Testing four candidate barcoding markers in temperate woody bamboos (Poaceae: Bambusoideae)

Abstract Bambusoideae is an important subfamily of the grass family Poaceae that has considerable economic, ecologic and cultural value. In addition, Bambusoideae species are important constituents of the forest vegetation in China. Because of the paucity of flower‐bearing specimens and homoplasies of morphological characters, it is difficult to identify species of Bambusoideae using morphology alone, especially in the case of temperate woody bamboos (i.e. Arundinarieae). To this end, DNA barcoding has shown great potential in identifying species. The present study is the first attempt to test the feasibility of four proposed DNA barcoding markers (matK, rbcL, trnH–psbA, and internal transcribed spacer [ITS]) in identifying 27 species of the temperate woody bamboos. Three plastid markers showed high levels of universality, whereas the universality of ITS was comparatively low. A single plastid marker provided low levels of discrimination success at both the genus and species levels (<12%). Among the combinations of plastid markers, the highest discriminatory power was obtained using the combination of rbcL+matK (14.8%). Using a combination of three markers did not increase species discrimination. The nuclear region ITS alone could identify 66.7% of species, although fewer taxa were included in the ITS analyses than in the plastid analyses. When ITS was integrated with a single or combination of plastid markers, the species discriminatory power was significantly improved. We suggest that a combination of rbcL+ ITS, which exhibited the highest species identification power of all combinations in the present study, could be used as a potential DNA barcode for temperate woody bamboos.     

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.)

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.     

DNA barcoding in Atlantic Forest plants: What is the best marker for Sapotaceae species identification?

The Atlantic Forest is a phytogeographic domain with a high rate of endemism and large species diversity. The Sapotaceae is a botanical family for which species identification in the Atlantic Forest is difficult. An approach that facilitates species identification in the Sapotaceae is urgently needed because this family includes threatened species and valuable timber species. In this context, DNA barcoding could provide an important tool for identifying species in the Atlantic Forest. In this work, we evaluated four plant barcode markers (matK, rbcL, trnH-psbA and the nuclear ribosomal internal transcribed spacer region - ITS) in 80 samples from 26 species of Sapotaceae that occur in the Atlantic Forest. ITS yielded the highest average interspecific distance (0.122), followed by trnH-psbA (0.019), matK (0.008) and rbcL (0.002). For species discrimination, ITS provided the best results, followed by matK, trnH-psbA and rbcL. Furthermore, the combined analysis of two, three or four markers did not result in higher rates of discrimination than obtained with ITS alone. These results indicate that the ITS region is the best option for molecular identification of Sapotaceae species from the Atlantic Forest.     

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.     

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.     

Lauraceae: A promising family for the advance of neotropical dendrochronology

The botanical family Lauraceae is ecologically and physiognomically very important in neotropical forests. It is one of the most frequent and distributed family both in number of individuals and species. Despite of this, we have noticed that a very few Lauraceae species have been considered in dendrochronological investigations. In order to analyze the potential of Lauraceae species in dendrochronology and to facilitate future studies we: (1) reviewed the literature on wood anatomy, cambial activity, tree growth and dendrochronology and compiled a list of species’ tree-ring features throughout the Neotropics; (2) Investigated wood anatomy, growth synchronism and climate-growth relationship using dendrochronological standard techniques in 14 species from subtropical forests of southern Brazil. Our review pointed out that the majority of Lauraceae forms distinct tree-rings in several biomes and climates in the Neotropics. Seasonal growth pattern related to water stress and to seasonal air temperature were identified in Amazonia and in subtropical high elevation sites, respectively. Time series of tree-ring width of Lauraceae species were successfully cross-dated and were already used in reconstruction of fire and vegetation dynamics. Our own dendrochronological investigations brought to light that all the 14 studied species form distinct tree-rings in seasonal or even rainforests. By analyzing time series of tree-ring width we found the same growth tendency within trees of Cinnamomum amoenum and Ocotea pulchella. Moreover, year-to-year variation in the growth time-series was linked to climate variations of temperature and precipitation, showing growth decreases when summer water stress occur. We evinced Lauraceae has distinct, synchronic and climate-sensitive tree-rings. Therefore, since Lauraceae has wide distribution and high frequency in the Neotropics and since many species become centenary, we strongly encourage the use of Lauraceae’s tree-rings in autoecology, climatology and on the reconstruction of vegetation and disturbance dynamics.     

Testing for the influence of niche and neutral factors on sapling community assembly beneath isolated woody plants in grasslands

Questions: Perching and nursing effects drive initial steps of forest expansion over grasslands. Nursing effect is obviously related to niche mechanisms, while perching effect is likely to result both from neutral and niche factors. This study assessed the effect of neutral and niche factors on species composition in sapling communities developing beneath isolated trees/shrubs (ITS) in grassland. Location: A mosaic of Campos grassland and Araucaria forest in São Francisco de Paula, southern Brazil (29°28′S, 50°13′W). Methods: We described sapling communities beneath 32 ITS using mean number of forest woody saplings of different species. We performed a stepwise canonical correspondence analysis (CCA) to select ITS traits that maximized the association with species composition. Then we evaluated the contribution of distance from seed source, ITS traits and distance‐structured ITS traits on sapling community assembly using a variation partitioning method based on CCA and partial CCA. Results: Sapling species composition was significantly explained by ITS traits (ITS dispersal mode, ITS growth form, crown area:ITS height ratio, crown area, ITS height and crown area:volume ratio). Distance from seed sources explained only a minor, non‐significant fraction of sapling species composition. Distance‐structured trait variation was negligible. Conclusions: Sapling community assembly beneath ITS was mostly explained by niche factors related to both nursing and perching effects. Dispersal limitation explained only a small fraction of variation in species composition beneath ITS, suggesting that neutral‐based perching effect had a minor role in community assembly.     

Evaluation of Four Commonly Used DNA Barcoding Loci for Chinese Medicinal Plants of the Family Schisandraceae

Many species of Schisandraceae are used in traditional Chinese medicine and are faced with contamination and substitution risks due to inaccurate identification. Here, we investigated the discriminatory power of four commonly used DNA barcoding loci (ITS, trnH-psbA, matK, and rbcL) and corresponding multi-locus combinations for 135 individuals from 33 species of Schisandraceae, using distance-, tree-, similarity-, and character-based methods, at both the family level and the genus level. Our results showed that the two spacer regions (ITS and trnH-psbA) possess higher species-resolving power than the two coding regions (matK and rbcL). The degree of species resolution increased with most of the multi-locus combinations. Furthermore, our results implied that the best DNA barcode for the species discrimination at the family level might not always be the most suitable one at the genus level. Here we propose the combination of ITS+trnH-psbA+matK+rbcL as the most ideal DNA barcode for discriminating the medicinal plants of Schisandra and Kadsura, and the combination of ITS+trnH-psbA as the most suitable barcode for Illicium species. In addition, the closely related species Schisandra rubriflora Rehder & E. H. Wilson and Schisandra grandiflora Hook.f. & Thomson, were paraphyletic with each other on phylogenetic trees, suggesting that they should not be distinct species. Furthermore, the samples of these two species from the southern Hengduan Mountains region formed a distinct cluster that was separated from the samples of other regions, implying the presence of cryptic diversity. The feasibility of DNA barcodes for identification of geographical authenticity was also verified here. The database and paradigm that we provide in this study could be used as reference for the authentication of traditional Chinese medicinal plants utilizing DNA barcoding.     

Multiple introductions and gene flow in subtropical South American populations of the fireweed,Senecio madagascariensis(Asteraceae)

Non-indigenous plants exhibit different attributes that make them aggressive competitors with indigenous plants and serious threats to biodiversity.Senecio madagascariensis (fireweed, Asteraceae), a native from southern Africa, is a strong competitor in agricultural activities and has toxic alkaloids that may result in high cattle mortality. In Brazil, this weed was collected for the first time in 1995 and has since spread quickly throughout the Pampas region. To better understand the invasion of the fireweed in South America, we used a genetic characterization with internal transcribed spacer (ITS) and microsatellite markers. Based on the ITS data, the southern Brazil populations of S. madagascariensis shared genetic homology with samples taken from the Hawaiian Islands and South Africa. Microsatellite analysis showed the genetic diversity split in two clusters, perhaps intimating the independent introduction of each species into South America. Although fireweed was introduced recently in southern Brazil, the considerable levels of genetic diversity, gene flow, and inbreeding may indicate success in the species establishment in this environment.     

Barcoding Poplars (Populus L.) from Western China

Background

Populus is an ecologically and economically important genus of trees, but distinguishing between wild species is relatively difficult due to extensive interspecific hybridization and introgression, and the high level of intraspecific morphological variation. The DNA barcoding approach is a potential solution to this problem.

Methodology/Principal Findings

Here, we tested the discrimination power of five chloroplast barcodes and one nuclear barcode (ITS) among 95 trees that represent 21 Populus species from western China. Among all single barcode candidates, the discrimination power is highest for the nuclear ITS, progressively lower for chloroplast barcodes matK (M), trnG-psbK (G) and psbK-psbI (P), and trnH-psbA (H) and rbcL (R); the discrimination efficiency of the nuclear ITS (I) is also higher than any two-, three-, or even the five-locus combination of chloroplast barcodes. Among the five combinations of a single chloroplast barcode plus the nuclear ITS, H+I and P+I differentiated the highest and lowest portion of species, respectively. The highest discrimination rate for the barcodes or barcode combinations examined here is 55.0% (H+I), and usually discrimination failures occurred among species from sympatric or parapatric areas.

Conclusions/Significance

In this case study, we showed that when discriminating Populus species from western China, the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions. Meanwhile, combining the ITS region with chloroplast regions may improve the barcoding success rate and assist in detecting recent interspecific hybridizations. Failure to discriminate among several groups of Populus species from sympatric or parapatric areas may have been the result of incomplete lineage sorting, frequent interspecific hybridizations and introgressions. We agree with a previous proposal for constructing a tiered barcoding system in plants, especially for taxonomic groups that have complex evolutionary histories (e.g. Populus).     

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.     

Evaluation of the DNA Barcodes in Dendrobium (Orchidaceae) from Mainland Asia

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera.     

Evolutionary Diversification of New Caledonian Araucaria

New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a ‘museum’ for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.     

Evaluating DNA barcoding criteria using African duiker antelope (Cephalophinae) as a test case

African duikers in the subfamily Cephalophinae (genera Cephalophus, Philantomba and Sylvicapra) constitute an important target for DNA barcoding efforts because of their importance to the bushmeat trade and protection under the Convention for International Trade in Endangered Species (CITES). Duikers also make a challenging test case of barcoding methods due to their recent diversification, substantial intra-specific genetic variation and high species richness. However, no study to date has evaluated how well DNA barcoding methods can be used to delineate all of the taxa within this group. To address this question, cytochrome c oxidase subunit 1 (COX1) sequences from all eighteen species within this subfamily and an outgroup taxon (genus Tragelaphus) were used to build a neighbor-joining tree, identify species-specific diagnostic synapomorphies, and determine whether species exceed a given pair-wise genetic distance threshold commonly employed in DNA barcoding studies. Tree-based analyses of the data indicate that several species within two clusters of closely related taxa consistently failed to form reciprocally monophyletic clades and similarly lack species-specific synapomorphies. Furthermore, one additional taxon failed to constitute a diagnosable clade and another occupied an unresolved position in the tree. Of the two genetic distance criteria evaluated, the 3% threshold was far more effective in delimiting species than a threshold level based on the ratio of inter- to intra-specific distances. However, neither approach could effectively delineate all sister species. While the taxonomy of this group might be open to question, the fact that barcodes consistently failed to differentiate several currently recognized sister taxa challenges the routine application of this approach in forensic studies of duiker species. Future barcoding work of this group should always include a complete taxonomic sampling and strive to include a broader geographic sampling of sequence diversity than has been carried out to date. Lastly, this work highlights the need to re-examine the taxonomy of this group, which may illuminate why some barcoding criteria fail to reliably differentiate species.     

The Use of DNA Barcoding on Recently Diverged Species in the Genus Gentiana (Gentianaceae) in China

DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes (trnH-psbA, ITS and ITS2) in 30 Gentiana species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating ‘Qin-Jiao’ Gentiana medicinal plants (G. macrophylla, G. crassicaulis, G. straminea, and G. dahurica), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.     

DNA barcoding Chinese medicinal Bupleurum

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.     

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 Barcode ITS Effectively Distinguishes the Medicinal Plant Boerhavia diffusa from Its Adulterants

Boerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant in India and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, a DNA barcoding technique has been applied to identify and distinguish B. diffusa from its closely-related species. The phylogenetic analysis was carried out for the four species of Boerhavia using barcode candidates including nuclear ribosomal DNA regions ITS, ITS1, ITS2 and the chloroplast plastid gene psbA-trnH. Sequence alignment revealed 26% polymorphic sites in ITS, 30% in ITS1, 16% in ITS2 and 6% in psbA-trnH, respectively. Additionally, a phylogenetic tree was constructed for 15 species using ITS sequences which clearly distinguished B. diffusa from the other species. The ITS1 demonstrates a higher transition/transversion ratio, percentage of variation and pairwise distance which differentiate B. diffusa from other species of Boerhavia. Our study revealed that ITS and ITS1 could be used as potential candidate regions for identifying B. diffusa and for authenticating its herbal products.     

DNA Barcoding of Rhodiola (Crassulaceae): A Case Study on a Group of Recently Diversified Medicinal Plants from the Qinghai-Tibetan Plateau

DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences — rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS) — for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau.