Lian-Ming Gao

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH–psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1–92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9–79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

High variation and strong phylogeographic pattern among cpDNA haplotypes in Taxus wallichiana (Taxaceae) in China and North Vietnam

We studied the phylogeography of Chinese yew (Taxus wallichiana), a tree species distributed over most of southern China and adjacent regions. A total of 1235 individuals from 50 populations from China and North Vietnam were analysed for chloroplast DNA variation using polymerase chain reaction–restriction fragment length polymorphism of the trnL‐F intron‐spacer region. A total of 19 different haplotypes were distinguished. We found a very high level of population differentiation and a strong phylogeographic pattern, suggesting low levels of recurrent gene flow among populations. Haplotype differentiation was most marked along the boundary between the Sino‐Himalayan and Sino‐Japanese Forest floristic subkingdoms, with only one haplotype being shared among these two subkingdoms. The Malesian and Sino‐Himalayan Forest subkingdoms had five and 10 haplotypes, respectively, while the relatively large Sino‐Japanese Forest subkingdom had only eight. The strong geography–haplotype correlation persisted at the regional floristic level, with most regions possessing a unique set of haplotypes, except for the central China region. Strong landscape effects were observed in the Hengduan and Dabashan mountains, where steep mountains and valleys might have been natural dispersal barriers. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggest the existence of several localized refugia during the last glaciation from which the present‐day distribution may be derived. The pattern of haplotype distribution across China and North Vietnam corresponded well with the current taxonomic delineation of the three intraspecific varieties of T. wallichiana.

Phylogeographic studies of plants in China: Advances in the past and directions in the future

Abstract  Phylogeography has been one major focus of evolutionary biology in recent years, with many important advances in Chinese species. In this issue, we collected 11 phylogeographic studies of plants by Chinese laboratories. We further synthesized the main findings and patterns emerging from these and previous phylogeographic studies in China and asked where phylogeographic research should be directed in the coming years. Numerous examples have shown that phylogeographic patterns in China did not show an expected expansion–contraction pattern at large scale, mirroring the geological records showing that no unified ice sheet had developed in China during the Quaternary Period. Instead, regional expansions and intraspecific divergences are very common in most studied species during the Quaternary oscillations. Different intraspecific lineages or alleles (haplotypes) were detected in multiple localized refugia, from where regional or local expansions are likely to have started. Hybridizations and introgressions are frequent between intraspecific lineages or between different species. We also reviewed computational methods for phylogeographic analyses. Despite the great progress made in recent years, there remains much to discover about the spatial–temporal dimensions and underlying speciation mechanisms of Chinese plants. Phylogeographic studies represent a key knot that connects the genus phylogeny (macroevolution) and speciation and adaptation (microevolution). Therefore, we advocate that: (i) phylogeographic studies of plants in China should be directed to the closely related species or a monophyletic group (for example, a genus or a section) in the coming years; and (ii) population genetic data based on direct sequencing multiple loci, especially those from nuclear genome and statistical tests should be widely adopted and enforced. The recovered intraspecific divergences and phylogeographic patterns of multiple‐species may allow us to better understand the high plant diversity in China and set up concrete hypotheses for studying plant speciation and diversification mechanisms in this region.

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species.

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH‐psbA, trnL‐F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear‐cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty‐seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL‐F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH‐psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH‐psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL‐F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH‐psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH‐psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH‐psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four‐marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH‐psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology‐based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.

Plant DNA barcoding in China

Identification is the keystone of biology (Bell,1986). However, to biologists and students of biology,the total numbers of species that must be identified faroutnumber the names commonly used in English, Chi-nese,orotherlivinglanguages.Inaddition,theidentifi-cation cues vary greatly between different taxonomicalgroups.Evenforthetaxonomistswithlongtrainingandexperience, it is difficult to remember all specific termsfor a given group, e.g., Orchidaceae or Poaceae, with-outhelpoffloristicbooksormonographs.Ittakesmuchtime and effort to train a taxonomist, at a time whenfewer and fewer young students are interested in this“classical”and“out-of-style”,butextremelyimportant,discipline. Many students elect to learn the more “ad-vanced” and “modern” biological disciples like molec-ular biology and biochemistry. Thus, in China and therest of the world, taxonomists are themselves becom-ing “endangered”. The rise of the DNA barcoding isexpected to mitigate, at least in part, this dilemma.The concept of DNA barcoding was proposed torapidly and accurately identify species by using short,standardized DNA markers (Arnot et al., 1993; Floydet al., 2002; Hebert et al., 2003). In fact, the idea ofspecies identification using molecular evidence datesback to 1982 for discerning the origin of fresh meats(Kang’ethe et al., 1982). Since 2003, the approachof DNA barcoding has been greatly promoted, mainlyby zoologists, to provide tools for the recognition ofspecies as an improvement on or supplement to tradi-tionalmorphology-basedtaxonomy(Hebertetal.,2003;

DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya-Hengduan Mountains.

The Himalaya–Hengduan Mountains encompass two global biodiversity hotspots with high levels of biodiversity and endemism. This area is one of the diversification centres of the genus Rhododendron, which is recognized as one of the most taxonomically challenging plant taxa due to recent adaptive radiations and rampant hybridization. In this study, four DNA barcodes were evaluated on 531 samples representing 173 species of seven sections of four subgenera in Rhododendron, with a high sampling density from the Himalaya–Hengduan Mountains employing three analytical methods. The varied approaches (nj, pwg and blast) had different species identification powers with blast performing best. With the pwg analysis, the discrimination rates for single barcodes varied from 12.21% to 25.19% with ITS < rbcL < matK < psbA‐trnH. Combinations of ITS + psbA‐trnH + matK and the four barcodes showed the highest discrimination ability (both 41.98%) among all possible combinations. As a single barcode, psbA‐trnH performed best with a relatively high performance (25.19%). Overall, the three‐marker combination of ITS + psbA‐trnH + matK was found to be the best DNA barcode for identifying Rhododendron species. The relatively low discriminative efficiency of DNA barcoding in this genus (~42%) may possibly be attributable to too low sequence divergences as a result of a long generation time of Rhododendron and complex speciation patterns involving recent radiations and hybridizations. Taking the morphology, distribution range and habitat of the species into account, DNA barcoding provided additional information for species identification and delivered a preliminary assessment of biodiversity for the large genus Rhododendron in the biodiversity hotspots of the Himalaya–Hengduan Mountains.

Paraphyly of Cyrtomium (Dryopteridaceae): evidence from rbcL and trnL-F sequence data

Cyrtomium is an Asiatic genus characterized by anastomosing veins with included veinlets, and comprises about 40 species. We sequenced rbcL and trnL-F sequences of 19 species of Cyrtomium and eight species from related genera in order to elucidate a molecular phylogeny of the genus using maximum-parsimony methods. The phylogenetic trees did not agree with traditional classifications. Cyrtomium was resolved as paraphyletic, and a clade including subseries Balansana of Cyrtomium, Cyrtogonellum, Polystichum subacutidens and Cyrtomidictyum (the BCPC clade) and a second one containing Cyrtomium sensu stricto were monophyletic. The results also implied that: (1) C. uniseriale was synonymous with C. balansae; (2) C. falcatum was likely the female parent of C. devexiscapulae; and (3) based on the rbcL and trnL-F sequence data, C. nephrolepioides and C. grossum were the female parents of C. shingianum and C. chingianum, respectively, although other evidence is needed for the confirmation of this hypothesis.

Using Morphological, Molecular and Climatic Data to Delimitate Yews along the Hindu Kush-Himalaya and Adjacent Regions

Background Despite the availability of several studies to clarify taxonomic problems on the highly threatened yews of the Hindu Kush-Himalaya (HKH) and adjacent regions, the total number of species and their exact distribution ranges remains controversial. We explored the use of comprehensive sets of morphological, molecular and climatic data to clarify taxonomy and distributions of yews in this region. Methodology/Principal Findings A total of 743 samples from 46 populations of wild yew and 47 representative herbarium specimens were analyzed. Principle component analyses on 27 morphological characters and 15 bioclimatic variables plus altitude and maximum parsimony analysis on molecular ITS and trnL-F sequences indicated the existence of three distinct species occurring in different ecological (climatic) and altitudinal gradients along the HKH and adjacent regions Taxus contorta from eastern Afghanistan to the eastern end of Central Nepal, T. wallichiana from the western end of Central Nepal to Northwest China, and the first report of the South China low to mid-elevation species T. mairei in Nepal, Bhutan, Northeast India, Myanmar and South Vietnam. Conclusion/Significance The detailed sampling and combination of different data sets allowed us to identify three clearly delineated species and their precise distribution ranges in the HKH and adjacent regions, which showed no overlap or no distinct hybrid zone. This might be due to differences in the ecological (climatic) requirements of the species. The analyses further provided the selection of diagnostic morphological characters for the identification of yews occurring in the HKH and adjacent regions. Our work demonstrates that extensive sampling combined with the analysis of diverse data sets can reliably address the taxonomy of morphologically challenging plant taxa.

High universality of matK primers for barcoding gymnosperms

Abstract  DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA region. A two‐marker combination of matK+rbcL was formally proposed as the core barcode for land plants by the Consortium for the Barcode of Life Plant Working Group. However, there are currently no barcoding primers for matK showing high universality in gymnosperms. We used 57 gymnosperm species representing 40 genera, 11 families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this study. Primer (1F/724R) of rbcL is proposed here as a universal primer for gymnosperms due to high universality. One of the nine candidate matK primers (Gym_F1A/Gym_R1A) is proposed as the best “universal”matK primer for gymnosperms because of high polymerase chain reaction success and routine generation of high quality bidirectional sequences. A specific matK primer for Ephedra was newly designed in this study, which performed well on the sampled species. The primers proposed here for rbcL and matK can be easily and successfully amplified for most gymnosperms.