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.     

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.     

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).     

中国葫芦科1新记录种——厚叶棒锤瓜

报道了葫芦科厚叶棒锤瓜[Neoalsomitra sarcophylla(Wall.)Hutch.]在中国的分布新记录。该物种形态上与N.balansae(Gagnep.)Hutch.近似,但蒴果长3~4cm;种子长6~7mm,宽3~4mm,两端呈短角状。本研究利用DNA条形码技术对该物种进行测序,获得matK、rbcL、psbA-trnH 3个基因序列。应用Blast法为该物种的分类处理提供佐证。凭证标本存放于广西药用植物园标本馆(GXMG)。     

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.     

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.     

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.     

Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes

Background

The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.

Methodology

Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.

Conclusion

The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.     

First large-scale DNA barcoding assessment of reptiles in the biodiversity hotspot of Madagascar, based on newly designed COI primers

Background

DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar.

Methodology/Principal Findings

Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7–100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41–48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family.

Conclusions/Significance

The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge.     

DNA barcoding and phylogeny in neotropical species of the genus Spondias

The genus Spondias belongs to the Anacardiaceae family, with about 18 species, having significant economic and social importance and with some species used in the agricultural industry, however, problems are encountered when trying to identify phylogenetic relationships among the species. The use of DNA barcoding is of importance to this group, allowing species identification at the molecular level and in determining the phylogenetic relationships within the group. The objective of this study is to obtain DNA barcoding and to determine the phylogenetic relationships among the species. For this, DNA from six species of the genus was extracted and amplified by PCR using sequences from the rbcL and matK genes and the trnH-psbA spacer gene, followed by sequencing using the Sanger method. The results show that the matK and rbcL genes cannot be used for DNA barcoding, because their discriminatory level between species is low. On the other hand, trnH-psbA shows a high level of discrimination, allowing most of the species to be identified. However it is not possible to separate Spondias venulosa and Spondias tuberosa. Phylogenetic analysis shows that Spondias mombim and S. tuberosa are distinct “umbucajá” clades, suggesting a non-hybrid origin for “umbucajá”.     

The architecture of the chloroplast psbA-trnH non-coding region in angiosperms

The psbA-trnH intergenic region is among the most variable regions in the angiosperm chloroplast genome. It is a popular tool for plant population genetics and species level phylogenetics and has been proposed as suitable for DNA barcoding studies. This region contains two parts differing in their evolutionary conservation: 1) the psbA 3′UTR (untranslated region) and 2) the psbA-trnH intergenic non-transcribed spacer. We compared the sequence and RNA secondary structure of the psbA 3′ UTR across angiosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TTAGTGTATA box. The psbA-trnH spacer exhibited patterns that can be explained by the independent evolution of large inversions in the psbA 3′UTR and mutational hot spots in the remaining portion of the psbA-trnH spacer. We conclude that a comparison of chloroplast UTRs across angiosperms offer clues to the identity of putative regulatory elements and information about selective constraints imposed on the chloroplast non-coding regions.     

Molecular phylogeny of the subgenus Ceratotropis (genus Vigna, Leguminosae) reveals three eco-geographical groups and Late Pliocene-Pleistocene diversification: evidence from four plastid DNA region sequences

Background and Aims

The subgenus Ceratotropis in the genus Vigna is widely distributed from the Himalayan highlands to South, Southeast and East Asia. However, the interspecific and geographical relationships of its members are poorly understood. This study investigates the phylogeny and biogeography of the subgenus Ceratotropis using chloroplast DNA sequence data.

Methods

Sequence data from four intergenic spacer regions (petA-psbJ, psbD-trnT, trnT-trnE and trnT-trnL) of chloroplast DNA, alone and in combination, were analysed using Bayesian and parsimony methods. Divergence times for major clades were estimated with penalized likelihood. Character evolution was examined by means of parsimony optimization and MacClade.

Key Results

Parsimony and Bayesian phylogenetic analyses on the combined data demonstrated well-resolved species relationships in which 18 Vigna species were divided into two major geographical clades: the East Asia–Southeast Asian clade and the Indian subcontinent clade. Within these two clades, three well-supported eco-geographical groups, temperate and subtropical (the East Asia–Southeast Asian clade) and tropical (the Indian subcontinent clade), are recognized. The temperate group consists of V. minima, V. nepalensis and V. angularis. The subtropical group comprises the V. nakashimae–V. riukiuensis–V. minima subgroup and the V. hirtella–V. exilis–V. umbellata subgroup. The tropical group contains two subgroups: the V. trinervia–V. reflexo-pilosa–V. trilobata subgroup and the V. mungo–V. grandiflora subgroup. An evolutionary rate analysis estimated the divergence time between the East Asia–Southeast Asia clade and the Indian subcontinent clade as 3·62 ± 0·3 million years, and that between the temperate and subtropical groups as 2·0 ± 0·2 million years.

Conclusions

The findings provide an improved understanding of the interspecific relationships, and ecological and geographical phylogenetic structure of the subgenus Ceratotropis. The quaternary diversification of the subgenus Ceratotropis implicates its geographical dispersal in the south-eastern part of Asia involving adaptation to climatic condition after the collision of the Indian subcontinent with the Asian plate. The phylogenetic results indicate that the epigeal germination is plesiomorphic, and the germination type evolved independently multiple times in this subgenus, implying its limited taxonomic utility.     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

Background

The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.

Methodology/Principal Findings

Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level.

Conclusions

The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.     

Internal transcribed spacer 2 (nu ITS2 rRNA) sequence-structure phylogenetics: towards an automated reconstruction of the green algal tree of life

Background

Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta.

Methodology/Principal Findings

Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses.

Conclusions/Significance

Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.     

山麦冬及其近缘种DNA条形码研究

通过分析山麦冬及其近缘种cpDNA trnL-F、psbA-trnH间隔区序列特点,探讨trnL-F、psbA-trnH序列作为山麦冬及其近缘种DNA条形码的潜力。分别对两者进行PCR扩增后,并进行纯化测序。其中山麦冬及其近缘种9个物种18个样品的trnL-F序列长度为355~356 bp,在山麦冬属内序列完全一致,但在沿阶草属中存在特异变异位点,可对麦冬与沿阶草两物种进行鉴别。而psbA-trnH序列长度为543~544 bp,仅在麦冬中存在特异鉴别位点。结果表明,trnL-F、psbA-trnH序列由于进化速率低、保守性强,仅适合麦冬类植物属间鉴别的DNA条形码,而在属下水平的应用有一定的局限。     

DNA barcoding for community ecology--how to tackle a hyperdiverse, mostly undescribed Melanesian fauna

Background

Trigonopterus weevils are widely distributed throughout Melanesia and hyperdiverse in New Guinea. They are a dominant feature in natural forests, with narrow altitudinal zonation. Their use in community ecology has been precluded by the “taxonomic impediment”.

Methodology/Principal Findings

We sampled >6,500 specimens from seven areas across New Guinea; 1,002 specimens assigned to 270 morphospecies were DNA sequenced. Objective clustering of a refined dataset (excluding nine cryptic species) at 3% threshold revealed 324 genetic clusters (DNA group count relative to number of morphospecies = 20.0% overestimation of species diversity, or 120.0% agreement) and 85.6% taxonomic accuracy (the proportion of DNA groups that “perfectly” agree with morphology-based species hypotheses). Agreement and accuracy were best at an 8% threshold. GMYC analysis revealed 328 entities (21.5% overestimation) with 227 perfect GMYC entities (84.1% taxonomic accuracy). Both methods outperform the parataxonomist (19% underestimation; 31.6% taxonomic accuracy). The number of species found in more than one sampling area was highest in the Eastern Highlands and Huon (Sørensen similarity index 0.07, 4 shared species); ⅓ of all areas had no species overlap. Success rates of DNA barcoding methods were lowest when species showed a pronounced geographical structure. In general, Trigonopterus show high α and β-diversity across New Guinea.

Conclusions/Significance

DNA barcoding is an excellent tool for biodiversity surveys but success rates might drop when closer localities are included. Hyperdiverse Trigonopterus are a useful taxon for evaluating forest remnants in Melanesia, allowing finer-grained analyses than would be possible with vertebrate taxa commonly used to date. Our protocol should help establish other groups of hyperdiverse fauna as target taxa for community ecology. Sequencing delivers objective data on taxa of incredible diversity but mostly without a solid taxonomic foundation and should help pave the road for the eventual formal naming of new species.     

Barcoding bugs: DNA-based identification of the true bugs (Insecta: Hemiptera: Heteroptera)

Background

DNA barcoding, the analysis of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene, has been shown to provide an efficient method for the identification of species in a wide range of animal taxa. In order to assess the effectiveness of barcodes in the discrimination of Heteroptera, we examined 344 species belonging to 178 genera, drawn from specimens in the Canadian National Collection of Insects.

Methodology/Principal Findings

Analysis of the COI gene revealed less than 2% intra-specific divergence in 90% of the taxa examined, while minimum interspecific distances exceeded 3% in 77% of congeneric species pairs. Instances where barcodes fail to distinguish species represented clusters of morphologically similar species, except one case of barcode identity between species in different genera. Several instances of deep intraspecific divergence were detected suggesting possible cryptic species.

Conclusions/Significance

Although this analysis encompasses 0.8% of the described global fauna, our results indicate that DNA barcodes will aid the identification of Heteroptera. This advance will be useful in pest management, regulatory and environmental applications and will also reveal species that require further taxonomic research.     

Herbarium tale: the utility of dry specimens for DNA barcoding Juncaceae

Phylogenetic and barcoding studies usually use fresh plant tissues as sources of DNA and have successfully amplified DNA for various loci. The use of dried samples, however, is often necessary due to the frequent inaccessibility of fresh rare plants or their parts for genetic analyses or barcoding. The difficulty in obtaining amplifiable DNA is a major restriction of the use of herbarium specimens for DNA analyses. Recent study has highlighted the crucial issues for comparing herbarium and fresh plants for barcoding. We analysed the performance of samples of the family Juncaceae from various herbarium specimens of different ages with fresh plant material in PCRs and the sequences of seven loci (rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, and psbA-trnH from chloroplast DNA; atp1 from mitochondrial DNA; and ITS1-5.8S-ITS2 from nuclear DNA) using a combination of 28 primers. The herbarium specimens amplified well and may thus be successfully applied for both phylogenetic analyses and barcoding for the Juncaceae family. Amplifying DNA was more difficult from dried herbarium specimens than fresh samples but could be successful in most cases when appropriate internal primers were designed or methods were optimised. Using the set of universal primers recommended by the Consortium for the Barcode of Life and designing specific primers for a particular group of interest were both useful. Specimen age and amplicon length had limited detrimental effects on amplification success for most of the Juncaceae loci tested.