Application of DNA Barcodes in Asian Tropical Trees – A Case Study from Xishuangbanna Nature Reserve,Southwest China

Background

Within a regional floristic context, DNA barcoding is more useful to manage plant diversity inventories on a large scale and develop valuable conservation strategies. However, there are no DNA barcode studies from tropical areas of China, which represents one of the biodiversity hotspots around the world.

Methodology and Principal Findings

A DNA barcoding database of an Asian tropical trees with high diversity was established at Xishuangbanna Nature Reserve, Yunnan, southwest China using rbcL and matK as standard barcodes, as well as trnH–psbA and ITS as supplementary barcodes. The performance of tree species identification success was assessed using 2,052 accessions from four plots belonging to two vegetation types in the region by three methods: Neighbor-Joining, Maximum-Likelihood and BLAST. We corrected morphological field identification errors (9.6%) for the three plots using rbcL and matK based on Neighbor-Joining tree. The best barcode region for PCR and sequencing was rbcL (97.6%, 90.8%), followed by trnH–psbA (93.6%, 85.6%), while matK and ITS obtained relative low PCR and sequencing success rates. However, ITS performed best for both species (44.6–58.1%) and genus (72.8–76.2%) identification. With trnH–psbA slightly less effective for species identification. The two standard barcode rbcL and matK gave poor results for species identification (24.7–28.5% and 31.6–35.3%). Compared with other studies from comparable tropical forests (e.g. Cameroon, the Amazon and India), the overall performance of the four barcodes for species identification was lower for the Xishuangbanna Nature Reserve, possibly because of species/genus ratios and species composition between these tropical areas.

Conclusions/Significance

Although the core barcodes rbcL and matK were not suitable for species identification of tropical trees from Xishuangbanna Nature Reserve, they could still help with identification at the family and genus level. Considering the relative sequence recovery and the species identification performance, we recommend the use of trnH–psbA and ITS in combination as the preferred barcodes for tropical tree species identification in China.     

Incorporating trnH-psbA to the core DNA barcodes improves significantly species discrimination within?southern African Combretaceae

Recent studies indicate that the discriminatory power of the core DNA barcodes (rbcLa + matK) for land plants may have been overestimated since their performance have been tested only on few closely related species. In this study we focused mainly on how the addition of complementary barcodes (nrITS and trnH-psbA) to the core barcodes will affect the performance of the core barcodes in discriminating closely related species from family to section levels. In general, we found that the core barcodes performed poorly compared to the various combinations tested. Using multiple criteria, we finally advocated for the use of the core + trnH-psbA as potential DNA barcode for the family Combretaceae at least in southern Africa. Our results also indicate that the success of DNA barcoding in discriminating closely related species may be related to evolutionary and possibly the biogeographic histories of the taxonomic group tested.     

DNA Barcoding Evaluation and Its Taxonomic Implications in the Species-Rich Genus Primula L. in China

The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA) and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.     

Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences

An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL–F, and psbA–trnH from the chloroplast genome, and ITS, ITS1, and ITS2 from the nuclear genome) and three combinations (rbcL + matK, psbA–trnH + ITS1, and trnL–F + ITS1). The present study collected 232 sequences from 27 Salvia species through DNA sequencing and 77 sequences within the same taxa from the GenBank. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intraspecific and interspecific divergence, DNA barcoding gaps, and identification efficiency via a tree-based method. ITS1 was superior to the other marker for discriminating between species, with an accuracy of 81.48%. The three combinations did not increase species discrimination. Finally, we found that ITS1 is a powerful barcode for identifying Salvia species, especially Salvia miltiorrhiza.     

Evaluation of the DNA barcoding approach to develop a reference data-set for the threatened flora of Sicily

The Mediterranean Basin is one of the most significantly altered World Biodiversity Hotspots with extensive habitat loss and fast genetic population erosion, for which urgent biodiversity reconnaissance and preservation actions are required. In particular, Sicily has about 600 taxa classified as threatened or near-threatened. The correct recognition and identification of such biodiversity is required for supporting further activities. The objective of this work is to assess the ability of the DNA barcoding approach to identify different taxonomic groups from a collection of the most threatened plant taxa, throughout natural Sicilian populations. The evaluation of the DNA barcoding core markers, rbcL and matK, was carried out on 30 taxa belonging to 13 families. DNA barcode fragments were recovered from all taxa (100%). The rbcL gene was recovered from 97% of the taxa and matK gene from 73%. In this test, 19 taxa overall (63%) were totally resolved at the specific or subspecific level, by at least one of the core markers. Fourteen of the 17 most threatened taxa (EN, CR) included in this work were totally discriminated. The matK and rbcL locus, respectively, resolved 64% and 48% of the taxa successfully sequenced. The matK gene expressed the highest genetic distance (K2P value), from 0.4% to 8.6%, against a range of 0.1–2% of rbcL gene. However, the rbcL gene appeared a good compromise between PCR, sequencing success and species-level resolution. Cryptic groups suggest the implementation of additional barcoding markers or different primer combinations, particularly for matK, in order to increase the performances. However, this preliminary result confirms the potential of the barcoding approach for quick identification of unknown and heterogeneous plant groups to generate a dedicated reference data-set of the threatened Sicilian flora for a wide range of applications.     

Establishing a community‐wide DNA barcode library as a new tool for arctic research

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to‐date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology‐based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology‐based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species‐level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community.     

Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Abstract Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB‐rbcL, trnH‐psbA, trnL‐F, trnS‐G, atpF‐H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spacer [ITS]) as candidate DNA barcodes for peaches, using samples from 38 populations, covering all the species in sect. Persica. On the whole, the primers worked well in this group and sequencing difficulties were met only in the case of ITS locus. Five loci (rbcL, matK, rpoB, rpoC, and nad1) have very low variation rates, whereas atpB‐rbcL, atpF‐H, trnH‐psbA, trnL‐F and trnS‐G show more variability. The most variable loci, atpB‐rbcL and trnH‐psbA, can distinguish three of the five species. Two two‐locus combinations, atpB‐rbcL+trnL‐F and atpB‐rbcL+atpF‐H, can resolve all five species. We also find that identification powers of the loci are method‐dependent. The NeighborNet method shows higher species identification power than maximum parsimony, neighbor joining, and unweighted pair group method with arithmetic mean methods.     

Species identification of the medicinal plant Tulipa edulis (Liliaceae) by DNA barcode marker

Tulipa edulis (Liliaceae) is the botanical origin of the traditional Chinese medicine (TCM) “Guangcigu”. Due to overexploitation that induced a decline in natural sources, many dried bulbs from other species of Tulipa have been used, adulterating the medicine in recent years. This practice may cause a series of inconsistent therapeutic effects and quality control problems in the herbal medicine industry. Hence, three DNA regions (matK, psbA-trnH and rbcL) were evaluated as barcodes for identifying T. edulis and its adulterants. All candidate DNA barcodes were successfully amplified from leaf samples. Based on the sequence divergences, rbcL and psbA-trnH can assign T. edulis and its adulterants to the correct genus, while matK can accurately differentiate T. edulis and its adulterants. Thus, at the DNA level, the matK intergenic region is a more suitable, accurate and applicable identification of T. edulis and its adulterants than rbcL and psbA-trnH.     

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.     

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.     

Phylogenetic relationships between disjunctly occurring groups of Tristicha trifaria (Podostemaceae)

Aim To reveal the phylogeographic relationship of disjunct specimens of Tristicha trifaria (Bory ex Willd.) Spreng., a member of the Podostemaceae river‐weed family, which is distributed exceptionally widely, but disjunctly, in Africa and the Americas. Location Brazil, Mexico, Ghana, Tanzania and Madagascar. Methods The chloroplast matK and rbcL genes, a trnK intron, the trnS‐trnG intergenic spacer (IGS), the two IGSs of trnT‐trnL‐trnF, a trnL intron, and nuclear ribosomal ITS regions were sequenced and analysed. Phylogenetic analyses were conducted using maximum likelihood and maximum parsimony methods. Results The T. trifaria samples analysed were separated into two groups in a rooted tree based on a combined matK/rbcL/ITS dataset; one contained the West African and all of the American samples, and the other contained the East African and Madagascan samples. An unrooted tree obtained from a combined analysis of all the chloroplast DNA and nuclear ITS data showed that a sample from West Africa was sister to an American T. trifaria group. Main conclusions The American and West African T. trifaria are closely related, despite the great distance between their locations. This observation, along with a tree of the whole Tristichoideae subfamily and estimated divergence times, suggests that an ancestor of T. trifaria migrated from Asia to Africa during the early Tertiary, and that this was followed by further westward migration to the Americas at the end of the Miocene or in the early Pliocene.     

Efficacy of the core DNA barcodes in identifying processed and poorly conserved plant materials commonly used in South African traditional medicine

Medicinal plants cover a broad range of taxa, which may be phylogenetically less related but morphologically very similar. Such morphological similarity between species may lead to misidentification and inappropriate use. Also the substitution of a medicinal plant by a cheaper alternative (e.g. other non-medicinal plant species), either due to misidentification, or deliberately to cheat consumers, is an issue of growing concern. In this study, we used DNA barcoding to identify commonly used medicinal plants in South Africa. Using the core plant barcodes, matK and rbcLa, obtained from processed and poorly conserved materials sold at the muthi traditional medicine market, we tested efficacy of the barcodes in species discrimination. Based on genetic divergence, PCR amplification efficiency and BLAST algorithm, we revealed varied discriminatory potentials for the DNA barcodes. In general, the barcodes exhibited high discriminatory power, indicating their effectiveness in verifying the identity of the most common plant species traded in South African medicinal markets. BLAST algorithm successfully matched 61% of the queries against a reference database, suggesting that most of the information supplied by sellers at traditional medicinal markets in South Africa is correct. Our findings reinforce the utility of DNA barcoding technique in limiting false identification that can harm public health.     

DNA barcoding as a complementary tool for conservation and valorisation of forest resources

Since the pre-historic era, humans have been using forests as a food, drugs and handcraft reservoir. Today, the use of botanical raw material to produce pharmaceuticals, herbal remedies, teas, spirits, cosmetics, sweets, dietary supplements, special industrial compounds and crude materials constitute an important global resource in terms of healthcare and economy. In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on a selected group of forest tree species with the aim of contributing to the identification, conservation and trade control of these valuable plant resources.The “core barcode” for land plants (rbcL, matK, and trnH-psbA) was tested on 68 tree specimens (24 taxa). Universality of the method, ease of data retrieval and correct species assignment using sequence character states, presence of DNA barcoding gaps and GenBank discrimination assessment were evaluated. The markers showed different prospects of reliable applicability. RbcL and trnH-psbA displayed 100% amplification and sequencing success, while matK did not amplify in some plant groups. The majority of species had a single haplotype. The trnH-psbA region showed the highest genetic variability, but in most cases the high intraspecific sequence divergence revealed the absence of a clear DNA barcoding gap. We also faced an important limitation because the taxonomic coverage of the public reference database is incomplete. Overall, species identification success was 66.7%.This work illustrates current limitations in the applicability of DNA barcoding to taxonomic forest surveys. These difficulties urge for an improvement of technical protocols and an increase of the number of sequences and taxa in public databases.     

DNA barcoding the Dioscorea in China, a vital group in the evolution of monocotyledon: use of matK gene for species discrimination

Background

Dioscorea is an important plant genus in terms of food supply and pharmaceutical applications. However, its classification and identification are controversial. DNA barcoding is a recent aid to taxonomic identification and uses a short standardized DNA region to discriminate plant species. In this study, the applicability of three candidate DNA barcodes (rbcL, matK, and psbA-trnH) to identify species within Dioscorea was tested.

Methodology/Principal Findings

One-hundred and forty-eight individual plant samples of Dioscorea, encompassing 38 species, seven varieties and one subspecies, representing majority species distributed in China of this genus, were collected from its main distributing areas. Samples were assessed by PCR amplification, sequence quality, extent of specific genetic divergence, DNA barcoding gap, and the ability to discriminate between species. matK successfully identified 23.26% of all species, compared with 9.30% for rbcL and 11.63% for psbA-trnH. Therefore, matK is recommended as the best DNA barcoding candidate. We found that the combination of two or three loci achieved a higher success rate of species discrimination than one locus alone. However, experimental cost would be much higher if two or three loci, rather than a single locus, were assessed.

Conclusions

We conclude that matK is a strong, although not perfect, candidate as a DNA barcode for Dioscorea identification. This assessment takes into account both its ability for species discrimination and the cost of experiments.     

Delineating closely related species with DNA barcodes for routine biological monitoring

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中国葫芦科1新记录种——厚叶棒锤瓜

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Detecting evolutionary rate heterogeneity among mangroves and their close terrestrial relatives

Mangroves form the dominant intertidal ecosystems and differ morphologically and physiologically from their close terrestrial relatives. We investigate the molecular evolutionary pattern of the typical mangrove family, i.e. Rhizophoraceae, and rate heterogeneity for the plastid matK and rbcL genes in different species of the family, as revealed by phylogenetic analyses and relative‐rate tests. Our study documents evolutionary rate heterogeneity in the Rhizophoraceae for the two genes: the mangrove genus Bruguiera has relatively slow substitution rates compared to the terrestrial genus Carallia at both synonymous and non‐synonymous sites in the matK sequences, and the synonymous and non‐synonymous substitution matrices are correlated. However, the rbcL non‐synonymous sites exhibit a high degree of rate heterogeneity among mangroves and related terrestrial groups, and uncoupling of rates with the synonymous sites. Selection is probably an important influence on the rate variation, suggesting further investigation for better understanding of various forces contributing to the rate heterogeneity and molecular adaptation in mangroves.     

Promise and Challenge of DNA Barcoding in Venus Slipper (Paphiopedilum)

Orchidaceae are one of the largest families of flowering plants, with over 27,000 species described and all orchids are listed in CITES. Moreover, the seedlings of orchid species from the same genus are similar. The objective of DNA barcoding is rapid, accurate, and automated species identification, which may be used to identify illegally traded endangered species from vegetative specimens of Paphiopedilum (Venus slipper), a flagship group for plant conservation with high ornamental and commercial values. Here, we selected eight chloroplast barcodes and nrITS to evaluate their suitability in Venus slippers. The results indicate that all tested barcodes had no barcoding gap and the core plant barcodes showed low resolution for the identification of Venus slippers (18.86%). Of the single-locus barcodes, nrITS is the most efficient for the species identification of the genus (52.27%), whereas matK + atpF-atpH is the most efficient multi-locus combination (28.97%). Therefore, we recommend the combination of matK + atpF-atpH + ITS as a barcode for Venus slippers. Furthermore, there is an upper limit of resolution of the candidate barcodes, and only half of the taxa with multiple samples were identified successfully. The low efficiency of these candidate barcodes in Venus slippers may be caused by relatively recent speciation, the upper limit of the barcodes, and/or the sampling density. Although the discriminatory power is relatively low, DNA barcoding may be a promising tool to identify species involved in illegal trade, which has broad applications and is valuable for orchid conservation.     

Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

DNA barcodes for discriminating the medicinal plant Isatis indigotica Fort. (Cruciferae) and its adulterants

Isatis indigotica Fort. (Cruciferae) is a biennial medicinal plant. In order to protect the decreasing natural genetic resources of I. indigotica, three candidate DNA barcodes (ITS2, trnL-F and rbcL) were employed to establish an accurate and effective identification system for I. indigotica. The results demonstrated that all three candidate DNA barcodes have performed very well in I. indigotica. The interspecific genetic distances were obviously greater than the intraspecific distance among I. indigotica as indicated by ITS2, trnL-F and rbcL. Sequence alignment analysis of I. indigotica genotypes revealed that four SNPs (54, 108, 146 and 181 bp) located in ITS2, three (2, 30, 709 bp) in trnL-F and one (531 bp) in rbcL, respectively. UPGMA phylogenetic tree constructed from trnL-F and rbcL could allote I. indigotica to the correct corresponding genus, whereas rbcL could not distinguish I. indigotica from its adulterants. Meanwhile, UPGMA tree of ITS2 could accurately identify I. indigotica from its adulterants according to the corresponding species. Consequently, it can be concluded that ITS2 is a more suitable and accurate DNA barcode for identifying I. Indigotica and its adulterants than trnL-F and rbcL.