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.     

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 DNA barcodes for identification of plant species in the family Araliaceae

An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.     

DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

DNA barcoding, an increasingly popular mean of species identification, has been widely used for global species identification despite a consensus not being reached regarding which DNA sequences can be used as the best plant barcodes. In this study, we tested the feasibility of five candidate DNA barcodes (nrITS, nrITS2, matk, rbcL and trnH-psbA) for identifying Uncaria species. We collected a total of 54 specimens of 10 Uncaria species across its distributional range. BLAST, barcoding gaps, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capability of the candidate DNA barcodes. The results showed that the ITS2 is most suitable as a candidate DNA barcode for identification of medicinal plants of the genus Uncaria.     

A molecular phylogeny of the marine mussel genus Perna (Bivalvia: Mytilidae) based on nuclear (ITS1&2) and mitochondrial (COI) DNA sequences

A molecular phylogeny is presented for marine mussels of the genus Perna, based on nuclear (ITS1,ITS2) and mitochondrial (COI) DNA sequence data. The three generally recognised species (Perna viridis, Perna perna and Perna canaliculus) and one putative species (Perna picta) were each sampled from several locations within their known geographic distributions. A range of phylogenetic analyses was used to investigate the current taxonomic assignments, evolutionary relationships and the biogeographical history of the genus. The different analyses produced similar, well supported topologies and verified the monophyly of the genus with respect to five mytilid outgroup species. P. perna (Atlantic), P. viridis (Indo-West Pacific), and P. canaliculus (New Zealand) each formed distinct clades, confirming their specific status. Putative P. picta from North Africa clustered within the P. perna clade and is not regarded as a separate species. P. perna and P. canaliculus were the most closely related of the three species. Possible biogeographic explanations for the present species distributions are evaluated.     

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention.Here,seven regions (psbA-trnH,matK,ycf5,rpoC1,rbcL,ITS2,and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae.To evaluate each barcode’s utility for species authentication,PCR amplification efficiency,genetic divergence,and barcoding gaps were assessed.We found that the ITS2 region exhibited the highest inter-specific divergence,and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests.The ITS2 locus had the highest identification efficiency among all tested regions.In a previous study,we found that ITS2 was able to discriminate a wide range of plant taxa,and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore,we propose that ITS2 is a promising candidate barcode for plant species identification.     

Genetic variation in native and introduced populations of the 'New Zealand flatworm', Arthurdendyus triangulatus

The internal transcribed spacer regions (ITS1 and ITS2) including the 5.8S region of the ‘New Zealand flatworm’, Arthurdendyus triangulates, are 1004 base pairs in length. Restriction fragment length polymorphism analysis of PCR products (PCR‐RFLP) was conducted on A. triangulates specimens from 45 locations in Northern Ireland, Scotland, England and New Zealand. Seven restriction endonucleases (Alu I, Rsa I, Sau3A I, Cfo I, Nde I, Dde I, and Mbo I) were used to reveal intraspecific variation. Analysis of molecular variance revealed the presence of population genetic substructuring, with most genetic heterogeneity present between populations rather than between individuals or geographical regions. No distinct differences were found between Northern Irish and Scottish populations but phylogenetic analysis supports the hypothesis of multiple introductions from New Zealand. There was no significant relationship between genetic distance and geographic distance, as would be expected for natural spread, indicating that this species is largely anthropochorous, even in parts of New Zealand.     

Molecular phylogenetic analysis of New Zealand mosquito species

ABSTRACT

Taxonomic information for New Zealand mosquito species is predominantly morphological with very few molecular data available to date. In this study, the mitochondrial COI gene and nuclear ITS1 were amplified and sequenced from DNA templates representing 17 species; 15 previously known New Zealand species, a newly discovered undescribed Aedes species from the Chatham Islands and a recently eradicated invader, Ae. camptorhynchus. This paper provides DNA barcoding sequences for the entire known New Zealand mosquito fauna, the first for the majority of these species. Phylogenetic analysis of COI and ITS1 indicated that the endemic species are all genetically distinct from the exotic species examined including vector species of pathogens of human diseases. The genus Opifex is distant from the genus Aedes based on analysis of ITS1 sequences, and Ae. chathamicus is more closely related to species within Aedes than to the genus Opifex. Results show Culex asteliae to be a valid species but Cx. rotoruae is not necessarily so. The Aedes species appears to be closely related to Ae. subalbirostris. The introduced Cx. quinquefasciatus was clearly shown to be related to Cx. pipiens; however, the endemic Cx. pervigilans was not. No evidence of population variation based on geographic location was detected.     

Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae)

Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

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。     

Molecular phylogenetics of the genus <Emphasis Type="Italic">Physalia</Emphasis> (Cnidaria: Siphonophora) in New Zealand coastal waters reveals cryptic diversity

Physalia is a genus of pelagic colonial hydrozoans often known by common names such as ‘Portuguese-man-of-war’ or ‘bluebottle’. Siphonophore systematists generally recognise only a single species in this genus, Physalia physalis, however the name Physalia utriculus is also still in common use, which has led to considerable taxonomic confusion. With some morphological variation between global regions there is the possibility that this genus holds a substantial amount of cryptic variation. We seek to examine the genetic structure of Physalia present in New Zealand coastal waters. Fifty-four specimens collected from 13 locations around New Zealand and Australia were sequenced for both mitochondrial cytochrome c oxidase I (COI) and the first internal transcribed spacer (ITS1) of the nuclear ribosomal cistron. Sequences were analysed using maximum likelihood and split decomposition neighbour networks to determine conflict between clans (the unrooted analog of clades). Three clans were identified from both the COI and ITS sequences. The results are complex and clans are not consistent between the two genes. Nevertheless, it seems that there is substantial cryptic diversity amongst Physalia present in New Zealand coastal waters.     

The molecular phylogeny of the genus Rhizopus based on rDNA sequences

In order to establish the molecular phylogeny of the genus Rhizopus, three molecules of the ribosomal RNA-encoding DNA (rDNA), complete 18S, internal transcribed spacer (ITS)1-5.8S-ITS2, and 28S D1/D2 regions of all the species of the genus were sequenced. Phylogenetic trees showed three major clusters corresponding to the three groups in the current morphological taxonomy, microsporus-group, stolonifer-group, and R. oryzae. R. stolonifer var. lyococcos was clustered independently from the major clusters. R. schipperae clustered differently in all trees. Strains of R. sexualis had multiple ITS sequences. A. rouxii clustered with R. oryzae. These results indicate the possibility of molecular identification of species groups using rDNA sequencing. Reclassification of the genus might be appropriate.     

nrDNA ITS和cpDNA rpl16基因测序法对六种鼠尾草的检测

为从鼠尾草属植物中鉴别丹参品种,采用基因测序方法,用核糖体核酸内转录间隔区基因(nrDNA ITS),编码核蛋白体大亚基多肽L16的基因(rpl16)及叶绿体DNA上包含trnL以及trnL和trnF间隔区的区域基因(trnL-trnF)的序列,检测六种鼠尾草属新鲜植物.由于nrDNA ITS和rpl16突变率较高,可以做为6种鼠尾草的基源鉴定标记,依此设计了两对特异引物,从6种鼠尾草中鉴定出丹参(Salvia miltiorrhiza)和云南鼠尾草(S.yunnanensis).但trnL-trnF突变率太低,未能用于鉴别.商品干燥中药材因加工和储藏的方式致使DNA降解严重,基因测序法难于应用.     

Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes

Angelica anomala Avé-Lall (Chuanbaizhi in Chinese) is an important medicinal plant which can be used in traditional Chinese medicines; however, there are no authentic and universal methods to differentiate this Sichuan famous-region drug of A. anomala from a large number of non-famous-region and false drugs. It has been demonstrated that DNA barcoding is a molecular diagnostic method for species identification, which uses a single standardized DNA fragment. In this study, we tested five DNA barcoding candidates (matK, ITS, ITS2, rbcL, and psbA-trnH), and we found that ITS was the best candidate to authenticate the famous-region drug of A. anomala. Moreover, through comparative analysis of these five DNA barcodes between A. anomala and Angelica dahurica, we found that ITS had the most and ITS2 had more variable regions, but the psbA-trnH, rbcL, and matK regions were identical. Hence, we suggest ITS as the DNA barcoding to identify A. anomala and A. dahurica. Moreover, we are determined to adopt the A. anomala as the accurate Latin name of Chuanbaizhi.     

Fifteen into Three Does Go: Morphology,Genetics and Genitalia Confirm Taxonomic Inflation of New Zealand Beetles (Chrysomelidae: Eucolaspis)

Eucolaspis Sharp 1886 is a New Zealand native leaf beetle genus (Coleoptera: Chrysomelidae: Eumolpinae) with poorly described species and a complex taxonomy. Many economically important fruit crops are severely damaged by these beetles. Uncertain species taxonomy of Eucolaspis is leaving any biological research, as well as pest management, tenuous. We used morphometrics, mitochondrial DNA and male genitalia to study phylogenetic and geographic diversity of Eucolaspis in New Zealand. Freshly collected beetles from several locations across their distribution range, as well as identified voucher specimens from major museum collections were examined to test the current classification. We also considered phylogenetic relationships among New Zealand and global Eumolpinae (Coleoptera: Chyrosomelidae). We demonstrate that most of the morphological information used previously to define New Zealand Eucolaspis species is insufficient. At the same time, we show that a combination of morphological and genetic evidence supports the existence of just 3 mainland Eucolaspis lineages (putative species), and not 5 or 15, as previously reported. In addition, there may be another closely related lineage (putative species) on an offshore location (Three Kings Islands, NZ). The cladistic structure among the lineages, conferred through mitochondrial DNA data, was well supported by differences in male genitalia. We found that only a single species (lineage) infests fruit orchards in Hawke’s Bay region of New Zealand. Species-host plant associations vary among different regions.     

DNA barcoding of Corydalis,the most taxonomically complicated genus of Papaveraceae

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya–Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA‐trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P‐distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia‐recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well‐supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.     

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.     

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.