Molecular identification of species in Juglandaceae: A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.     

Molecular identification of species in Juglandaceae:A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.     

悬钩子属DNA条形码通用序列的初步筛选

为了建立悬钩子属（Rubus）植物的DNA条形码分子鉴定技术,筛选获得适用于悬钩子属植物的通用条形码序列。该研究基于GenBank数据对ITS、ITS2、matK、rbcL、trnH-psbA、trnL-trnF 6个DNA条形码序列进行了遗传变异、barcoding gap、建树等评估分析。结果显示,trnH-psbA、matK、rbcL、rtnL-trnF的种内变异与种间变异差异较大,变异分辨率分别为97.32%、83.33%、79.07%、64.95%,存在较大的barcoding gap;NJ一致树分析显示,matK的单系性比例最高（67%）,其次为trnH-psbA（64%）,rtnL-trnF（43%）,rbcL（30%）。结果表明,悬钩子属植物的matK与trnH-psbA序列种内变异与种间变异差异较大,能较好地区分不同物种,具有较大的鉴定潜力。建议将matK和trnH-psbA作为悬钩子属植物鉴定的核心条形码序列,rtnL-trnF、rbcL作为辅助条形码序列。     

DNA barcoding of Pedicularis L.(Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus

One application ofDNA barcoding is species identification based on sequences of a short and standardized DNA region.In plants,various DNA regions,alone or in combination,have been proposed and investigated,but consensus on a universal plant barcode remains elusive.In this study,we tested the utility of four candidate barcoding regions (rbcL,matK,trnH-psbA,and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae).Amplification and sequencing was successful using single primer pairs for rbcL,trnH-psbA,and ITS,whereas two primer pairs were required for matK.Patterns of sequence divergence commonly showed a “barcoding gap”,that is,a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species,respectively Considering primer universality,ease of amplification and sequencing,and performance in discriminating species,we found the most effective single-region barcode for Pedicularis to be ITS,and the most effective two-region barcode to be rbcL +ITS.Both discriminated at least 78％ of the 88 species and correctly identified at least 89％ of the sequences in our sample,and were effective in placing unidentified samples in known species groups.Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis,a species-rich cosmopolitan clade much in need of revision,as well as ecological studies in its center of diversity,the Hengduan Mountains region of China.     

DNA barcoding of the main cultivated yams and selected wild species in the genus Dioscorea

Distinguishing yam species based on morphological traits is extremely difficult and unreliable, posing a challenge to breeders and genebank curators. Development of a molecular assay based on DNA barcoding can facilitate rapid and accurate identification of important Dioscorea species. To develop a DNA barcoding system forDioscorea species identification, the rbcL and matK loci (in unison and in combination), the non-coding intergenic spacer trnH-psbA of the chloroplast genome, and the nuclear ITS regions were investigated using criteria for developing candidate DNA barcodes. All DNA barcoding sequences were assessed for ease of PCR amplification, sequence quality and species discriminatory power. Amongst the markers investigated, the matK locus performed well in terms of species identification (63.2%), in addition to detecting high interspecific variation with mean divergence of 0.0196 (SD=0.0209). The combination of the two coding regions (rbcL + matK) was determined to be the optimal (76.2%) DNA barcoding approach as 16 out of 21 species could be defined. While the rbcL exhibited good PCR amplification efficiency and sequence quality, its species discriminatory power was relatively poor with 47.6% identification. Similarly, the trnH-psbA region had a weak discrimination efficiency of only 36.8%. While the development of more robust DNA barcoding systems is an ongoing challenge, our results indicate that therbcL + matK combination can be utilized as multi-locus DNA barcode regions for Dioscorea species identification.     

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trn H-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.     

中国秋海棠属（秋海棠科）植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH-psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH-psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限：ITS／ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100％／96％,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS／ITS2纳人种子植物DNA条形码核心片段中的观点。     

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.     

藓类植物DNA条码的初步研究——以蔓藓科部分属为例

选取7个叶绿体基因片段,其中3个编码区基因片段（matK,rps4 and rbcL-a）和4个非编码区基因片段（atpB-rbcL,atpF-H,psbK-Iandtrn H-psbA）,一个线粒体基因片段（nad5）和一个核基因片段（ITS2）,材料包括5个属14个种的74份样品。分析比较了6个基因片段的多样性、种内和种间的遗传距离、鉴别率等。结果显示,在使用单个片段时ITS2的鉴定效果最好,而atpB-rbcL则是叶绿体基因片段中鉴别能力最高的。使用组合片段的结果发现,两个基因片段组合的鉴别效果最好。通过NJ树的方法检验,atpB-rbcL＋trn H-psbA和rbcL-a＋＋trn H-psbA的鉴定成功率是64%,当再增加一个或两个基因片段时其鉴定效果并没有提高。本研究表明在植物中应用DNA条码需要用质体基因和核基因联合。     

DNA barcoding in closely related species: A case study of Primula L. sect. Proliferae Pax (Primulaceae) in China

DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoC1, trnH-psbA, psbK-psbI, atpF-atpH, and internal transcribed spacer (ITS)). The core combination rbcL+matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL+matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

DNA barcoding in closely related species: A case study of Primula L.sect.Proliferae Pax (Primulaceae) in China

DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoCl, trnH-psbA, psbK-psbI, atpFatpH, and internal transcribed spacer (ITS)). The core combination rbcL + matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL +matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.     

DNA barcodes identify Chinese medicinal plants and detect geographical patterns of Sinosenecio (Asteraceae)

DNA barcodes have proved to be efficient for plants species discrimination and identification using short and standardized genomic regions. The genus Sinosenecio(Asteraceae) is used for traditional medicinal purposes in China. Most species of the genus occur in restricted geographical regions and exhibit a wide range of morphological variations within species, making them difficult to differentiate in the field. Previously, taxonomic revisions have been made based on morphological and cytological evidence. In the present study, barcoding analysis was performed on 107 individuals representing 38 species in this genus to evaluate the performance of four candidate barcoding loci (matK, rbcL, trnH-psbA and internal transcribed spacer [ITS]) and detect geographical patterns. Three different methods based on genetic distance, sequence similarity, and the phylogenetic tree were used. Comparably high species discrimination power was detected in species-level taxonomic process by the ITS dataset alone or combined with other loci, which was suggested to be the most suitable barcode for Sinosenecio. Our results are congruent with previous taxonomic studies concerning the monophyly of the S. oldhamianus group. The present study provides an empirical paradigm for the identification of medicinal plant species and their geographical patterns, ascertaining the congruence between taxonomical studies and barcoding analysis inSinosenecio.     

姜科砂仁属植物DNA条形码序列的筛选

砂仁属（Amomum）隶属于姜科,全属约150种,我国有39种。该属多种植物可作药物或香料,但目前砂仁属的分类还不清楚,准确鉴定物种有很大难度。本研究利用DNA barcoding技术,对砂仁属50种121个个体的matK、rbcL-a、trnH-psbA序列及其不同组合进行比较,用Taxon DNA计算种间、种内bar-coding gap,运用相似法的BLASTn计算条码的正确鉴定率,筛选适合砂仁属的条码片段。结果显示：所有条码的barcoding gap均不存在;matK的正确鉴定率高于trnH-psbA和rbcL-a,联合片段的条码正确鉴定率高于单片段条码,三个片段联合条码的正确鉴定率最高。因此,推荐matK＋rbcL-a＋trnH-psbA作为砂仁属物种鉴定的候选条码。     

Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers

One nuclear and three chloroplast DNA regions (ITS, rbcL, matK and trnH-psbA) were used to identify the species of Alnus (Betulaceae). The results showed that 23 out of all 26 Alnus species in the world, represented by 131 samples, had their own specific molecular character states, especially for three morphologically confused species (Alnus formosana, Alnus japonica and Alnus maritima). The discriminating power of the four markers at the species level was 10% (rbcL), 31.25% (matK), 63.6% (trnH-psbA) and 76.9% (ITS). For ITS, the mean value of genetic distance between species was more than 10 times the intraspecific distance (0.009%), and 13 species had unique character states that differentiated them from other species of Alnus. The trnH-psbA region had higher mean values of genetic distance between and within species (2.1% and 0.68% respectively) than any other region tested. Using the trnH-psbA region, 13 species are distinguished from 22 species, and seven species have a single diagnostic site. The combination of two regions, ITS and trnH-psbA, is the best choice for DNA identification of Alnus species, as an improvement and supplement for morphologically based taxonomy. This study illustrates the potential for certain DNA regions to be used as novel internet biological information carrier through combining DNA sequences with existing morphological character and suggests a relatively reliable and open taxonomic system based on the linked DNA and morphological data.     

亚热带森林乔木树种DNA条形码研究——以哀牢山自然保护区为例

作为新一代植物志iFlora的重要组成部分,DNA条形码已经成为物种鉴定中重要且有效的方法。本研究以亚热带森林的乔木树种为研究对象,开展了DNA条形码的尝试性工作。为评估DNA条形码对鉴定亚热带森林树种的有效性,收集并研究了来自哀牢山自然保护区内5l科111属中204个树种的525个乔木个体。结果显示,所选4个DNA片段（rbcL,matK,trnH-psbA和ITS）的PCR扩增成功率都超过90％;测序成功率rbcL和matK最高,分别为90．7％和90．5％,trnH-psbA次之（83．6％）,ITS最低（73．5％）,表明4个片段在亚热带森林乔木中都具有较好的通用性。应用BLAST与NJ Tree两种方法,对物种和属水平的鉴别成功率进行统计,发现单片段中ITS最高,分别为68．4％-81．3％和99．0％～100％,核心条码rkL和matK组合的成功率是52．8％～60．2％和86．7％～90．5％,再与补充条码trnH-psbA和ITS联合,可以成功鉴别74．7％～79．6％哀牢山自然保护区亚热带森林中的乔木物种。由于ITS片段在亚热带森林部分重要树种类群（樟科和壳斗科等）中的测序成功率较差,所以对这些植物类群采用trnH-psbA作为DNA条形码是一个更好的选择。     

DNA barcoding of European Herbertus (Marchantiopsida, Herbertaceae) and the discovery and description of a new species

DNA barcoding of a group of European liverwort species from the genus Herbertus was undertaken using three plastid (matK, rbcL and trnH-psbA) and one nuclear (ITS) marker. The DNA barcode data were effective in discriminating among the sampled species of Herbertus and contributed towards the detection of a previously overlooked European Herbertus species, described here as H. norenus sp. nov. This species shows clear-cut differences in DNA sequence for multiple barcode regions and is also morphologically distinct. The DNA barcode data were also useful in clarifying taxonomic relationships of the European species with some species from Asia and North America. In terms of the discriminatory power of the different barcode markers, ITS was the most informative region, followed closely by matK. All species were distinguishable by ITS alone, rbcL + matK and various other multimarker combinations.     

DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates

Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid.