中国科技论文在线 http://www.paper.edu.cn Journal of Systematics and Evolution 46 (2): 142–154 (2008) doi: 10.3724/SP.J.1002.2008.07107 (formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com Phylogenetic inference of the genus Bupleurum (Apiaceae) in Hengduan Mountains based on chromosome counts and nuclear ribosomal DNA ITS sequences Qi-Zhi WANG * Xing-Jin HE ** Song-Dong ZHOU* Yun-Ke WU Yan YU Yun-Li PANG (Laboratory of Systematic and Evolutionary Botany, College of Life Science, Sichuan University, Chengdu 610064, China) Abstract Hengduan Mountains are the diversity center for the genus Bupleurum of Apiaceae in China. This paper reports chromosome numbers of six species and two varieties of Bupleurum, and for four species and two varieties their chromosome numbers are reported for the first time. The phylogeny of Bupleurum was investigated based on the ITS region of the nuclear ribosomal DNA (nrDNA) of 14 taxa from the Hengduan Mountains, 3 taxa from the North China (Hebei and Heilongjiang), and 16 taxa from Africa and the Mediterranean region. Varia- tions in chromosome numbers and the ITS sequences were used to infer phylogenetic relationships between Bupleurum species in Hengduan Mountains. The results showed that the Hengduan Mountains might represent one of the frequency and diversity centers for Bupleurum. The ancestors of Bupleurum species in the Hengduan Mountains may be related to the woody B. fruticosum in North Africa, or the species in the western Mediterranean region. It is postulated that the ancestral population migrated into Hengduan Mountains through the Middle East and the Caucasus. Furthermore, the neo-endemic B. mundtii in South Africa appeared to be a close relative of the species in the Hengduan Mountains. In the trend of basic chromosome number evolution, x = 8 should be regarded as the ancestral basic number, while x = 6, 7 as the derived ones. The Bupleurum species in the Hengduan Moun- tains have been undergoing changes in the basic chromosome numbers or the ploidy level. The ITS phylogenetic tree showed that the Chinese species were divided into two clades: one with the basic chromosome number x = 8, and the other with x = 6, 7. The results rejected the previous infrageneric classification of Bupleurum in China. We further suggested to raise B. marginatum var. stenophyllum to species rank based on the combined evidence from morphology, karyology, pollen morphology, and the ITS phylogenetic tree. Key words Bupleurum, chromosome, Hengduan Mountains, ITS, phylogeny, taxonomy. The genus Bupleurum L., with about 180–190 phylogeny and classification are incomplete. species spreading in the North Temperate Zone Traditionally, the woody Araliaceae is regarded (mainly in Eurasia, Mediterranean and North Africa), as the ancestor of the herbaceous Apiaceae (Calviňo et is one of the largest genera of the family Apiaceae al., 2006). Woody B. fruticosum L. in North Africa is (Liu et al., 2003; Neves & Watson, 2004). Species the possible ancestor of the species in genus Bupleu- within Bupleurum are easily recognized by the parallel rum (Gruas-Cavagnetto & Cerceau-Larrival, 1978). A venation of the simple leaves, conspicuous bracts and hypothesis on the origin of Apiaceae suggests that B. bracteoles on the inflorescences. Pollen morphology fruticosum might have migrated northward into Eura- exhibits little variation in this genus. The fruit is sia through the Middle East (Plunkett et al., 1996b). oblong to ovoid-oblong or ellipsoid, slightly laterally However, Neves and Watson (2004) suggested that compressed and the mericarp is subpentagonal (rarely the genus Bupleurum originated in the western Medi- rounded) in cross section. Bupleurum is a primitive terranean. Although B. fruticosum was nested within genus in Apioideae in recent molecular studies the western Mediterranean clade, it was not the most (Plunkett et al., 1996a, b, 1997; Downie & basal taxon, thus casting doubt regarding the origin of Katz-Downie, 1996, 1999; Downie et al., 1998, this genus. On the other hand, most Bupleurum spe- 2000a, b; Plunkett & Downie, 1999, 2000). Despite cies in the Hengduan Mountains in China exhibit the intensive research on Bupleurum, the infrageneric primitive pollen type of subrhomboidity to subround to subellipse that appeared in the early Palaeocene ——————————— (Shu & Sheh, 2001). The entire region of the Heng- Received: 27 August 2007 Accepted: 2 February 2008 duan Mountains harbors high Bupleurum diversity in * Co-First authors. Asia. Thus, the Hengduan Mountains are another ** Author for correspondence. E-mail: [email protected]; Tel.: 86-28- 85415006; Fax: 86-28-85415006. possible center of origin for Bupleurum (Shu & Sheh, 转载 中国科技论文在线 http://www.paper.edu.cn WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains 143 2001). By including Bupleurum species from Africa from the Hengduan Mountains in South China, repre- and the western Mediterranean region, we aim to senting two sections in the subgenus Eubupleura investigate the origin and evolution of Bupleurum in (Ranunculoidea and Falcata, sensu Su et al., 1998), as the Hengduan Mountains with a broad taxon sampling well as B. longiradiatum Turcz. from the monotypic throughout the entire genus. subgenus Longifolia. We also sequenced B. chinense Bupleurum was placed in the subtribe Carinae, DC. and B. scorzonerifolium Willd. from North China tribe Apieae, subfamily Apioideae (Drude, 1898). in subgenus Eubupleura. Our samplings represent all Wolff (1910) and Linczevsky (1950) briefly discussed subgenera and sections in China (sensu Su et al., the taxonomy of Chinese species. Su et al. (1998) 1998) (Fig. 1). studied fourteen species, two varieties and one form of Karyotype, including the basic chromosome Chinese Bupleurum in an infrageneric classification numbers and ploidy level, is often used to investigate based on morphology, anatomy and palynology. Two interspecific evolution between organisms. Previous subgenera of the genus were then proposed: Longifo- karyotype study for Bupleurum species in the Heng- lia (Wolff) Yuan and Eubupleura (Briq.) Yuan. duan Mountains was inadequate. Only two species Subgenus Eubupleura includes two sections: Ranun- and one variety have had their chromosome numbers culoidea (Wolff) Yuan and Falcata (Wolff) Yuan. reported (Pan & Qin, 1981; Pan et al., 1985; Qin et al., The ITS region of the nrDNA evolves at a rela- 1989). We herein report the chromosome numbers of tively rapid rate and is rich in variable and informative six species and two varieties, and the data from four sites. It has been proven to be a valuable source of species and two varieties were reported for the first information in evaluating phylogenetic relationships at time (i.e., B. chaishoui R. H. Shan & M. L. Sheh, B. the genus and species level (Baldwin, 1992; Baldwin gracilipes Diels, B. rockii H. Wolff, B. yunnanense et al., 1995; Tian & Li, 2002). It has also been suc- Franch., B. hamiltonii var. humile (Franch.) R. H. cessfully used to study intraspecific relationships in Shan & M. L. Sheh and B. longicaule var. franchetii Chinese Bupleurum species (Wu et al., 2005; Xie et H. Boiss.). al., 2006). However, so far no phylogenetic study has The questions of this study are as following: (1) included species from the Hengduan Mountains. What are the relationships between the Bupleurum Therefore, based on previous work, we sequenced the species in the Hengduan Mountains and the species in ITS region of ten Bupleurum species and four varieties Africa and the western Mediterranean region? Did the Bupleurum species in our experiment Subgenus Eubupleura Subgenus Longifolia B. longiradiatum Section Ranunculoidea Section Falcata B. commelynoideum B. chinense B. longicaule var. amplexicaule B. chaishoui B. longicaule var. franchetii B. gracilipes B. petiolulatum B. hamiltonii B. rockii B. hamiltonii var. humile B. yunnanense B. marginatum B. marginatum var. stenophyllum B. microcephalum B. scorzonerifolium B. wenchuanense Fig. 1. Infrageneric classification of the Chinese Bupleurum (sensu Su et al., 1998). All taxa are included in our sampling. 中国科技论文在线 http://www.paper.edu.cn 144 Journal of Systematics and Evolution Vol. 46 No. 2 2008 genus Bupleurum originate in the Hengduan Moun- reactions were performed for confirmation. Molecular tains? (2) Is the previous infrageneric classification of cloning was conducted on two species: B. longiradia- Chinese Bupleurum (Su et al., 1998) well supported tum and B. scorzonerifolium, to examine the extent of by the molecular and karyological data? and (3) How sequence homogenization among reiterated ITS to delimitate of Bupleurum species in the Hengduan copies. The purified ITS templates were ligated to the Mountains (e.g., treatment of B. marginatum var. pGM-T Easy vector (Tiangen Biotech, Beijing, China) stenophyllum (H. Wolff) R. H. Shan & Yin Li). and subsequently transformed into competent cells following the manufacturer’s protocol. The trans- formed cells were cultured overnight at 37 ℃ on the 1 Material and methods LB ampicillin plates. Colonies carrying the ITS insert were identified by colors (white) and were further 1.1 Material and methods of nrDNA ITS verified by PCR and sequencing. For each sample, up Sample materials of Bupleurum species were ob- to 10 white colonies were checked for inserts. All ITS tained from fresh leaves in the field, which were sequences were aligned manually using sequential collected individually in the Hengduan Mountains and pairwise comparisons. The boundaries of