Molecular systematics and biogeography of Wisteria inferred from nucleotide sequences of nuclear and plastid genes

Previous molecular phylogenetic studies of Fabaceae indicated that species of Wisteria, an intercontinental disjunct genus between eastern Asia and eastern North America, formed a clade derived from within Callerya. However, interspecific relationships were not well resolved or supported. In this study, we used sequences of the nuclear ribosomal DNA internal transcribed spacer region and the chloroplast gene matK to examine interspecific relationships and explore implications of the phylogeny for the systematics and biogeography of Wisteria. Our results showed that Wisteria with deciduous leaves and racemose inflorescences formed a strongly supported clade derived from within the paraphyletic Callerya. Afgekia was also found to be included within Callerya. Therefore, our data support the merger ofAfgekia, Callerya, and Wisteria. The phylogenetic pattern suggested that the deciduousness in Wisteria may be a derived trait likely in response to temperate climate, and the racemose inflorescences in the Afgekia–Callerya–Wisteria clade may have evolved from panicles. Our study also provided strong support for the sister relationship of the North American and eastern Asian species of Wisteria. In the Asian clade, Wisteria brachybotrys Siebold & Zucc. of Japan was sister to the clade containing W. floribunda (Willd.) DC of Japan and Korea, and W. sinensis (Sims) Sweet of China. However, our data offered weak support for the sister relationship ofW. floribunda and W. sinensis. Our divergence time and biogeographic analyses suggested that the eastern Asian–North American disjunction in Wisteria may have occurred through a dispersal event in the middle Miocene (13.4 Mya) from the Old World to the New World across the Bering land bridge followed by vicariance in the late Miocene (6.8 Mya). This study added another example to the “out of Asia” migration for the eastern Asian–eastern North American disjunction.     

Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae)

DNA barcoding is a new technology which can identify species rapidly based on short and standardized DNA sequences. Ligularia, a genus of Asteraceae with about 140 species, exhibits high morphological and ecological diversity, which makes the classification and species delimitation difficult, especially in the cases of closely related taxa. In this study, we tested four DNA core barcoding regions (ITS, matK, psbA trnH and rbcL) in 144 samples representing 35 species of Ligularia. The results revealed that the chloroplast regions (matK, psbA trnH and rbcL) have extremely low species identification rate due to low interspecific variation. Conversely, ITS sequence showed higher species identification rate (60%) and could discriminate the species which are difficult to identify. The combination of these four gene fragments did not improve the ability of species discrimination.     

Molecular identification of species in Juglandaceae: A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.     

Sectional relationships in the genus Rhododendron (Ericaceae): evidence from matK and trnK intron sequences

 Phylogenetic relationships among all eight subgenera and 12 sections in Rhododendron as well as its related genera were inferred from matK and trnK intron sequences. The results of this study provided the following insights: (1) Rhododendron is paraphyletic because Menziesia is nested within Rhododendron. (2) Subgenus Therorhodion forms a basal lineage of tribe Rhodoreae. (3) Subgenera Hymenanthes and Tsutsusi are monophyletic. (4) Subgenera Azaleastrum and Pentanthera are polyphyletic. (5) Subgenus Rhododendron is monophyletic, if section Rhododendron subsection Ledum is excluded. Received January 8, 1999, in definite form December 22, 2000 Accepted April 12, 2001     

Phylogenetic relationships of the Magnoliaceae inferred from cpDNA matK sequences

The coding region of the matK gene was sequenced to infer the phylogeny of the family Magnoliaceae. Phylogenetic analyses of 21 matK sequences representing ten genera of Magnoliaceae and three outgroups suggest relationships among both subfamilies and genera. Monophyly of the subfamily Liriodendroideae (the genus Liriodendron) and the subfamily Magnolioideae is strongly supported, respectively. Within the subfamily Magnolioideae, three clades are formed: (1) the genus Magnlietia, (2) the subgenus Magnolia, and (3) the subgenus Yulania, with the genera Michelia, Paramichelia, Tsoongiodendron, Alcimandra, Kmeria, Parakmeria and Manglietiastrum. However, the genus Magnolia is shown to be a polyphyletic group, and the genus Michelia a paraphyletic group. Relatively low sequence divergences are detected among genera of the the subfamily Magnolioideae, ranging from 0.14% to 1.70%, especially in the tribe Micheliinae (0.14–0.98%). Molecular evidence from matK sequence data suggests that the phylogenetic positions and the delimitation of the eight genera Magnolia, Michelia, Tsoongiodendron, Paramichelia, Alcimandra, Kmeria, Parakmeria and Manglietiastrum need to be reconsidered. Received: 2 January 2000 / Accepted: 12 February 2000     

Phylogenetic analyses of Andromedeae (Ericaceae subfam. Vaccinioideae)

Phylogenetic relationships within the Andromedeae and closely related taxa were investigated by means of cladistic analyses based on phenotypic (morphology, anatomy, chromosome number, and secondary chemistry) and molecular (rbcL and matK nucleotide sequences) characters. An analysis based on combined molecular and phenotypic characters indicates that the tribe is composed of two major clades—the Gaultheria group (incl. Andromeda, Chamaedaphne, Diplycosia, Gaultheria, Leucothoe¨, Pernettya, Tepuia, and Zenobia) and the Lyonia group (incl. Agarista, Craibiodendron, Lyonia, and Pieris). Andromedeae are shown to be paraphyletic in all analyses because the Vaccinieae link with some or all of the genera of the Gaultheria group. Oxydendrum is sister to the clade containing the Vaccinieae, Gaultheria group, and Lyonia group. The monophyly of Agarista, Lyonia, Pieris, and Gaultheria (incl. Pernettya) is supported, while that of Leucothoe¨ is problematic. The close relationship of Andromeda and Zenobia is novel and was strongly supported in the molecular (but not morphological) analyses. Diplycosia, Tepuia, Gaultheria, and Pernettya form a well-supported clade, which can be diagnosed by the presence of fleshy calyx lobes and methyl salicylate. Recognition of Andromedeae is not reflective of our understanding of geneological relationships and should be abandoned; the Lyonia group is formally recognized at the tribal level.     

Evolution and biogeography of the austral genus Phyllocladus (Podocarpaceae)

Aim To infer evolutionary relationships within the genus Phyllocladus and among its close relatives by phylogenetic analysis of DNA sequences. Interpret the inferred relationships in association with the fossil record to examine the origin and diversification of the genus. Location Australasia. Methods Phylogenetic analyses of rbcL, matK and internal transcribed spacer (ITS) sequences representing all of the extant species of Phyllocladus and a selection of outgroups from Podocarpaceae and Araucariaceae. Results The rbcL and matK sequences exhibit little variation within Phyllocladus, but ally its members to Podocarpaceae although its immediate sister remains unclear. The ITS sequences resolve all five species of Phyllocladus and two intraspecific ecotypes of P. alpinus. Main conclusions Phyllocladus forms a distinct lineage that diverged early in the evolutionary history of Podocarpaceae. The fossil record indicates that the genus was more widely distributed and morphologically diverse during the early Tertiary than at present. Although of Mesozoic origin, the level of sequence variation within Phyllocladus suggests that the extant species radiated during the late Tertiary c. 6.3 ± 0.9 Ma. New Zealand is the present centre of species diversity.     

植物DNA条形码促进系统发育群落生态学发展

系统发育群落生态学是近年兴起的一个重要牛态学研究分支,它以群落生态学为基础并引入了系统发育的分析方法,全面动态地反映了群落中物种内和物种间的相互作用关系,揭示了群落格局形成的生态学过程,研究了生物多样性的形成及维持机制.巴拿马BCI(Barro Colorado Island)样地的成功例子说明,在固定样地进行长期的群落生态与系统发育研究切实可行且极具意义;DNA条形码的快速兴起对这一研究发挥着重要作用.本文先列举了群落生态与系统发育综合分析能解决的群落系统发育结构、群落生态位结构、生物地理学和性状进化等生态学问题;接着介绍了标准植物DNA条形码以及利用片段组合(rbcL+matK+trnH-psbA)进行快速物种识别和近缘种区分、精确群落系统发育关系的构建以及群落生态学研究;随后提出DNA条形码研究在类群水平上需注意两片段的条形码组合(matK+rbcL)在同属种鉴别能力上的不足,而在较大尺度群落水平上需对实验设计进行优化.DNA条形码将为探讨物种多样性及其维持机理、系统发育beta多样性以及群落水平上功能性状进化研究提供新的思路.     

DNA barcoding and genetic fidelity assessment of micropropagated Aenhenrya rotundifolia (Blatt.) C.S. Kumar and F.N. Rasm.: a critically endangered jewel orchid

Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N⁶-(Δ²-isopentyl) adenine, thidiazuron, zeatin and meta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12 weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06 cm shoot length, 1671 ± 2.82 mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20 µM meta-topolin and 2.25 µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to 5.37 µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8–10 months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.