De-Zhu Li

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 Pedicularis L. (Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus

Abstract  One application of DNA barcoding is species identification based on sequences of a short and standardized DNA region. In plants, various DNA regions, alone or in combination, have been proposed and investigated, but consensus on a universal plant barcode remains elusive. In this study, we tested the utility of four candidate barcoding regions (rbcL, matK, trnH‐psbA, and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae). Amplification and sequencing was successful using single primer pairs for rbcL, trnH‐psbA, and ITS, whereas two primer pairs were required for matK. Patterns of sequence divergence commonly showed a “barcoding gap”, that is, a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species, respectively. Considering primer universality, ease of amplification and sequencing, and performance in discriminating species, we found the most effective single‐region barcode for Pedicularis to be ITS, and the most effective two‐region barcode to be rbcL + ITS. Both discriminated at least 78% of the 88 species and correctly identified at least 89% of the sequences in our sample, and were effective in placing unidentified samples in known species groups. Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis, a species‐rich cosmopolitan clade much in need of revision, as well as ecological studies in its center of diversity, the Hengduan Mountains region of China.

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae): implications for its conservation

Omphalogramma souliei Franch. is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. souliei in NW Yunnan, China. The genetic diversity at the species level is low with P=42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Neis genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenogamy predominated and autogamy played an assistant role in O. souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.

Use of DNA barcode sensu lato to identify traditional Tibetan medicinal plant Gentianopsis paludosa (Gentianaceae)

Abstract  Gentianopsis paludosa (Hook. f.) Ma (Gentianaceae) is an important species in Tibetan folk medicine commonly used to clear away the “heat evils” and toxic materials. A survey of market samples revealed that nine adulterant species, Gentianopsis barbata, G. contorta, G. grandis, Halenia elliptica, Lomatogonium macranthum, L. rotatum, Swertia angustifolia, S. bifolia and S. erythrosticta, are often marketed as G. paludosa. Methods to distinguish G. paludosa from its adulterants are limited by current morphological and chemical methods. DNA sequence analysis of the nuclear ribosomal DNA internal transcribed spacer region (ITS) was used in the differentiation of G. paludosa from the adulterant species. The data showed that the internal transcribed spacer regions differ significantly between G. paludosa and all nine adulterant species, so that they could be easily distinguished at the DNA level.

Efficiency of DNA barcodes for species delimitation: A case in Pterygiella Oliv. (Orobanchaceae)

Abstract  DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re‐assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS‐1 and ITS‐2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS‐2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.

Highly efficient pollination by bumblebees ensures seed production in Pedicularis lachnoglossa (Orobanchaceae), an early-flowering Himalayan plant

Abstract  Pedicularis (Orobranchaceae) is a common high altitude genus of the Himalayas that may be affected by pollination limitation. Using Pedicularis lachnoglossa from Yulong (Jade Dragon) Snow Mountain in Lijiang (Yunnan Province, southwest China), we investigated the effects of high altitude habitats on the process of pollination and seed production. Floral biology, pollinator foraging behavior, breeding system, and pollination efficiency were examined using observation and exclusionary techniques. Pedicularis lachnoglossa was found to be entomophilous and exclusively pollinated by Bombus friseanus and B. yunnanicola. Our results indicated that pollination limitation in P. lachnoglossa was not significant. Under open pollination, approximately 80% of flowers were successfully pollinated and developed to fruits, and about 38% of ovules developed to mature seeds. Bumblebee pollination is highly precise and efficient in P. lachnoglossa, because its flowering phenology and floral characters enhance the foraging of bumblebees on flowers. This study supports that animal pollination plays a crucial role in the outbreeding of the early flowering Pedicularis. The evolution of floral specification in Pedicularis has the advantages of adaptation to bumblebee pollination in adverse high altitude habitats.

Molecular evidence for natural hybridization between Rhododendron spiciferum and R. spinuliferum (Ericaceae)

Rhododendron ×duclouxii is morphologically intermediate between Rhododendron spiciferum and Rhododendron spinuliferum, and was suspected to be a natural hybrid between these species. In this study, the nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL‐F regions of 34 accessions of R. ×duclouxii and its putative parents were analyzed for evidence of hybridization. The results from molecular data supported by morphology demonstrated that R. ×duclouxii represents a natural hybrid between R. spiciferum and R. spinuliferum. It also implies that the hybridization between R. spiciferum and R. spinuliferum is bidirectional, and that populations of R. ×duclouxii represent hybrid swarms from backcrossing or selfing of F1 plants. The study is important for understanding the speciation processes in the genus since it demonstrates for the first time the existence of a natural homoploid hybrid in Rhododendron subgenus Rhododendron.

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.

Nujiangia (Orchidaceae: Orchideae): A new genus from the Himalayas

Abstract  The phylogenetic positions of the enigmatic “wildcard” taxon, Habenaria griffithii, were inferred from molecular data and morphological evidence. Morphologically, H. griffithii is quite “isolated” in Habenaria; instead, it is close to Gennaria, Diphylax, Peristylus, and Platanthera. It can be distinguished from these four genera by its slender staminodes conspicuously longer than the anther, rostellum having two relatively long arms, lip and lateral sepals connate in the basal part, and a transverse cushion‐shaped stigma. An analysis of combined plastid and nuclear data (rbcL, matK, and internal transcribed spacer) using Bayesian and parsimony methods revealed that H. griffithii is closest to Gennaria, a monotypic genus restricted to the western Mediterranean and Canary Islands. On this basis, a new genus, Nujiangia, is tentatively proposed to accommodate this taxon. In addition to the taxonomic treatment of this genus, the delimitation of many related genera in Orchideae is discussed.

DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae)

Abstract  Four DNA barcoding loci, chloroplast loci rbcL, matK, trnH‐psbA, and nuclear locus internal transcribed spacer (ITS), were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P‐distance, Wilcoxon signed rank test, and tree‐based analyses. This study included 186 individuals from 89 populations representing 30 species. For all individuals, single locus markers showed high levels of sequencing universality but were ineffective for species resolvability. Polymerase chain reaction amplification and sequencing were successful for all four loci. Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH‐psbA. A combination of matK and ITS was the most efficient DNA barcode among all studied regions, however, they do not represent an appropriate candidate barcode for Chinese Gaultheria, by which only 11 out of 30 species can be separated. Loci rbcL, matK, and trnH‐psbA, which were recently proposed as universal plant barcodes, have a very poor capacity for species separation for Chinese Gaultheria. DNA barcodes may be reliable tools to identify the evolutionary units of this group, so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.