Taiwania, 54(4): 338-342, 2009 The Relationship of Fagaropsis and Luvunga in Rutaceae Ka-Ho Ling(1), Yanli Wang(1), Wing-Sem Poon(1), Pang-Chui Shaw(2) and Paul Pui-Hay But(1,3*) 1. Food and Drug Authentication Laboratory, Department of Biology, The Chinese University of Hong Kong, 48-52 Tai Po Road, Shatin, N.T., Hong Kong, P.R. China. 2. Department of Biochemistry, The Chinese University of Hong Kong, 48-52 Tai Po Road, Shatin, N.T., Hong Kong, P.R. China. 3. Yunnan Institute of Materia Medica, 24 Lengshuitang, Bijizhen, Kunming, Yunnan, P.R. China. * Corresponding author. Tel: +852-2609-6299; Email: [email protected] (Manuscript received 12 November 2008; accepted 25 June 2009) ABSTRACT: Molecular analyses of ITS1 and trnL-F sequences confirmed that Fagaropsis falls in the same clade with Phellodendron, Tetradium, Toddalia, and Zanthoxylum, thus offering support for the ‘proto-Rutaceae’ hypothesis based on chemotaxonomic interpretation. On the other hand, Luvunga naturally fits in subfamily Aurantioideae. The results also indicate that Orixa is closer to Casimiroa and Skimmia, but not to Tetradium and Zanthoxylum. KEY WORDS: Aurantioideae, Proto-Rutaceae, Rutaceae, Fagaropsis, Luvunga, Orixa. Disagreement with Engler’s (1931) classification was INTRODUCTION independently raised by the biochemical studies of Rutaceae Juss. are a large family of great economic Waterman (1975) and Waterman & Khalid (1981). The importance, as many species are sources of foods, spices, genera Zanthoxylum (including Fagara L.) and essential oil, herbal medicines, horticultural items, Fagaropsis Mildbr. in Rutoideae and the genera pharmaceuticals, and lumbar (Wiersema and Leon, Phellodendron and Toddalia Juss. in Toddalioideae were 1999). A proper appreciation of the true phylogenetic found to have retained the primitive relationship in this family will definitely be helpful to the 1-benzyltetrahydroisoquinoline (1-BTIQ) alkaloid selection of taxa for breeding, searching for genes for pathway and produce 1-BTIQ alkaloids. Waterman (1983, biotechnological designs, and exploration for bioactive 2007) hypothesized that Rutaceae and related families leads in drug development. Regretfully, the classification switched from production of 1-BTIQ alkaloids to the of Rutaceae is in a fluid state. production of advanced Rutalean metabolites: coumarins, Engler (1931) provided the most comprehensive limonoids, and furoquinoline alkaloids. Based on classification and divided the family into seven chemotaxonomic analyses, he further proposed to place subfamilies. Three of the subfamilies, Rutoideae, these four genera in a tentative ‘proto-Rutaceae’ group. Toddalioideae K. Koch, and Aurantioideae Eaton, None of these four genera, however, contain all the four covering almost all genera in Rutaceae, are represented types of metabolites. So Waterman (1983) suggested that with follicles, capsules and berries, respectively. members of Euodia sensu lato with pinnately compound However, morphological studies by Hartley (1974, 1981 leaves (i.e., Tetradium) would be good candidates to and 2001) questioned the validity in dividing Rutoideae contain both 1-BTIQ and all three types of advanced and Toddalioideae based on differences in fruit Rutalean metabolites. Studies following this suggestion characters, as he concluded that some genera in indeed found in two Tetradium species 1-BTIQ and all Toddalioideae are more closely related to genera in three types of advanced Rutalean metabolites (Ng et al., Rutoideae. Hartley (1981) further indicated that 1987a, 1987b; Quader et al., 1990; Waterman, 2007). Tetradium Lour. (Rutoideae) and Phellodendron Rupr. These findings lend support to the ‘proto-Rutaceae’ (Toddalioideae) are closely related, so close that it is suggestion, which includes Fagaropsis, Phellodendron, impossible to distinguish them using vegetative or Tetradium, Toddalia, and Zanthoxylum. staminate material. He even suggested that Tetradium is Recently, Poon et al. (2007) undertook cladistic likely the immediate ancestor of Phellodendron. He also analyses of subfamilies Rutoideae and Toddalioideae in considered Zanthoxylum L. (Rutoideae), Tetradium, and the Rutaceae. Their findings demonstrated that the two Phellodendron to be related to one another in a linear subfamilies are not natural. Moreover, four of the sequence. Similarly, he suggested that Acronychia J. R. ‘proto-Rutaceae’ genera included in their study, Forst. & G. Forst. of Toddalioideae is closely related to Phellodendron, Tetradium, Toddalia, and Zanthoxylum, Melicope J. R. Forst. & G. Forst. of Rutoideae (Hartley, were resolved as a natural (monophyletic) group, lending 1974, 1981). Thorne (1992, 2000) and Thorne and support to Waterman’s suggestion of a ‘proto-Rutaceae’ Reveal (2007) agreed with this suggestion and merged group. Since this group will bear relevance to future Toddalioideae into Rutoideae. classifications of Rutaceae and also possibly directions for 338 December, 2009 Ling et al.: The relationship of Fagaropsis and Luvunga in Rutaceae search for bioactive leads (Waterman, 2007), we Melia azedarach analyzed the ITS1 and trnL-F sequences of Fagaropsis 69 Tetradium ruticarpu glabra Capuron to test if it is also a member of the 77 Tetradium trichotom ‘proto-Rutaceae’ clade. 92 Tetradium austrosin On the other hand, Luvunga Ham. ex Wight & Arn. 53 is traditionally considered a member of Aurantioideae Tetradium glabrifol (Huang, 1997). However, Chase et al. (1999) reported Phellodendron amure that a specimen identified as 'Luvunga eleutherandra 97 Fagaropsis glabra Dalzell' paired with Zanthoxylum (of ‘proto-Rutaceae’), 100 Zanthoxylum piperit but then added in proof that the 'Luvunga eleutherandra' Zanthoxylum simulan specimen was actually a Zanthoxylum. So far, no further 93 Zanthoxylum schinif clarification of the alliance of Luvunga with molecular data has been made; but Thorne (2000) and Thorne and Toddalia asiatica Acronychia peduncul Reveal (2007) went ahead to place this genus in 65 98 Rutoideae. We found it necessary to include L. scandens 68 Melicope ternata Roxb. in the analysis so as to clarify its true position in 100 Melicope rubra Rutaceae. Euodia hortensis 95 72 Euodia pubifolia 83 MATERIALS AND METHODS Halfordia kendack 83 Bosistoa medicinali 1. Plant materials 89 Leaf fragments of Fagaropsis glabra and Luvunga Dinosperma melanoph scandens were sampled from herbarium specimens 95 Skimmia japonica collected by R. Capmon 24502-SF and by SK Lau 6331, 64 Skimmia laureola respectively, kept in Harvard University Herbaria. 98 Skimmia reevesiana 2. DNA extraction,,sequencing and data analysis 63 59 Skimmia anquetilia Casimiroa edulis Sample preparation and DNA extraction, 93 70 amplification and sequencing, as well as cladistic Ptelea trifoliata analysis followed those described in Poon et al. (2007). Orixa japonica ITS1 and trnL-F sequences of the taxa from Rutoideae Citrus maxima and Toddalioideae reported in Poon et al. (2007) were 99 100 Citrus reticulata included in this analysis. In addition, seven DNA sequences were downloaded from GenBank for tree Luvunga scandense construction. They include Melia azedarach L. Ruta graveolens (AY695595, AB057481 and EF489265), Citrus maxima (Burm. ex Rumph.) Merr. (EU178124 and AM398228) Fig. 1. ITS1 MP jackknife tree. Tree was rooted using Melia azedarach as an outgroup. The numbers on the branches and Citrus reticulata Blanco (EU178123 and represent JK values larger than 50. AM398230). based our phylogenetic inferences on the RESULTS simultaneous-analysis tree (Fig. 3). Our results confirmed that Fagaropsis glabra, as A total of six DNA sequences (FJ440571– suggested by Waterman (1983), clustered with FJ440576) from Fagaropsis glabra and Luvunga Phellodendron, Tetradium, Toddalia, and Zanthoxylum scandens were uploaded to GenBank. The most (Figs. 1–3). This “proto-Rutaceae” group (including parsimony JK trees for the nuclear ribosomal regions, Fagaropsis, Phellodendron, Tetradium, Toddalia and plastid regions, and the simultaneous analysis are Zanthoxylum) was well-supported (97% & 71% JK) in both presented in Figs. 1–3. Data-matrix and tree statistics for the nuclear and plastid trees without conflict; this group was all analyses are presented in Table 1. Conflicts between also strongly supported (100% JK) in the the nuclear and plastid JK trees (Figs. 1 & 2) were simultaneous-analysis tree. It may be possible to group these attributed to stochastic error rather than the process five genera in a re-circumscribed tribe Zanthoxyleae (A. partitions having different histories in our previous study Juss.) Dumort., with Zanthoxylum as the type genus. This (Poon et al., 2008). Because there were no well group probably is close to an ancestral one in Rutaceae as supported conflicts between the process partitions, we fossil records revealed that all five genera were already 339 Taiwania Vol. 54, No. 4 Melia azedarach Melia azedarach Skimmia reevesiana 100 Citrus maxima Skimmia laureola 96 100 Citrus reticulata Skimmia anquetilia 67 Luvunga scandense 79 Skimmia japonica Ruta graveolens Orixa japonica Skimmia japonica Casimiroa edulis 94 55 Skimmia laureola 63 Melicope rubra 100 99 Melicope ternata Skimmia reevesian 76 Acronychia peduncu 77 Skimmia anquetili 100 Euodia hortensis 67 Casimiroa edulis 76 Euodia pubifolia Orixa japonica 56 Halfordia kendack 81 72 Ptelea trifoliata Bosistoa medicinal Zanthoxylum piper Dinosperma melanop 100 Tetradium ruticarp Zanthoxylum simul 100 85 Tetradium glabrifo Zanthoxylum schin 98 76 78 Tetradium trichoto