蒟蒻薯属 (薯蓣科) 植物DNA条形码研究

蒟蒻薯属（Tacca）植物种间在形态上差别不大,导致分类上存在一定的困难。DNA条形码是一种利用短的DNA标准片段来鉴别和发现物种的方法。本研究利用核基因ITS片段和叶绿体基因trnH psbA, rbcL, matK片段对蒟蒻薯属6个种的DNA条形码进行研究,对4个DNA片段可用性,种内种间变异,barcode gap进行了分析,采用Tree based和BBA两种方法比较不同序列的鉴定能力。结果显示：单片段ITS正确鉴定率最高,片段组合rbcL+matK正确鉴定率最高。支持CBOL植物工作组推荐的条码组合rbcL+matK可作为蒟蒻薯属物种鉴定的标准条码,建议ITS片段作为候选条码。丝须蒟蒻薯Tacca integrifolia采自西藏的居群与马来西亚居群形成了2个不同的遗传分支,且两者在形态上也存在一定的差异,很可能是一个新种。     

The D1‐D2 region of the large subunit ribosomal DNA as barcode for ciliates

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1‐D2 region of the ribosomal DNA large subunit (LSU‐rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1‐D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1‐D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1‐D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour‐joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1‐D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.     

DNA barcoding and morphological identification of neotropical ichthyoplankton from the Upper Paraná and São Francisco

The identification of fish larvae from two neotropical hydrographic basins using traditional morphological taxonomy and DNA barcoding revealed no conflicting results between the morphological and barcode identification of larvae. A lower rate (25%) of correct morphological identification of eggs as belonging to migratory or non‐migratory species was achieved. Accurate identification of ichthyoplankton by DNA barcoding is an important tool for fish reproductive behaviour studies, correct estimation of biodiversity by detecting eggs from rare species, as well as defining environmental and management strategies for fish conservation in the neotropics.     

DNA barcodes of eight species in genus Sebastes

DNA barcode is effective for biological taxonomy and is able to identify species from any life-history stage. In the present study, eight species which belong to four different subgenera of genus Sebastes found in China sea waters were identified by cytochrome c oxidase I (COI) barcode. The results indicated that the intra-species variation in DNA barcode was less than inter-species variation. When the phylogenetic trees were reconstructed by neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods, all the species clustered in their groups distinguishable by high bootstrap values, which proved that COI barcode is a powerful means to differentiate species of Sebastes and supports their identification. When the molecular tree was compared to the morphological tree, only Sebastes trivittatus in subgenus Sebastocles settled in the different positions. It is suggested that S. trivittatus is one of the shallowest occurring species in the Northwest Pacific due to its life characters.     

DNA Barcoding as an Effective Tool in Improving a Digital Plant Identification System: A Case Study for the Area of Mt. Valerio, Trieste (NE Italy)

Background

Identification keys are decision trees which require the observation of one or more morphological characters of an organism at each step of the process. While modern digital keys can overcome several constraints of classical paper-printed keys, their performance is not error-free. Moreover, identification cannot be always achieved when a specimen lacks some morphological features (i.e. because of season, incomplete development or miss-collecting). DNA barcoding was proven to have great potential in plant identification, while it can be ineffective with some closely related taxa, in which the relatively brief evolutionary distance did not produce differences in the core-barcode sequences.

Methodology/Principal Findings

In this paper, we investigated how the DNA barcoding can support the modern digital approaches to the identification of organisms, using as a case study a local flora, that of Mt. Valerio, a small hill near the centre of Trieste (NE Italy). The core barcode markers (plastidial rbcL and matK), plus the additional trnH-psbA region, were used to identify vascular plants specimens. The usefulness of DNA barcoding data in enhancing the performance of a digital identification key was tested on three independent simulated scenarios.

Conclusions/Significance

Our results show that the core barcode markers univocally identify most species of our local flora (96%). The trnH-psbA data improve the discriminating power of DNA barcoding among closely related plant taxa. In the multiparametric digital key, DNA barcoding data improves the identification success rate; in our simulation, DNA data overcame the absence of some morphological features, reaching a correct identification for 100% of the species. FRIDA, the software used to generate the digital key, has the potential to combine different data sources: we propose to use this feature to include molecular data as well, creating an integrated identification system for plant biodiversity surveys.     

Comparing the usefulness of distance, monophyly and character-based DNA barcoding methods in species identification: a case study of neogastropoda

Background

DNA barcoding has recently been proposed as a promising tool for the rapid species identification in a wide range of animal taxa. Two broad methods (distance and monophyly-based methods) have been used. One method is based on degree of DNA sequence variation within and between species while another method requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree. Nevertheless, some issues complicate the use of both methods. A recently applied new technique, the character-based DNA barcode method, however, characterizes species through a unique combination of diagnostic characters.

Methodology/Principal Findings

Here we analyzed 108 COI and 102 16S rDNA sequences of 40 species of Neogastropoda from a wide phylogenetic range to assess the performance of distance, monophyly and character-based methods of DNA barcoding. The distance-based method for both COI and 16S rDNA genes performed poorly in terms of species identification. Obvious overlap between intraspecific and interspecific divergences for both genes was found. The “10× rule” threshold resulted in lumping about half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI could distinguish all species studied. However, the 16S rDNA tree could not distinguish some closely related species. In contrast, the character-based barcode method for both genes successfully identified 100% of the neogastropod species included, and performed well in discriminating neogastropod genera.

Conclusions/Significance

This present study demonstrates the effectiveness of the character-based barcoding method for species identification in different taxonomic levels, especially for discriminating the closely related species. While distance and monophyly-based methods commonly use COI as the ideal gene for barcoding, the character-based approach can perform well for species identification using relatively conserved gene markers (e.g., 16S rDNA in this study). Nevertheless, distance and monophyly-based methods, especially the monophyly-based method, can still be used to flag species.     

Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.     

Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns: an ecological perspective

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes--while playing an essential role in fern colonization and reproduction--generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.     

基于三个分子标记的粉毛蚜亚科DNA条形码

【目的】粉毛蚜亚科昆虫是重要的林业害虫,但是由于蚜虫体型较小,形态特征趋于简化,可用于物种鉴定的有效特征非常有限,因此一般基于外部形态特征难以对蚜虫物种实现快速准确的鉴定。本研究获取该亚科2属10种的DNA条形码标准序列,解决部分物种的分类问题,同时比较了3种标记对粉毛蚜亚科（Pterocommatinae）物种快速鉴定的效率。【方法】基于蚜虫的线粒体细胞色素氧化酶C亚基I（cytochrome oxidase subunit I, COI）基因、细胞色素b（cytochrome b, Cytb）基因和蚜虫初级内共生菌Buchnera 6-磷酸葡萄糖酸脱氢酶（gluconate-6-phosphate dehydrogenase, gnd）基因,对2属10种共197号样品进行NJ分析、遗传距离的计算以及基于相似性的物种鉴定分析。【结果】与K-2P模型相比,基于p-distance模型计算得到的遗传距离更小,序列差异频次图上种内距离与种间距离的重叠区域也小于前者;COI序列的物种鉴定成功率最高。获取了粉毛蚜亚科近200条DNA条形码标准序列,并建立了基于3个标记的该亚科物种DNA条形码序列库。【结论】在粉毛蚜亚科DNA条形码研究中,p-distance模型要优于K-2P模型;COI序列具有最高的条形码分析效率;增毛卷粉毛蚜Plocamaphis assetacea可能为蜡卷粉毛蚜Plocamaphis flocculosa的同物异名。     

DNA barcoding to map the microbial communities: current advances and future directions

During the last two decades, the DNA barcode development towards microbial community has increased dramatically. DNA barcode development is related to error-free and quick species identification which aid in understanding the microbial biodiversity, as well as the diseases related to microbial species. Here, we seek to evaluate the so-called barcoding initiatives for the microbial communities and the emerging trends in this field. In this paper, we describe the development of DNA marker-based DNA barcoding system, comparison between routine species identification and DNA barcode, and microbial biodiversity and DNA barcode for microbial communities. Two major topics, such as the molecular diversity of viruses and barcode for viruses have been discussed at the same time. We demonstrate the current status and the maker of DNA barcode for bacteria, algae, fungi, and protozoa. Furthermore, we argue about the promises, limitations, and present and future challenges of microbial barcode development.     

地黄属植物的DNA条形码研究

地黄属(Rehmannia)为玄参科(Scrophulariaceae)药用植物,广泛分布于中国中东部及北部地区。由于地黄属植物经历了快速成种,导致其属内物种间形态性状差异较小,运用传统的形态学分类方法已难以准确地鉴定物种,近年来迅速发展起来的DNA条形码技术为快速、准确地鉴别物种提供了新思路。本研究选用3个叶绿体DNA非编码区片段(trn L-trn F、trn M-trn V和trn S-trn G)及核基因ITS片段,运用PWG-distance和TreeBuilding两种方法对地黄属5个物种75个个体进行了DNA条形码分析。结果表明:单个叶绿体DNA片段或核基因ITS片段对地黄属物种的鉴别率较低(0%~20%),组合的叶绿体DNA片段分辨能力虽然高于单个DNA片段,但并不能将地黄属5个物种完全区分开;trn S-trn G+ITS片段组合的分辨率可达100%,能够将地黄属5个物种准确区分,与所有叶绿体DNA片段和核基因ITS片段组合(trn L-trn F+trn M-trn V+trn S-trn G+ITS)的辨别率相同,因此推荐trn S-trn G+ITS作为地黄属植物的标准条形码。此外,利用DNA条形码鉴别物种时,可采用叶绿体DNA片段和核DNA片段组合的方法来提高物种鉴定的成功率。     

After 7 years and 1000 citations: comparative assessment of the DNA barcoding and the DNA taxonomy proposals for taxonomists and non-taxonomists

In 2003, two different approaches-DNA taxonomy and DNA barcoding-were simultaneously proposed to overcome some of the perceived intrinsic weaknesses of the traditional morphology-based taxonomical system, and to help non-taxonomists to resolve their crucial need for accurate and rapid species identification tools. After 7 years, it seems unlikely that a completely new taxonomical system based on molecular characters only (DNA taxonomy) will develop in the future. It is more likely that both morphological and molecular data will be simultaneously analyzed, developing what has been coined as "integrative taxonomy". Concerning DNA barcoding, it is now clear that it does not focus on building a tree-of-life nor to perform DNA taxonomy, but rather to produce a universal molecular identification key based on strong taxonomic knowledge that is collated in the barcode reference library. The indisputable success of the DNA barcoding project is chiefly due to the fact that DNA barcoding standards considerably enhance current practices in the molecular identification field, and standardization offers virtually endless applications for various users.     

Using DNA Barcodes to Identify Bird Species Involved in Birdstrikes

Abstract: We determined effectiveness of using mitochondrial DNA barcodes (cytochrome c oxidase subunit 1 [CO1]) to identify bird-aircraft collision (birdstrike) cases that lacked sufficient feather evidence for morphological diagnosis. From September through December 2006, 821 samples from birdstrike events occurring in the United States were submitted for DNA analysis. We successfully amplified a CO1 DNA barcode product from 554 (67.5%) of the samples; 267 (32.5%) did not contain viable DNA and depended on morphological methods (microscopy) for Order or Family level identification. We deemed 19 cases inconclusive either because the DNA barcode recovered from the sample did not meet our 98% match criteria when compared to the Barcode of Life Database (BoLD) or because the DNA barcode matched to a set of ≥ 2 closely related species with overlapping barcodes, preventing complete species identification. Age of the sample (≤6 months) did not affect DNA viability, but initial condition of the sample and the collection method was critical to DNA identification success. The DNA barcoding approach has great potential in aiding in identification of birds (and wildlife) for airfield management practices, particularly in regions of the world that lack the vast research collections and individual expertise for morphologic identifications.     

Congruence between cytochrome oxidase I (COI) and morphological data in Anuraphis spp. (Hemiptera,Aphididae) with a comparison between the utility of the 5’ barcode and 3’ COI regions

The discrimination of species in the genus Anuraphis is particularly difficult due to the overlap of morphological characters. In this study, we used the 5’ (barcode) and 3’ regions of cytochrome oxidase I (COI) to test their utility in the identification of species in this genus as well as closely related species. Both regions were useful to discriminate all the species tested. However the non-barcode 3’ region resulted in higher resolution and support for species relationships when the data were analyzed using both Maximum Likelihood and MrBayes. We propose the development of an integrated database that encompasses morphological, molecular, life-cycle, host plant and bibliographic information to facilitate and increase the accuracy of aphid identification.     

DNA BARCODING OF CHLORARACHNIOPHYTES USING NUCLEOMORPH ITS SEQUENCES1

Chlorarachniophytes are a small group of marine photosynthetic protists. They are best known as examples of an intermediate stage of secondary endosymbiosis: their plastids are derived from green algae and retain a highly reduced nucleus, called a nucleomorph, between the inner and outer pairs of membranes. Chlorarachniophytes can be challenging to identify to the species level, due to their small size, complex life cycles, and the fact that even genus‐level diagnostic morphological characters are observable only by EM. Few species have been formally described, and many available culture collection strains remain unnamed. To alleviate this difficulty, we have developed a barcoding system for rapid and accurate identification of chlorarachniophyte species in culture, based on the internal transcribed spacer (ITS) region of the nucleomorph rRNA cistron. Although this is a multicopy locus, encoded in both subtelomeric regions of each chromosome, interlocus variability is low due to gene conversion by homologous recombination in this region. Here, we present barcode sequences for 39 cultured strains of chlorarachniophytes (>80% of currently available strains). Based on barcode data, other published molecular data, and information from culture records, we were able to recommend names for 21 out of the 24 unidentified, partially identified, or misidentified chlorarachniophyte strains in culture. Most strains could be assigned to previously described species, but at least two to as many as five new species may be present among cultured strains.     

Evaluating the Species Boundaries of Green Microalgae (Coccomyxa,Trebouxiophyceae, Chlorophyta) Using Integrative Taxonomy and DNA Barcoding with Further Implications for the Species Identification in Environmental Samples

Integrative taxonomy is an approach for defining species and genera by taking phylogenetic, morphological, physiological, and ecological data into account. This approach is appropriate for microalgae, where morphological convergence and high levels of morphological plasticity complicate the application of the traditional classification. Although DNA barcode markers are well-established for animals, fungi, and higher plants, there is an ongoing discussion about suitable markers for microalgae and protists because these organisms are genetically more diverse compared to the former groups. To solve these problems, we assess the usage of a polyphasic approach combining phenotypic and genetic parameters for species and generic characterization. The application of barcode markers for database queries further allows conclusions about the ‘coverage’ of culture-based approaches in biodiversity studies and integrates additional aspects into modern taxonomic concepts. Although the culture-dependent approach revealed three new lineages, which are described as new species in this paper, the culture-independent analyses discovered additional putative new species. We evaluated three barcode markers (V4, V9 and ITS-2 regions, nuclear ribosomal operon) and studied the morphological and physiological plasticity of Coccomyxa, which became a model organism because its whole genome sequence has been published. In addition, several biotechnological patents have been registered for Coccomyxa. Coccomyxa representatives are distributed worldwide, are free-living or in symbioses, and colonize terrestrial and aquatic habitats. We investigated more than 40 strains and reviewed the biodiversity and biogeographical distribution of Coccomyxa species using DNA barcoding. The genus Coccomyxa formed a monophyletic group within the Trebouxiophyceae separated into seven independent phylogenetic lineages representing species. Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.     

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota): Parmelia sensu stricto as a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen‐forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species‐level lineages. Here, we provide a DNA barcode reference library with well‐identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 21–29.