DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae)

We evaluated the utility of the core barcode regions (matK and rbcL) and the plastid intergenic spacer trnH‐psbA to distinguish between Thymus spp. This is a taxonomically complex group that has been investigated so far mainly using morphological approaches. Thirty‐six samples representing nine different morphospecies were collected and used for molecular analysis. The three markers showed clear amplification and sequencing. However, the genetic variation and the resulting haplotype networks showed that only Thymus capitatus forms a well‐defined ‘barcoding gap’ compared with the other taxa. The identification problems observed in the other Thymus spp. may be related to reduced gene flow among populations, resulting in high intraspecific and low interspecific genetic variation. This situation does not permit the definition of species‐specific barcodes. A second hypothesis suggests that morphological traits used for the delimitation of Thymus spp. do not reflect real biological and molecular species boundaries. If this is the case, the taxonomy of Thymus should be revised through extensive sampling and analyses with different tools (i.e. molecular variability, morphology, geographical distribution, etc.) to define the natural units at the species level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013 , 171 , 687–699.     

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.     

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co‐evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH‐psbA, psbK‐psbI, atpF‐atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra‐ and inter‐specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK‐psbI and trnH‐psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF‐atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH‐psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.     

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.     

DNA barcoding in a biodiversity hot spot: potential value for the identification of Malagasy Euphorbia L. listed in CITES Appendices I and II

The island of Madagascar is a key hot spot for the genus Euphorbia, with at least 170 native species, almost all endemic. Threatened by habitat loss and illegal collection of wild plants, nearly all Malagasy Euphorbia are listed in CITES Appendices I and II. The absence of a reliable taxonomic revision makes it particularly difficult to identify these plants, even when fertile, and thereby compromises the application of CITES regulations. DNA barcoding, which can facilitate species‐level identification irrespective of developmental stage and the presence of flowers or fruits, may be a promising tool for monitoring and controlling trade involving threatened species. In this study, we test the potential value of barcoding on 41 Euphorbia species representative of the genus in Madagascar, using the two widely adopted core barcode markers (matK and rbcL), along with two additional DNA regions, nuclear internal transcribed spacer (ITS) and the chloroplastic intergenic spacer psbA‐trnH. For each marker and for selected marker combinations, inter‐ and intraspecific distance estimates and species discrimination rates are calculated. Results using just the ‘official’ barcoding markers yield overlapping inter‐ and intraspecific ranges and species discrimination rates below 60%. When ITS is used, whether alone or in combination with the core markers, species discrimination increases to nearly 100%, whereas the addition of psbA‐trnH produces less satisfactory results. This study, the first ever to test barcoding on the large, commercially important genus Euphorbia shows that this method could be developed into a powerful identification tool and thereby contribute to more effective application of CITES regulations.     

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

Abstract One application of DNA 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.     

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, matK, 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.     

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 supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.     

Karyological and molecular characterisation of subgenus Vicia (Fabaceae)

In the present report, we have analysed the subgenus Vicia by karyological and molecular approaches with the aim to clarify the relationships among Vicia species included in this subgenus by previously evidenced morphological investigations. Multivariate analysis using several karyomorphological parameters in addition to symmetry indices has allowed the construction of a dendrogram of linkage distances very useful to compare and to include in a phylogenetic tree obtained by internal transcribed spacer (ITS) rDNA sequences. Moreover, a separate analysis was performed combining our molecular data on ITS sequences with those reported in the literature for the section Vicilla. Our analyses partly confirm the monophyletic status of the various sections in which the subgenus Vicia has been divided, however questioning, in some cases, the real need to maintain all the nine sections so far accepted and the placement of some individual species in the two subgenera Vicia and Vicilla.     

RAPD and morphological analysis of the rare plant species Vicia pisiformis (Fabaceae)

The amount and pattern of genetic variation was surveyed in two Swedish and three Czech populations of the rare perennial forest plant Vicia pisiformis. This species has a mainly easterly-continental European distribution and has few and small populations in Sweden. It is classified as 'vulnerable' on the Swedish Red Data list. Seeds from natural populations were collected and grown under controlled conditions in growth chambers. The variation was estimated in growth and fecundity traits and with Random Amplified Polymorphic DNA, RAPD. Low inter- and intra-population variation in RAPD-markers was found using 11 primers, with a similarity index (Wetton) for the families of 0.98. In contrast, multivariate analysis of variance showed significant morphological differences within and between populations. Also in the univariate ANOVAs, a number of the traits showed significant between-and/or within population differentiation. Cluster analysis for the morphological traits and RAPDs (UPGMA) did not structure the variation of families in accordance with their geographical distance. A Mantel test based on comparisons between Mahalonobfs and Jaccard distance for morphological and RAPD data, respectively, did not reveal any significant correlation between the two matrices. It is concluded that if a genetic conservation program is to be applied on Vicia pisiformis , different sampling strategies are needed to capture morphological vs RAPD variation. This is, to our knowledge, the first investigation that compares RAPD and morphological variation in a threatened plant species.     

中国东南沿海弹涂鱼科常见鱼类的遗传多样性和DNA条形码

为了探究中国东南沿海海岸线经济开发、海水污染等对该区域生物遗传多样性的潜在影响,本文使用COI基因作为DNA条形码,对从中国东南沿海广东汕头、福建东山、福建云霄、福建泉州、福建霞浦、上海崇明6个地点采集的大弹涂鱼(Boleophthalmus pectinirostris)、弹涂鱼(Periophthalmus modestus)和青弹涂鱼(Scartelaos histophorus)进行种间和种内的遗传学分析。遗传多样性分析显示,青弹涂鱼的核苷酸多样性(0.0018)在3个种中最低;青弹涂鱼广东汕头种群的核苷酸多样性(0.0018)比福建云霄种群低;大弹涂鱼的上海崇明种群核苷酸多样性(0.0021)低于其他5个地理种群。物种水平分析表明,3个物种间分化明显,种间与种内遗传距离有明显差异;不同种的单倍型在系统发育树上聚成3个高度支持的独立分支。种群水平分析显示,大弹涂鱼的6个不同地理种群之间有共享单倍型,地理种群间与地理种群内的遗传距离重叠;各地理种群之间的基因流较频繁(Nm>4);青弹涂鱼的分析情况与大弹涂鱼基本一致。大弹涂鱼和青弹涂鱼的单倍型在系统发育树上均呈现多系或并系分布格局,并未按地理位置分成不同的单系群。本研究表明,应及时加强3种鱼类及其野生种群遗传多样性的保护,且COI基因对种一级的识别明确可靠,而在地理种群水平的识别则比较困难,有待进一步深入研究。     

Generic recircumscriptions of Oncidiinae (Orchidaceae: Cymbidieae) based on maximum likelihood analysis of combined DNA datasets

Phylogenetic relationships within the orchid subtribe Oncidiinae sensu Chase were inferred using maximum likelihood analyses of single and multilocus DNA sequence data sets. Analyses included both nuclear ribosomal internal transcribed spacer DNA and plastid regions (matK exon, trnH‐psbA intergenic spacer and two portions of ycf1 exon) for 736 individuals representing approximately 590 species plus seven outgroup taxa. Based on the well resolved and highly supported results, we recognize 61 genera in Oncidiinae. Mimicry of oil‐secreting Malpighiaceae and other floral syndromes evolved in parallel across the subtribe, and many clades exhibit extensive variation in pollination‐related traits. Because previous classifications heavily emphasized these floral features, many genera recognized were not monophyletic. Our classification based on monophyly will facilitate focused monographs and clarifies the evolution of morphological and biochemical traits of interest within this highly diverse subtribe. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 117–146.     

Selection of candidate coding DNA barcoding regions for use on land plants

An in silico screen of 41 of the 81 coding regions of the Nicotiana plastid genome generated a shortlist of 12 candidates as DNA barcoding loci for land plants. These loci were evaluated for amplification and sequence variation against a reference set of 98 land plant taxa. The deployment of multiple primers and a modified multiplexed tandem polymerase chain reaction yielded 85–94% amplification across taxa, and mean sequence differences between sister taxa of 6.1 from 156 bases of accD to 22 from 493 bases of matK. We conclude that loci should be combined for effective diagnosis, and recommend further investigation of the following six loci: matK, rpoB, rpoC1, ndhJ, ycf5 and accD. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 1–11.     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

Molecular phylogenetics of Vanda and related genera (Orchidaceae)

The genus Vanda and its affiliated taxa are a diverse group of horticulturally important species of orchids occurring mainly in South‐East Asia, for which generic limits are poorly defined. Here, we present a molecular study using sequence data from three plastid DNA regions. It is shown that Vanda s.l. forms a clade containing approximately 73 species, including the previously accepted genera Ascocentrum, Euanthe, Christensonia, Neofinetia and Trudelia, and the species Aerides flabellata. Resolution of the phylogenetic relationships of species in Vanda s.l. is relatively poor, but existing morphological classifications for Vanda are incongruent with the results produced. Some novel species relationships are revealed, and a new morphological sectional classification is proposed based on support for these groupings and corresponding morphological characters shared by taxa and their geographical distributions. The putative occurrence of multiple pollination syndromes in this group of taxa, combined with complex biogeographical history of the South‐East Asian region, is discussed in the context of these results. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 549–572.     

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.     

Phylogenetics of Annona cherimola (Annonaceae) and some of its closest relatives

Annona cherimola is a woody perennial species in the Annonaceae family that produces edible fruits and has economic importance in several regions of the world with subtropical climates. Together with other 10‐12 species, A. cherimola belongs to the section Atta of the Annona genus with a center of origin in Central America and the Caribbean. Species of the section Atta produce soft skin ripe fruits with raised areoles bounded by recessed furrows. Annona cherimola is the only species of the section naturally found in the Andean region of South America. Currently, no information is available at the molecular level on the phylogenetic relationships of most of the species of Atta and closely related sections in Annona. In order to fill this gap, in this work a phylogenetic approach was performed using five coding and non‐coding plastid DNA regions, to determine the phylogenetic relationships between A. cherimola and other related species included in Atta and other sections of the genus. The results obtained support recent studies that demonstrated the likely Mesoamerican origin of A. cherimola based on biogeographical analysis with SSR markers, rather than the previously considered South American origin hypothesis. In addition, the species belonging to the Atta section did not show monophyly. Finally, A. cherimola and A. pruinosa seem to be phylogenetically close species and additional studies are needed to discern the relations between them.