Biologia 65/6: 997—1003, 2010 Section Botany DOI: 10.2478/s11756-010-0105-8 Molecular phylogeny of Ranunculaceae based on rbc L sequences Ying-fan Cai1*†, Sheng-wei Li2,MinChen2,Ming-fengJiang2†,YiLiu1, Yong-fang Xie1, Quan Sun1,Huai-zhongJiang1,Neng-wenYin1,LingWang1,RuiZhang1, Cheng-lin Huang1 &KairongLei3 1Chongqing University of Posts and Telecommunications, Chongqing 400065, People’s Republic of China; e-mail: [email protected] 2Southwest University for Nationalities, Chengdu 610041, People’s Republic of China 3Chongqing Key Laboratory of Adversity Agriculture, Chongqing 401329,People’s Republic of China Abstract: A phylogenetic tree was constructed by sequencing rbcL genes of 33 species representing 19 genera of Ranuncu- laceae, and three related species, Mahonia bealei, Mahonia fortunei and Nandina domestica. The results showed that the rbcL sequences of these Ranunculaceae range from 1,346 bp to 1,393 bp. The results based on the phylogenetic tree indi- cated that Caltha and Trol lius should not be put in the same tribe, and a close relationship betweenAdonis and Trol lius is supported by our research, while Aquilegia should be in Thalictroideae. In combination with the morphological and chemical evidence, the generic classification of Ranunculaceae should be revised into five subfamilies: Hydrastidoideae, Coptidoideae, Helleboroideae, Thalictroideae and Ranunculoideae. We demonstrate that the rbcL gene is of great value for investigating generic to subfamilial relationships in Ranunculaceae. Key words: phylogeny; Ranunculaceae; rbcL Abbreviations: rbcL, ribulose-1,5-bisphosphate carboxylase/oxygenase; IPTG, isopropyl β-D-1-thiogalactopyranoside; X- Gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside Introduction boroideae, Coptidoideae and Isopyroideae. Based on nuclear 26S ribosomal DNA, Ro et al. (1997) sug- The Ranunculaceae comprises about 2,500 described gested classification into four subfamilies, Hydrasti- species distributed amongst 59 genera throughout the doideae Rafinesque, Coptidoideae Tamura, Thalic- world, but mostly in temperate and cold areas of the troideae Rafinesque and Ranunculoideae Arnott. Peng’s northern hemisphere (Tamura 1993; Wu et al. 2003). (2006a) results, based on pharmaphylogenetic research, Ranunculaceae, which is considered pharmaceutically were in accordance with the phylogenetic analysis of important, is also of phylogenetic importance (Tamura Tamura (1966, 1993), and also supported the establish- 1993). More than 30 genera and about 220 species ment of Cimifugoideae in light of their chemical com- have been used as herbal medicine in China, and in position, with Isopyrum as a transitional group (Peng many other countries for a variety of uses (e.g. antibio- et al. 2006b). Other studies have used cytology (Yang sis, congestion, fever, cancer,arrhythmia,malaria;Xiao 2001; Lehnebach 2007), serological approaches (Jensen 1980). In China, Ranunculaceae distribute mainly in 1966, 1968) and cladistic analyses (Leconte & Estes the southwest (Delectis Florae Reipublicae Popularis 1989; Hoot 1991). Some of these studies are roughly Sinicae Agendae, Academiae Sinicae Edita 1979). The compatible with the current classification of Ranuncu- chemistry and taxonomy of Ranunculaceae is varied laceae, but this information should be carefully eval- and complex within and amongst species. uated in light of independent phylogenetic estimates, To clarify the relationships amongst subfamilies especially molecular sequencing. of Ranunculaceae, and correlations amongst their phy- In recent years, plant molecular systematics and logeny, chemistry and pharmacology, many studies have phylogenetics have been used to supplement classical examined taxonomic characters. According to chromo- taxonomy. Because of differences in evolutionary rates, some number and floral characteristics, Tamura et al. plant DNA sequences can be used to examine clas- (1966, 1993) recognised six subfamilies, namely Hy- sification. However, only a small number of molecu- drastidoideae, Thalictroideae, Ranunculoideae, Helle- lar markers have currently proved useful for phylo- * Corresponding author † Ying-fan Cai and Sheng-wei Li contributed equally to this work. c 2010 Institute of Botany, Slovak Academy of Sciences 998 Y.-F. Cai et al. Table 1. RbcL sequences identified in this study. Species Locality Voucher Specimen No. GenBank Accession No. Aconitum carmichaeli Mt. JINFO, Chongqing, China S. Q. Sun SM 0322 FJ449849 Aconitum racemulosum Mt. JINFO, Chongqing, China T. Y. Zhang SM 1043 FJ449850 Aquilegia vulgaris Mt. JINFO, Chongqing, China T. Y. Zhang SM 793 FJ449851 Clematis armandi Mt. JINFO, Chongqing, China T. Y. Zhang SM 0061 FJ449852 Clematis finetiana Mt. JINFO, Chongqing, China S. Y. Chen SM 2653 FJ449853 Clematis gratopsis Mt. JINFO, Chongqing, China W. T. Wang SM 0574 FJ449854 Clematis montona Mt. JINFO, Chongqing, China C. Ho SM 96482 FJ449855 Coptis chinensis Shizhu Chongqing, China W. T. Wang SM 1137 FJ449856 Mahonia bealei Chongqing Academy of Chinese Materia Medica Z. O. Gu SM 543 FJ449858 Mahonia fortunei Chongqing Academy of Chinese Materia Medica Z. O. Gu SM 589 FJ449857 Nandina domestica Chongqing Academy of Chinese Materia Medica T. Y. Zhang SM 0348 FJ449859 Ranunculus cantoniensis Mt. JINFO Chongqing, China H. X. Luo SM 0027 FJ449861 Ranunculus japonicus Chongqing Academy of Chinese Materia Medica J. L. Li SM 26297 FJ449862 Ranunculus sieboldii Mt. JINFO, Chongqing, China C. Ho SM 1751 FJ449860 Thalictrum simplex Jianyang, Sichuan, China S. Y. Chen SM 6572 FJ449863 genetic inference within flowering plants (Ro 1997). Genomic DNA extraction and PCR amplification The ribulose-1,5–bisphosphate carboxylase/oxygenase Genomic DNA was extracted from freshly frozen leaf ma- (rbcL) gene from the chloroplast genome has proved terial using Genomic DNA extraction reagent kits. Accord- suitable for phylogenetic analyses (Ritland & Clegg ingtotherbcL gene sequences in GenBank data of rela- 1987; Zurawski & Clegg 1987), with information now tive plants the primer of rbcL sequences were designed with Primer 5 software. which are primer rbcL-F: 5’-TTC AAA available on its structure and function (Kellogg & Ju- GCG GGT GTT AAA GAT TA-3’ and primer rbcL-R: 5’- liano 1997), evolutionary rate (Bousquet et al. 1992) GAT TGG GCC GAG TTT AAT TGC-3’. The amplifica- and systematic significance in classification (Kellogg & tion reactions were carried out in a final volume of 20 µL Juliano 1997). rbcL sequences are now commonly ap- containing 2 µL DNA (100 ng), 5 pmol each primer and 0.5 plied to study molecular plant phylogeny. Coding genes U Taq DNA polymerase in 1.8 mM MgCl2, 100 mM dNTP ◦ such as rbcL are likely informative to resolve phylo- and 10× reaction buffer. The cycling parameters were: 94 C for 5 min followed by 35 cycles of 94 ◦C1min,55◦C1.5min, genetic issues ranging from higher taxonomic ranks to ◦ ◦ relationships amongst seed plant lineages (Tian & Li 72 C 1min, and a final extension for 5 min at 72 C. 2002). In this research, we analysed the cpDNA rbcL T/A clone and sequencing sequences of Ranunculaceae and the related genera Ma- Using the Agarose Gel DNA Purification Kit (TaKaRa) to honia, Nandina and Hydrastis. A preliminary molec- purify the gene fragments, isolated by 1.0% agarose gel elec- ular basis was provided to distinguish Ranunculaceae trophoresis, quantitative PCR purification products insert containing different chemical components, such as mag- to the pMD18-T cloning vector and transform into the com- noflorine and ranunculin. The research will be useful to petent E. coli DH5α, then the cells were incubated in a find new drugs, as well as new information on relation- growth medium and finally spread on an agar LB medium ships within the Ranunculaceae. plate which contained ampicillin, IPTG and X-gal then in- cubated at 37 ◦C for 12 ∼ 16 h. Picking the white mono- clonal colony to incubate in the LB liquid culture medium, Material and methods as well, extracting the plasmid which was digested by re- striction enzymes and sequenced. Plasmid DNA sequence Plant materials was completed by Beijing Sunbiotech co., Ltd. (ABI3730XL The materials used in the present study are listed in Table 1. DNA Sequencer). Coptis Chinensis was from Shizhu of Chongqing provided by Chen Daxia, Ranunculus japonicus, Mahonia fortunei, Analysis of DNA sequence data and construction of a phy- Mahonia bealei and Nandina domestica were collected from logenetic tree the Medical Plants Garden of Chongqing Academy of Chi- The NCBI website and Blastn online searches were used nese Materia Medica, and all the other species were collected to analyse the target gene, and sites with missing data or from Mt. Jinfo, Chongqing. In addition, rbcL sequences of 21 gaps were excluded from all following analyses. Neighbour- species were downloaded from GenBank. Totally 36 species joining (NJ) analysis (Saitou & Nei 1987) was performed out of 19 genera were included in the final analyses (Tables using the MEGA software (Kumar et al. 1993). The Jukes– 1, 2). Cantor model of nucleotide substitution was selected for analyses based on Nei’s (1991) guidelines for choosing the Strain and agent most appropriate distance measure. Maximum parsimony Escherichia coli DH5α were preserved in the labora- (MP) analysis was used to implement the heuristic search tory, pMD18-T Vector purchased from TaKaRa com- procedure, with 100 replications with random addition of pany. Genomic DNA extraction reagent kits, Taq DNA taxa to reduce possible bias from the input order. The relia- polymerase,