工作尚需继续、深入地开展.