American Journal of Botany 92(4): 625±633. 2005. MOLECULAR PHYLOGENY OF INCARVILLEA (BIGNONIACEAE) BASED ON ITS AND TRNL-F SEQUENCES1 SHAOTIAN CHEN,2 KAIYUN GUAN,2 ZHEKUN ZHOU,2,3 RICHARD OLMSTEAD,4 AND QUENTIN CRONK5 2Kunming Institute of Botany, Chinese Academy of Sciences, Heilongtan, Kunming, Yunnan, 650204, P. R. China; 4Department of Biology, University of Washington, Hitchcock Hall Rm 423, Seattle, Washington 98195 USA; 5UBC Botanical Garden & Centre for Plant Research, Faculty of Agricultural Sciences, University of British Columbia, 6804 Southwest Marine Drive, Vancouver, British Columbia V6T 1Z4 Canada Incarvillea is a herbaceous and temperate member of Bignoniaceae, previously divided into four subgenera, Niedzwedzkia, Amphi- come, Incarvillea, and Pteroscleris. Niedzwedzkia and Amphicome have in the past been treated as independent genera. Different relationships have been proposed for the four subgenera. Here, maximum parsimony analysis using ITS and trnL-F sequences resulted in similar trees and showed that the genus is monophyletic. Analysis of the combined data resulted in a single tree with ®ve major clades highly supported and well resolved. The relationships of the ®ve major clades are (subgenus Niedzwedzkia (Incarvillea olgae (subgenus Amphicome (subgenus Incarvillea, subgenus Pteroscleris)))). All four subgenera are well supported for monophyly, with the exception of subgenus Incarvillea, represented here by I. sinensis and I. olgae. Incarvillea olgae is not closely related to I. sinensis, a conclusion supported by morphology. The two basal monotypic subgenera are found in Central Asia. The most species-rich subgenus, Pteroscleris, has 10 species in the Himalaya-Hengduan Mountains and may have dispersed early from central Asia to eastern Asia. Short branch lengths on the molecular trees within Pteroscleris suggest a recent and rapid radiation of this rosette-forming subgenus, perhaps connected with the uplift of the Himalaya-Hengduan massif. Key words: Bignoniaceae; classi®cation; Himalaya-Hengduan Mountains; Incarvillea; ITS; phylogenetics; radiation; trnL-F. Incarvillea Juss. is notable for being a temperate and her- was put into Bignoniaceae as an independent genus (Vassil- baceous genus of the primarily tropical and woody family Big- czenko, 1957, 1958). noniaceae (Fig. 1). It is composed of 16 species (Grierson, The geographical distribution of the genus is interesting. 1961; Zhao, 1988; Wang et al., 1991; Grey-Wilson, 1998), and Most of the species occur in the Himalayas and S. W. China, the modern classi®cation of Incarvillea was established by where the diversity of the mainly herbaceous species appears Grierson (1961) who divided the genus into four subgenera, to be related to the uplift of the Himalaya-Hengduan Moun- Amphicome (Royle) R. Br. apud Royle, Incarvillea, Pteros- tains (Chen et al., 2003). However, a number of species (all cleris Baillon, and Niedzwedzkia (B. Fedtsch.) Grierson. Al- with basal woodiness) are distributed through Central Asia and though the subgenera differ considerably in many characters into Mongolia (Fig. 3). Subgenera Amphicome and Incarvillea (including habit, calyx, stamens, capsule texture, and seeds), are found in eastern and central Asia, whereas subgenus Pter- morphological classi®cation is complicated due to extensive oscleris is endemic to the Himalaya-Hengduan region (E. apparent character convergence. Therefore, Grierson (1961) Asia). Subgenus Niedzwedzkia and the distinctive I. olgae Re- drew only tentative conclusions about evolutionary relation- gel are endemic to central Asia. ships within Incarvillea (Fig. 2A). Three subgenera, Incarvil- In this paper, we used ITS and trnL-F sequences to produce lea, Amphicome, and Pteroscleris are found in China, and in a phylogenetic framework on which to assess previous work Flora Reipublicae Sinicae (Wang et al., 1990), a different re- on the classi®cation of Incarvillea, particularly the number and lationship between them (Fig. 2B) was postulated from that of relationships of natural groups in the genus. Grierson. Furthermore, subgenus Amphicome was once placed MATERIALS AND METHODS variously in Gesneriaceae or Bignoniaceae as a genus (Don, 1837; Bureau, 1864; Burtt and Grierson, 1953). Based on the Samples and DNA extractionÐWe sampled 13 species of Incarvillea rep- karyomorphology and morphology, Chen et al. (2004) sug- resenting all four subgenera (Grierson, 1961). The three species not sampled gested that the three subgenera, Amphicome, Incarvillea, and are two that may be extinct (I. altissima G. Forrest and I. forrestii Fletcher, Pteroscleris, should all be treated as genera. The subgenus both in subgenus Pteroscleris) and I. potaninii Batalin from Mongolia, which Niedzwedzkia was originally described in Pedaliaceae and then was not obtainable for this study. Considering the historically unstable posi- tions of subgenera Amphicome and Niedzwedzkia in Bignoniaceae, Gesneri- aceae, and Pedaliaceae in the history of the genus, we selected one species 1 Manuscript received 21 January 2004; revision accepted 16 December from each of these families in order to be certain of the placement of Incar- 2004. villea. Tecomaria capensis (Thunb.) Spach, Sesamum indicum L., and Ne- The authors thank Dr. Mark Chase (Royal Botanic Gardens, Kew) for gift- matanthus strigillosus (Mart.) H. E. Moore were thus added into the analysis ing the DNA sample of Incarvillea emodi. We are grateful to the National Key Basic Research Program (973) (2003CB415102), National Natural Sci- as outgroups, and the tree was rooted on Scrophularia ningpoensis Hemsl. ence Foundation (40332021), and the Chinese Academy of Science Innovation In addition, we selected six further outgroups in Bignoniaceae for the trnL- Project (KSCX2±1±09) for support. F analyses to test the monophyly of the genus. These are Campsidium val- 3 Author for correspondence (e-mail: [email protected]) phone: 86± diviana (Phil.) Bull, Campsis radicans Seem., Pandorea jasminoides Schum., 871±5219932, fax: 86±871±5150227 Podranea ricasoliana Sprague, Tecoma stans Juss. and Tecomanthe volubilis 625 626 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 1. Growth habits of Incarvillea arguta, I. zhongdianensis, and I. mairei var. mairei. Incarvillea arguta has the much branched habit characteristic of subgenus Amphicome and contrasts with I. zhongdianensis and I. mairei var. mairei with the herbaceous habit characteristic of subgenus Pteroscleris. (A, B) I. arguta. (C) I. zhongdianensis. (D) I. mairei var. mairei. L.S. Gibbs subsp. volubilis, all related Incarvillea, following the results of a gel-dried leaves using a DNA extraction kit (Watson Biotechnologies, Shang- study of the family based on three genes by Olmstead et al. (2002). hai, P.R. China). The Incarvillea semiretschenskia Grierson sample was collected from a herbarium specimen (E), and DNA of I. emodi (Lindl.) Chatterjee was pro- Ampli®cation, sequencing and sequence alignmentÐThe whole internal vided by Dr. Mark Chase. Sequences of Sesamum indicum and Nematanthus transcribed spacer region was ampli®ed using primers ITS4 and ITS5 (White strigillosus were obtained from GenBank (Beardsley and Olmstead, 2002; et al., 1990) for all materials except I. semiretschenskia, for which four prim- Bremer et al., 2002; Zimmer, 2002). Other materials were collected from the ers (ITS2, ITS3, ITS4, and ITS5) were used. The universal primers trnc and ®eld and Kunming Botanic Garden (Appendix, see Supplemental Data accom- trnf of Taberlet et al. (1991) were used for the trnL-F ampli®cation, according panying the online version of this article). DNA was extracted from silica- to the following protocol: 948C for 3 min; 35 cycles of 948C for 30 s, 538C Fig. 2. Comparison between three kinds of phylogenetic arrangements of groups in Incarvillea. (A) based on the revision of Grierson. (B) based on Flora Reipublicae Sinicae. (C) based on combined analysis of ITS and trnL-F sequences. April 2005] CHEN ET AL.ÐMOLECULAR PHYLOGENY OF INCARVILLEA 627 Fig. 3. Distribution of the genus Incarvillea, showing the E Asian distribution of subgenus Pteroscleris (solid line) and Incarvillea sinensis (dotted line). Other species are scattered through Central Asia and Mongolia with the exception of I. arguta (subgenus Amphicome) in E Asia. for 30 s, 728C for 1 min; 728C for 7 min. PCR products were subsequently imum parsimony (MP) approach. Characters were weighted equally and were visualized on a 1% low-melting temperature agarose gel, then were cut from unordered. Gaps were treated as missing data rather than as a ®fth character. the gel and puri®ed using a gel extraction kit (Watson Biotechnologies). A heuristic research was conducted using 1000 random taxon addition repli- Puri®ed products were sequenced in both directions, using the same con- cates, and 10 trees were held in each step during stepwise addition. Other tree ditions as the PCR. ITS sequences of all species were obtained using primer search options included tree-bisection-reconnection (TBR) branch swapping, ITS4 and ITS5, except for I. semiretschenskia for which ITS2 and ITS3 were steepest descent off, MulTrees on. To estimate the support for individual used. Four primers, trnc, trnd, trne and trnf were used for trnL-F sequencing clades, heuristic bootstrapping (1000 resampling replicates) was performed. of all material. Sequencing was carried out on an ABI 3100 automated se- Decay indices (Bremer, 1988) were calculated manually. quencer (Applied Biosystems, Foster City, California, USA)