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Home , Actaea (plant), Adonis amurensis, Aquilegia brevistyla, Aquilegia ecalcarata, Aquilegia vulgaris, Beesia

Biologia 65/6: 997—1003, 2010 Section Botany DOI: 10.2478/s11756-010-0105-8

Molecular phylogeny of 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, 400065, People’s Republic of ; 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 was constructed by sequencing rbcL genes of 33 representing 19 genera of Ranuncu- laceae, and three related species, 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 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 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 in China, and in position, with 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 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, 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 racemulosum Mt. JINFO, Chongqing, China T. Y. Zhang SM 1043 FJ449850 Mt. JINFO, Chongqing, China T. Y. Zhang SM 793 FJ449851 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 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 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 simplex Jianyang, , China S. Y. Chen SM 6572 FJ449863

genetic inference within flowering (Ro 1997). Genomic DNA extraction and PCR amplification The ribulose-1,5–bisphosphate carboxylase/oxygenase Genomic DNA was extracted from freshly frozen 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 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). 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 . The relia- polymerase, dNTPs and MgCl2 were purchased from bility of clustering patterns in trees was tested by bootstrap- HUASHUN Biotechnology Company in SHANGHAI; DNA ping, in the case of both NJ and MP trees, and by standard Ladder was purchased from TIANGEN Biotechnology Com- error testing for internal branches of NJ trees (Rzhetsky & pany in Beijing. Nei 1992). In total, 2,000 bootstrap replications were used Molecular phylogeny of Ranunculaceae based on rbcL sequences 999

Fig. 1. Ranunculaceae group relationships inferred from Neighbor-Joining tree using Jukes-Cantor distances based on all pairwise comparison of rbcL sequences (1389bp). Integers above branches are bootstrap support values. for the NJ and MP trees. Ranunculaceae group relationships than the expected ratio of 0.5 when all types of substi- were inferred from the NJ tree using Jukes–Cantor distances tution are assumed to be equally likely. based on all pair-wise comparisons of rbcL sequences from 33 species and three out-group taxa in the . Phylogenetic analysis Berberidaceae was chosen as the out-group because it con- Clustal X software was applied to align the sequences tains several protoberberine compounds that are common of rbcL and a phylogenetic tree was constructed us- to both families (Xiao 1980; Ro et al. 1997). ing the rbcL sequences and MEGA software. Ranun- culaceae group relationships were inferred from the NJ Result tree using Jukes–Cantor distances based on all pair- wise comparisons of rbcL sequences from 33 species of PCR amplification and sequencing of the rbcL gene Ranunculaceae and three out-group taxa in Berberi- The amplified rbcL sequences of all species were sub- daceae. The inferred phylogenies with NJ were nearly mitted to GenBank (Table 1). The length of the rbcL consistent (Fig. 1); interior branch P values (Pc) from sequences included in the final data matrix ranged the standard error test tended to be higher than boot- from 1,346 to 1,393 bp. For the 36 species studied, strap P values (Pb), and we considered values greater we obtained 1,395 aligned sequences. Amongst these, than or equal to 95% as statistically significant. Pb 289 were variable and 203 informative for parsimony values above 70% are generally considered informa- analysis. The average A:T:C:G was 27.3:28.1:19.9:24.7, tive (Hillis & Bull 1993; Ro et al. 1997). In Ranucu- with a narrow standard error around the means. There loideae groups, nearly all the species were concentrated were 1,330 identical pairs and the average transi- together into genera (Figs 1, 2). Caltha and tion/transversion (ts/tv) ratio was 2.6, which is higher should not be put in the same tribe, according to NJ 1000 Y.-F. Cai et al.

Fig. 2. Ranunculaceae group relationships inferred from Maximum parsimony tree based on all pairwise comparison of rbcL sequences (1389bp). Integers above branches are bootstrap support values. and MP trees. Groups 13, 14 and 15 were placed in Differences were found between our rbcL phylogeny Ranunculoideae, and group 18 was placed in the cen- and previous morphology-based taxonomies (Figs 1, 2). tre, between groups 17 and 19 (Fig. 2). However, all For the following discussion, Tamura’s (1993) classifica- distance measures (P distance, Jukes–Cantor, Kimura tion (Table 3) and nomenclature are followed through- two-parameter, Tajama–Nei and maximum composite out. Most found in NJ analyses (numbered in likelihood) generated identical NJ topologies. Further- numerals) can be characterized by morphological or more, the NJ tree topologies were closest to the Tamura karyological features reported in the literature and classification (Table 3), so we chose the NJ method for might represent a basis for future revised classifica- further analysis (Fig. 1). tion. Many species of the Ranunculaceae have inter- esting morphology and chemical structure, which could Discussion be mapped on an inferred topology derived from rbcL sequences. Analysis of rbcL sequences with in-groups Phylogenetic studies are based mainly on morpholog- and out-groups has proven useful for their classifica- ical and molecular characters. In this research, we se- tion. quenced and analysed rbcL genes of 36 species of Ra- The result showed that the genetic distance be- nunculaceae and the related Mahonia bealei, Mahonia tween Hydrastis and Ranunculoideae is large. However, fortunei and Nandina domestica in the Berberidaceae. the nearest to Hydrastis is Coptis in Ranuncu- We thus obtained an initial understanding of the evolu- loideae (Fig. 1); these have similar chemical composi- tionary relationship and process in Ranunculaceae and tion, both contain berberine and isoquinoline alkaloids related plants. and other common chemical constituents, suggesting Molecular phylogeny of Ranunculaceae based on rbcL sequences 1001

Table 2. GenBank accession numbers of downloaded DNA se- port that place at the base of phylogenetic trees. These quences in this study. authors consider that Hydrastis is a highly autapomor- phic lineage that should be retained within the Ranun- Species Author GenBank Accession No culaceae, while Hoot (1991) advocates placement of Hy- drastis in the monotypic Hydrastidaceae, closely laciniata Anderson C.L. DQ099449 related to the Ranunculaceae (Delectis Florae Reipub- amurensis Wang W. AY954487 Anderson C.L. DQ099444 licae Popularis Sinicae Agendae, Academiae Sinicae Wang W. AY954495 Edita 1979). Combining pharmaphylogeny analysis, we Asteropyrum cavaleriei Wang X.Q. AF079453 also believe that Hydrastis should be retained within calthifolia Wang X.Q. AF079452 the Ranunculaceae, congruent with the results of Ro Wardle P. AF307908 Silvertown J. AY395532 and colleagues (1997). Hoot S.B. AF093730 Based on chromosome and floral characteristics, Dichocarpum sutchuenense Wang W. AY954493 Tamura (1993) recognised five subfamilies in the Ra- raddeanum Wang W. AY954494 nunculaceae (Hydrastidoideae, Thalictroideae, Isopy- cymbalaria Wang W. AY954490 Anderson C.L. DQ099436 roideae, Ranunculoideae, Helleboroideae). The results Helleborus thibetanus Wang W. AY954485 of Peng (2006a) using pharmaphylogenetic analysis are Hydrastis canadensis Hoot S.B. AF093725 in accordance with the phylogenetic system presented minimus Anderson C.L. DQ099441 by Tamura (1993), and support the establishment of cernua Wang W. AY954492 Ranunculus macranthus Leebens-Mack J. DQ069502 Cimifugoideae. However, our phylogenetic tree based Thalictrum cultratum Anderson C.L. DQ099447 on rbcL sequences shows that Actaea and Beesia were Thalictrum javanicum Wang W. AY954496 in a low bootstrap value branch, with only 15% sup- Wang W. AY954486 port in the NJ tree and 25% in the MP tree. Hence, these results do not support the establishment of Cim- ifugoideae.Beesia, which is more primitive than Actaea, is included in our data and other research (Wang et al. Table. 3. The classification by Tamura (1993) for Ranunculaceae. 1993, 1998; Wang 1999). Caltha is generally considered the most original (Ranunculaceae) Tribe Genus Group No. group of Helleboroideae (Smith 1928; Tamura 1987, Subfamily 1990), as also supported in later research (Loconte & Helleboroideae Trollieae Caltha Group 17 Estes 1989; Hoot 1991), and the two trees used in our re- Trol lius Group 8 search gave similar results.Calthais not closely related Cimicifugeae Beesia Group 15 to Trollius and these two genera should not be assigned Actaea Group 14 to the same tribe (Xi et al. 1993). Furthermore, our phy- Helleboreae Helleborus Group 16 Delphineae Aconitum Group 6 logenetic tree supported a distance genetic relationship between Caltha and Trollius, consistent with Song et Ranunculoideae Anemoneae Clematis Group 5 al. (2007) who used floral morphology. Our trees sug- Pulsatilla Group 4 gested that Adonis and Trollius are sister groups and Ranunculeae Adonis Group 7 that they have a close genetic distance (Ro et al. 1997), Ranunculus Group 1 Myosurus Group 3 confirming Tamura’s (1966, 1995) classification. How- Halerpestes Group 2 ever, results from traditional taxonomy and molecular phylogeny are still disputed and further work is needed Thalictroideae Thalictreae Thalictrum Group 9 to clarify the relationship between Adonis and Trollius. A closely genetic relationship was observed among Isopyroideae Isopyreae Aquilegia Group 12 Thalictrum, Enemion and Aquilegia, strongly consistent Dichocarpum Group 10 with the of Ro et al. (1997) and the accepted Enemion Group 11 Thalictroideae classification (Delectis Florae Reipub- Coptidoideae Coptideae Coptis Group 18 licae Popularis Sinicae Agendae, Academiae Sinicae Asteropyrum Group 13 Edita 1979). Most Isopyroideae contain benzylisoquino- line and bisbenzylisoquinoline alkaloids, berberine and Hydrastidoideae Hydrastis Group 19 saponin. However, Thalictroideae mainly contain ben- zylisoquinoline and bisbenzylisoquinoline alkaloids, and most Coptidoideae contain berberine. Isopyroideae sits between Thalictroideae and Coptidoideae from an evo- lutionary viewpoint, and is considered a transitional they may have the same origin (Xiao 1980; Bill et al. group between subfamilies (Peng et al. 2006b). Com- 2007). bined with chemical constituents and the result of phy- Ro et al. (1997) used 26S ribosomal DNA to study logenetic trees, we infer that Isopyroideae should not intrafamilial relationships of the Ranunculaceae. Gu & be treated as a subfamily and should be retained in Ren (2007) found that Coptidoideae was an early Thalictroideae. next to Hydrastis in Ranunculaceae and our results sup- Most Aquilegia species contain flavonoids, saponins 1002 Y.-F. Cai et al. and fewer benzylisoquinoline alkaloids and may be re- Drummond J.R. & Hutchinsom J. 1920. A revision of Isopyrum lated to Thalictrum, which also contains saponins (Peng (Ranunculaceae) and its nearer allies. Kew Bull. 1920: 145– et al. 2006a). Our results supported that they are sister 169. Fu D.Z. 1990. Phylogenetic considerations on the subfamily Thal- groups, with a bootstrap value of 99%, but the appear- ictroideae (Ranunculaceae). Cathaya 2: 181–190. ance of cyanogenic in Aquilegia showed that Gu T.Q. & Ren Y. 2007. Floral Morphogenesis of Coptis (Ranun- the genetic location of this genus is closer to Ranuncu- culaceae) 24: 80–86. loideae. However, our results did not infer this relation- Hillis D.M. & Bull J.J. 1993. 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