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Systematic Study on Guttiferae Juss. of Peninsular Malaysia Based on Plastid Sequences

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Systematic Study on Guttiferae Juss. of Peninsular Malaysia Based on Plastid Sequences TROPICS Vol. 16 (2) Issued March 31, 2007 Systematic study on Guttiferae Juss. of Peninsular Malaysia based on plastid sequences 1, 2,＊ 3 1 Radhiah ZAKARIA , Chee Yen CHOONG and Ibrahim FARIDAH-HANUM 1 Faculty of Forestry, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2 SEAMEO BIOTROP, Jl. Raya Tajur Km. 6 Bogor-Indonesia 3 Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia ＊ Corresponding author: Radhiah ZAKARIA ABSTRACT Twenty-one taxa in 4 genera of knowledge for this family was published in the last (Calophyllum, Mammea, Mesua s.l. and Garcinia) century by Planchon and Triana (1862). Kostermans of Guttiferae from several areas in Peninsular (1961) published a monograph of the Asiatic and Pacific Malaysia were used to investigate the status and species of Mammea, Stevens (1980) published a revision relationships of taxa within the family Guttiferae of the old world species of Calophyllum, and Jones (1980) using the chloroplast DNA trn L-trn F sequence published a revision of the genus Garcinia worldwide. For data. Molecular phylogeny results indicated Peninsular Malaysian genera, Ridley (1922) made the first that Calophyllum , Mammea and Garcinia are treatment of the family Guttiferae followed by Henderson monophyletic genera. However, the genus and Wyatt-Smith (1956) and Whittmore (1973). The Mesua appeared to be polyphyletic as Mesua status of some taxa in Guttiferae of Peninsular Malaysia fer rea did not form a cluster with the other before and after the current study is presented in Table 1. Mesua taxa. Therefore, the molecular phylogeny In Guttiferae, one of the taxonomic problems is the supports the morphological classification that status of the closely related genera Kayea and Mesua. Mesua taxa in Peninsular Malaysia other than Linnaeus first introduced the genus Mesua L. in 1753 M. ferrea, be transferred back into genus Kayea. when he described the species Mesua ferrea in his Species On the other hand, the molecular phylogeny Plantarum. In 1832, Wallich introduced Kayea Wall. which disagrees with the morphological classification of was closely related to Mesua. For more than a century, Calophyllum wallichianum var. wallichianum and Kayea and Mesua remained as two distinct genera and C. wallichianum var. incrassatum as varieties of C. Mesua was a monotypic genus. Bentham and Hooker wallichianum . Therefore, the status of these two (1862) distinguished Kayea and Mesua by ovary/stigma varieties should be reinstated to distinct species as structure. Kayea has a one-celled ovary with one seed and C. wallichianum and C. incrassatum respectively. 4-fid stigma whereas Mesua has a two-celled ovary and peltate stigma (Bentham and Hooker, 1862). However, Key words: Guttiferae, Mesua, trnL-trnF, cpDNA, Kostermans (1969) later observed that one and two- phylogeny celled fruit may be found with one or two seeds on the same individual tree of Mesua ferrea. Kayea species with Guttiferae Juss. (Clusiaceae Lindl. (nom. Altern.)), two-seeded fruits have been observed (Kostermans,1969). a medium sized and varied tropical family, plays an Therefore, based on the fruit structure, Kostermans important role as a main canopy component of the (1969) abolished the genus Kayea, and placed the entire Malaysian rainforest (Whitmore, 1973). There are Kayea taxa into Mesua. This classification has since been 40 genera with ca. 1000 species of Guttiferae found followed by many other authors such as Whitmore (1973), throughout the tropics, and in Peninsular Malaysia 4−5 Keng (1978), Corner (1988), Chua (1995), Turner (1995) genera with 121 species occur in several habitats, from and Kochummen (1997). Nevertheless, Stevens (1993) sea level to mountain (Keng, 1969; Whitmore, 1973; and Turner (2000) placed Kayea from Mesua separately. Corner, 1988; Turner, 1995). More recently, however, To address these inconsistencies in the classification Stevens (2005) reduced the number of Guttiferae genera of the Guttiferae, we have conducted a molecular to 27 and the number of species to1050. phylogenetic study using plastid trnL-trnF sequence data. A comprehensive description laying the foundation 142 Radhiah ZAKARIA, Choong CHEE YEN and Ibrahim FARIDAH-HANUM Table 1. Status of some taxa in Guttiferae of Peninsular Malaysia before and after this study. No. Before this study Family/species After this study Family/species Guttiferae Guttiferae 01. Calophyllum depressinervosum Henderson et Wyatt-Smith Calophyllum depressinervosum 02. C. dioscurii P. F. Stevens C. dioscurii 03. C. macrocarpum Hook. f. C. macrocarpum 04. C. rupicolum Ridl. C. rupicolum 05. C. soulattri Burm. f. C. soulattri 06. C. tetrapterum Miq. C. tetrapterum 07. C. wallichianum var. wallichianum (Planch. et Triana) P. F. Stevens C. wallichianum Planch. et Triana 08. C. wallichianum var. incrassatum (Henderson et Wyatt-Smith) P. F. Stevens C. incrassatum Henderson et Wyatt-Smith 09. Garcinia atroviridis Griff. ex T. Anderson Garcinia atroviridis 10. G. malaccensis Hook.f. G. malaccensis 11. G. nervosa Miq. G. nervosa 12. G. opaca King G. opaca 13. G. parvifolia (Miq.) Miq. G. parvifolia 14. Mammea brevipes (Craib) Kosterm. Mammea brevipes 15. M. odorata (Rafin.) Kosterm. M. odorata 16. M. siamense (Miq.) Anders. M. siamense 17. Mesua cornerii Kochummen Kayea cornerii P. F. Stevens 18. M. ferrea L. Mesua ferrea 19. M. kunstleri (King) Kosterm. Kayea kunstleri King 20. M. lepidota Anders. Kayea lepidota Anders. 21. M. racemosa (Planch. et Triana) Kosterm. Kayea racemosa Planch. et Triana Molecular approaches MATERIALS AND METHODS Total DNA was isolated from 0.1−0.2 g silica-dried leaf Taxon sampling materials and from fresh leaf materials following the Twenty-one ingroup taxa consisting of 4 Guttiferae genera CTAB method of Doyle and Doyle (1987 and 1990). The (Calophyllum, Garcinia, Mammea and Mesua) were trnL-trnF spacer of the chloroplast genome was amplified examined (Table 2). Samples were collected from Pasoh using universal primers e and f according to Taberlet et Forest Reserve and Langkawi in Malaysia, and the Bogor al. (1991). The PCR amplification was performed in a total Botanic Garden in Indonesia. Herbarium specimens were volume of 25 µl with 100 ng of template DNA, 1X PCR made for all taxa collected from the field except Garcinia buffer, 1.5−3 mM MgCl2, 0.2 mM dNTPs mixture (200 taxa and Mesua racemosa. These were deposited in the µM each of dATP, dTTP, dGTP and dCTP), 0.2 µM each Faculty of Forestry Herbarium, Universiti Putra Malaysia primer (forward and reverse) and 1 unit Taq AmpliTaq® (UPMF) and the SEAMEO-BIOTROP Herbarium DNA polymerase (Invitrogen). The PCR amplification (BIOT) Bogor, Indonesia. Leaf materials of Garcinia was performed as follows: 1 min pre-heating at 94˚C, 1 taxa and Mesua racemosa were not collected as the min denaturation at 94˚C, 1 min annealing at 49−58˚C, sequence data of these taxa were obtained from Nazre and 2 min extension at 72˚C. The denaturation-annealing- (2000). Ternstroemia impressa (Theaceae) which is very extension steps were repeated 30 cycles before the final closely related to Guttiferae was chosen as an outgroup extension step at 72˚C for 10 min. The PCR amplification for comparison (Vestal, 1937; Whitmore, 1973). All the was performed using the GeneAmp PCR System 2400 sequence data generated in this study were deposited in (Perkin Elmer) or Eppendorf Mastercycler. GenBank. Voucher information, literature citations and Aliquots of 3 µl of each PCR product were checked database accession numbers are listed in Table 2. on 1.5% agarose gel for quality, and the balance of the product was purified using QIAquick® PCR Purification Kit (QIAGEN) following the supplier’s instructions. Systematic study on Guttiferae Juss. of Peninsular Malaysia based on plastid sequences 143 Table 2. Location and detail of taxa used in the molecular study. GenBank accession No. Taxon Voucher Collection site number/source 01. Calophyllum rupicolum R. Zakaria 184, BIOT, UPMF Pasoh Forest Reserve AY389781 02. C. depressinervosum R. Zakaria 183, BIOT, UPMF Pasoh Forest Reserve AY389787 03. C. macrocarpum R. Zakaria 177, BIOT, UPMF Pasoh Forest Reserve AY389788 04. C. dioscurii R. Zakaria 178, BIOT, UPMF Pasoh Forest Reserve AY389783 05. C. soulattri R. Zakaria 187, BIOT, UPMF Pasoh Forest Reserve AY389782 06. C. tetrapterum R. Zakaria 179, BIOT, UPMF Pasoh Forest Reserve AY389784 07. C. wallichianum var. wallichianum R. Zakaria 185, BIOT, UPMF Pasoh Forest Reserve AY389786 08. C. wallichianum var. incrassatum R. Zakaria 182, BIOT, UPMF Pasoh Forest Reserve AY389785 09. Garcinia atroviridis M. Nazre 193, UKMB Pasoh Forest Reserve Nazre, 2000 10. G. malaccensis M. Nazre 68, UKMB Pasoh Forest Reserve Nazre, 2000 11. G. nervosa M. Nazre 97, UKMB Pasoh Forest Reserve Nazre, 2000 12. G. opaca M. Nazre 79, UKMB Pasoh Forest Reserve Nazre, 2000 13. G. parvifolia M. Nazre 109, UKMB Pasoh Forest Reserve Nazre, 2000 14. Mammea brevipes A. Zainudin 4385, UKMB Langkawi, Kedah AY389790 15. M. odorata Annon, KRB1, BIOT, UPMF Bogor Bot. Garden AY389789 16. M. siamense Annon, KRB2, BIOT, UPMF Bogor Bot. Garden AJ606679 17. Mesua cornerii R. Zakaria 173, BIOT, UPMF Pasoh Forest Reserve AY389799 18. M. ferrea R. Zakaria 174, BIOT, UPMF Pasoh Forest Reserve AY389792 19. M. racemosa A.R.Khalid 011, BIOT, UPMF Pasoh Forest Reserve Nazre, 2000 20. M. kunstleri R. Zakaria 181, BIOT, UPMF Pasoh Forest Reserve AJ606678 21. M. lepidota R. Zakaria 188, BIOT, UPMF Pasoh Forest Reserve AJ606677 22. Ternstroemia impressa ＊ − − AF396228 ＊ Ternstroemia impressa was used as an outgroup. Aliquot of 2 µl of the purified PCR product was checked Navigator™ (Applied Biosystems). by 1.5% agarose gel electrophoresis for the quality and quantity prior to cycle sequencing. Phylogenetic Analyses An amount of 100 ng of the purified PCR product The sequence data obtained from this study was compared was used in cycle sequencing adopting dideoxy chain to DNA sequences in the GenBank database using the terminator method using the dRhodomine Terminator BLAST programme (Altschul et al.
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