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. 1997) to conﬁrm that Cycle Sequencing Ready Reaction Kit (Applied the relevant sequences had been obtained. The trnL- Biosystems). The cycle sequencing was done on both trnF sequences were aligned using CLUSTAL W 1.82 the forward and reverse sequence readings. The primers (Thompson et al., 1994), and then corrected visually with used in the cycle sequencing were the same as that in the the assistance of Seqpup® Ver. 0.6 software (Applied PCR amplification. Biosystems). Ambiguous regions in the alignment were The cycle sequencing reaction mixture contained excluded from the phylogenetic analyses. Insertions and 3.2 pmol primer, 4 µl Terminator Ready Reaction mixture deletions (indels) from the sequence alignment were and 2.4 µl double distilled water. The cycle sequencing treated as gaps and were not included in the phylogenetic was subjected to 25 cycles of denaturation at 96˚C for 30 analyses. Pairwise distances among the ingroups as well seconds, annealing at 50˚C for 30 seconds and extension as between the ingroup and the outgroup were obtained at 60˚C for 4 min. The cycle sequencing product was then using PAUP beta ver. 4.0 b10 (Swofford, 2000). HKY85 precipitated in 1 µl 3 M sodium acetate (pH 4.6) and 25 µl evolution model was used to generate the pairwise 100% ethanol before being analyzed in ABIPRISM ™ 310 distances. This pairwise distance matrix (Table 3) Genetic Analyser. Electropherogram data were edited by was then used to generate phylogenetic tree using the comparing forward and reverse readings using Sequence Neighbor-joining (NJ) approach (Saito and Nei, 1987) in 144 Radhiah ZAKARIA, Choong CHEE YEN and Ibrahim FARIDAH-HANUM

Table 3. Pairwise distances of the trnL-trnF spacer among taxa in the families Guttiferae. 1 2 3 4 5 6 7 8 9 10 11 01 G. malacensis − 02 G. opaca 0.00578 − 03 G. parvifolia 0.01746 0.01154 − 04 G. atroviridis 0.01453 0.00865 0.00863 − 05 G. nervosa 0.00868 0.01460 0.02350 0.01181 − 06 M. lepidota 0.15738 0.14973 0.14952 0.14589 0.15302 − 07 M. kunstleri 0.15738 0.14973 0.14952 0.14589 0.15302 0.00000 − 08 M. racemosa 0.16122 0.15345 0.15327 0.14956 0.15694 0.01997 0.01997 − 09 M. corneri 0.16872 0.16091 0.16076 0.15706 0.16424 0.02583 0.02583 0.01144 − 10 C. tetrapterum 0.19179 0.18388 0.19144 0.18031 0.18623 0.10352 0.10352 0.11328 0.12023 − 11 C. rupicolum 0.18018 0.17254 0.18005 0.16897 0.17500 0.10616 0.10616 0.10937 0.11642 0.03229 − 12 C. soulattri 0.16887 0.16116 0.16861 0.15760 0.16368 0.08384 0.08384 0.08711 0.09400 0.03785 0.03765 13 C. depressinervosum 0.13926 0.13180 0.13894 0.12826 0.13824 0.04975 0.04975 0.04664 0.05312 0.06946 0.06332 14 C. macrocarpum 0.13926 0.13180 0.13894 0.12826 0.13824 0.04975 0.04975 0.04664 0.05312 0.06946 0.06332 15 C. dioscurii 0.13910 0.13166 0.13883 0.12813 0.13813 0.05272 0.05272 0.04960 0.05611 0.07237 0.06048 16 C. wall. var. incrass 0.15275 0.14500 0.15235 0.14135 0.15158 0.05666 0.05666 0.05962 0.06649 0.06461 0.05257 17 C. wall. var. wallich 0.13910 0.13166 0.13883 0.12813 0.13813 0.05272 0.05272 0.04960 0.05611 0.07237 0.06048 18 Mesua ferrea 0.13549 0.12817 0.13170 0.12454 0.13149 0.04642 0.04642 0.04345 0.04357 0.07170 0.06820 19 Mammea odorata 0.14292 0.13543 0.13517 0.13160 0.13892 0.04654 0.04654 0.04361 0.04978 0.09384 0.09011 20 M. brevipe 0.17541 0.16750 0.16733 0.16369 0.17096 0.07725 0.07725 0.07436 0.08076 0.13057 0.12663 21 M. siamense 0.14292 0.13543 0.13517 0.13160 0.13892 0.04654 0.04654 0.04361 0.04978 0.09384 0.09011 22 Ternstroemia impressa 0.32944 0.32263 0.31237 0.30700 0.31601 0.29568 0.29568 0.28505 0.29558 0.37189 0.36411

12 13 14 15 16 17 18 19 2 21 22 10 C. tetrapterumn − 13 C. depressinervosum 0.03510 − 14 C. macrocarpum 0.03510 0.00000 − 15 C. dioscurii 0.03786 0.00263 0.00263 − 16 C. wall. var. incrass 0.03001 0.01064 0.01064 0.00796 − 17 C. wall. var. wallich 0.03786 0.00263 0.00263 0.00000 0.00796 − 18 Mesua ferrea 0.04697 0.00869 0.00869 0.01147 0.02066 0.01147 − 19 Mammea odorata 0.06212 0.03198 0.03198 0.03488 0.04472 0.03488 0.02873 − 20 M. brevipes 0.09719 0.06245 0.06245 0.06546 0.07294 0.06546 0.05877 0.03453 − 21 M. siamense 0.06212 0.03198 0.03198 0.03488 0.04472 0.03488 0.02873 0.00000 0.03453 − 22 Ternstroemia impressa 0.34595 0.30113 0.30113 0.29604 0.31016 0.29604 0.29569 0.28657 0.30792 0.28657 −

PAUP. PAUP was also employed to construct Maximum carried out with 500 replicates. Only those groups with Parsimony (MP) cladograms by applying unordered greater than 50% frequency were reported. parsimony (Fitch, 1971) with equal weight. Heuristic search with tree bisection-reconnection (TBR) branch swapping algorithm, random stepwise addition and RESULTS ‘MulTrees’ option were set. In Maximum Likelihood Characteristics of the trnL-trnF sequences (ML) analysis, the HKY85 evolution model was selected The pairwise sequence divergence of the trnL-trnF ranged based on the transition/transversion (ti/tv) ratio and from 0 to 19.2% among the ingroup taxa whilst between gamma distribution. To assess the internal support of the the ingroup and outgroup taxa it ranged from 28.5 to phylogenetic trees (NJ, MP and ML), statistical analysis 37.2% (Table 3). This level of variation was sufficiently by the bootstrap method of Felsenstein (1985) was high for a non-coding chloroplast region to be used in Systematic study on Guttiferae Juss. of Peninsular Malaysia based on plastid sequences 145

��

�� 63 � �� 100 ��

97 �� 99 �� 87 � � ��

�� 98

69 100 �� 84 � � �� 89 � � �� 100�� 95 � ��

��

�� 62 � � 94 � ��

Fig. 1. One of the 9 equally most parsimonious trees based on the trnL-trnF sequence data. Number above branch indicates bootstrap percentage.

phylogenetic inference. Numerous length mutations used for phylogenetic analyses contained 503 characters. with sizes ranging from 1 to 24 bp were observed in the However, 103 characters were excluded from the DNA region. The occurrence of length mutations is more phylogenetic analyses. These characters included the end prominent across the genera but remained minimum portions of the DNA sequences and the two ambiguous within genus. Two zones with a poly-T motif contributing zones caused by poly-T motifs. Of the 400 characters to length mutations were found within the DNA region. analyzed, 145 characters were variable, and 75 of them However, none of the length mutations in the DNA were parsimonious informative characters. regions were scored for phylogenetic analyses as their origin and evolutionary course were uncertain. Phylogenetic relationships The alignment matrix of the trnL-trnF sequences One of the nine equally most parsimonious (MP) trees 146 Radhiah ZAKARIA, Choong CHEE YEN and Ibrahim FARIDAH-HANUM

��

�� 53 � � �� 100 � �� 97 ��

98 � �� 86 ��

�� 99��

100 �� 60 � 91 �� 55 �� 86 � 95�� 94��

��

66 �� 95 ��

Fig. 2. Maximum Likelihood tree based on the trnL-trnF sequence data. Number above branch indicates bootstrap percentage.

with a length of 184, a CI value of 0.8913 and an RI value bootstrap value (BV). M. ferrea was observed to cluster of 0.9324 is shown in Fig. 1, the Maximum Likelihood with the Calophyllum clade (BV=81−89%) with a high (ML) tree with the best score of 1482.68 is shown in Fig. bootstrap support (90−95%). All the Mammea taxa formed 2, and the Neighbour-Joining (NJ) tree is shown in Fig. 3. a well supported clade (BV=94−95%) whilst the Mesua The topologies of MP, ML and NJ are generally congruent clade and Calophyllum clade formed a sister relationship to each other, with some differences on some collapsed in MP (BV=69%) and ML (BV=60%) trees. In contrast, in branches due to poor bootstrap support particularly the NJ tree the Mammea clade and Calophyllum clade among some of the Calophyllum taxa, and the position of formed a cluster (BV=<50%). The Garcinia clade sat at the Mammea and Mesua clades. the base of the trees with high bootstrap support (100%). The phylogenetic trees (MP, ML and NJ) produced Within the Calophyllum clade, Calophyllum wallichianum 4 clades according to genus designation with high var. incrassatum and C. wallichianum var. wallichianum bootstrap support. With the exception of Mesua ferrea, did not form a cluster by themselves. C. wallichianum var. the Mesua taxa formed a clade supported by 99−98% incrassatum was found closely related to C. tetrapterum, Systematic study on Guttiferae Juss. of Peninsular Malaysia based on plastid sequences 147 �� 97 F sequence data. Number above branch indicates bootstrap percentage. �� trn �� L- � ��

trn �� 57 50 100 �� 76 � �� 98 97 81 61 90 95 98 100 . Neighbour-joining tree based on the Fig. 3 . 148 Radhiah ZAKARIA, Choong CHEE YEN and Ibrahim FARIDAH-HANUM

C. rupicolum and C. soulattri (in MP, ML and NJ) while C. phylogenetic trees has shaken the foundation of wallichianum var. wallichianum was close to C. dioscuri classifying M. ferrea and the other Mesua taxa together (in NJ). under a single genus. Based on the rbcL sequences, Kayea and Mesua formed a clade with low support (Gustafsson et al. 2002). However, the phylogenetic DISCUSSION relationship between Kayea and Mesua in the study was The amount of variation in the trnL-trnF sequences far from conclusive due to the limited number of Kayea among the genera in Guttiferae was unexpectedly taxa used in the phylogenetic inference. Morphologically, high with an observed pairwise sequence divergence M. ferrea is different from the other Mesua taxa. The between genera up to 19.2%. This should generate crown of M. ferrea is entirely whitish pink with a new sufﬁcient variation/characters for phylogenetic analyses, flush of drooping young leaves for a few days several even though the trnL-trnF region is relatively short times in a year, whereas the other Mesua taxa do not (400−490 bp) in these taxa. Inclusion of other Guttiferae/ have this characteristic. The leaf of M. ferrea is intensely Hypericaceae genera such as Ploiarium and Cratoxylum white below (glaucous) with shiny pale brown above in the alignment has been attempted, however, this and the leaf nerves are indistinct on both sides with was unable to produce sufficiently confident alignment unrecurved margin. In contrast, the other Mesua taxa do (unpublished data). We believe that the non-coding not have glaucous or shiny leaves, but the leaf margin is trnL-trnF region has undergone rapid evolution among slightly recurved with distinct nerves on both sides of the genera in the family Guttiferae/Hypericaceae. We, the leaf. The current proposed genus Mesua is actually a therefore, decided to exclude Ploiarium and Cratoxylum combination of taxa between Mesua ferrea and the closely from our phylogenetic analyses and focused on 4 related former genus Kayea. Kostermans (1969) merged genera (Calophyllum, Mammea, Mesua and Garcinia) of Kayea under Mesua based on the number of seeds in the Guttiferae. fruit cell. Consequently, all the taxa previously identified The MP, ML and NJ analyses based on the trnL-trnF as Kayea have been renamed as Mesua. This taxonomic sequences produced very consistent phylogeny. Garcinia, treatment of Mesua/Kayea received strong support by Calophyllum, Mesua (except M. ferrea) and Mammea many authors (Whitmore, 1973; Keng, 1978; Corner, 1988; were successfully circumscribed as different individual Chua, 1995; Kochummen, 1997) whilst others expressed clades. Calophyllum, Mesua and Mammea were found their disagreement (Stevens, 1993; Turner, 2000). In to cluster together indicating their close relatedness as fact, Bentham and Hooker (1862), Ridley (1910 and these genera are from the subfamily Kielmeyeroidea and 1922) and Melchior (1964) used generative characters to tribe Calophylleae. On the other hand, Garcinia is from distinguish Kayea and Mesua. The generative characters the subfamily Clusioidea and tribe Garcinieae. Members appear to be more consistent than the fruit character in of the subfamily Kielmeyeroidea have cotyledons of more Mesua and Kayea. The molecular phylogeny based on than 1/3 length of the seed with superficial phellogen in the trnL-trnF sequences indicated that M. lepidota, M. stem and in deep-seated root. In the subfamily Clusioidea, kunstleri, M. racemosa and M. corneri (all formerly placed however, veins are usually embedded with phellogen in in Kayea) are distinct from M. ferrea. The separation of stem, and the root is usually superficial. Members of the the Mesua and other Mesua taxa (Kayea) was strongly tribe Calophyllea have terminal buds usually functional supported by bootstrap values in the ML, MP and NJ and a large embryo predominantly made up of two trees. Therefore, we strongly believe that Mesua and swollen cotyledons (Stevens, 1980). On the other hand, Kayea are two distinct genera. The molecular phylogeny members of the tribe Garcinieae have terminal buds supports the classification of Stevens (1993) and Turner which are usually eperulate and a large embryo almost (2000) to separate Kayea from Mesua. Consequently, we entirely made up of swollen hypocotyls. Therefore, recommend the reinstall of genus Kayea, and the transfer it is clear that Garcinia is distantly related to Mesua, of Mesua species other than M. ferrea, back to the genus Calophyllum and Mammea, and its position at the base of Kayea. the phylogenetic trees is expected. Henderson and Wyatt-Smith (1956) treated Mesua ferrea was found to cluster with the Calophyllum wallichianum and C. incrassatum as two Calophyllum clade while the rest of the Mesua taxa distinct species whereas Stevens (1980) placed C. grouped together in one clade (Mesua clade). The incrassatum and C. wallichianum as varieties of C. exclusion of M. ferrea from the Mesua clade in the wallichianum based on petiole and twig characters. Systematic study on Guttiferae Juss. of Peninsular Malaysia based on plastid sequences 149

Stevens (1980) argued that the limits in C. wallichianum important families. and C. incrassatum complex have been unclear. Henderson and Wyatt-Smith (1956) also realized that C. ACKNOWLEDGEMENTS We would like to thank wallichianum has a separated distribution in northern Dr. Nur Supardi and Pasoh staff for allowing us to collect and western (Kedah, Penang and Perak), and southern samples and use the field station in Pasoh Forest Reserve and eastern (Trengganu, Johore and Singapore) regions and the Bogor Botanical Garden for providing silica-dried of Peninsular Malaysia. The northern population of leaf materials and herbarium specimens of Mammea C. wallichianum is clearly intermediate between the odorata and M. siamense. We also thank Dr. Jennifer southern C. willichianum population and C. incrassatum. Ann Harikrishna for reading the manuscript and giving Nevertheless, the molecular phylogeny did not show valuable comments. The first author would like to thank the clustering of C. wallichianum var. wallichianum SEAMEO-SEARCA for financial support during her two- and C. wallichianum var. incrassatum. Instead, C. year study in Universiti Putra Malaysia. wallichianum var. inscrassatum was found to be closely related to C. tetrapterum, C. rupicolum and C. soulattri while C. wallichianum var. wallichianum was close to C. REFERENCES dioscuri. Generally, C. wallichianum var. wallichianum Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., and C. wallichianum var. incrassatum show distinct leaf Zhang, Z., Miller, W. & Lipman, D.J. 1997. Gapped morphology. The leaf of Calophyllum wallichianum var. BLAST and PST-BLAST: a new generation of protein wallichianum frequently has folding on falling and the database search programs. Nucleic Acids Research, inner bark is red with white, darkening to yellowish- 25: 3389−3402. cream, exudates. On the other hand, the leaf of C. Bentham, G. & Hooker, J.D. 1862. Hypericineae, wallichianum var. incrassatum does not have folding on Guttiferae. In: Genera Plantarum, Vol. 1 (eds. falling and the inner bark is yellow with cloudy white Bentham, G. & Hooker, J.D.), pp. 163−177. Reeve & exudates turning slightly yellowish on exposure. Thus, Co. London. these two taxa differ from each other morphologically to Chua, L. 1995. Mesua. In R. H. M. J. Lemmens, I. a certain extent and the molecular data does not support Soerianegara & W. C. Wong, eds., Timber Trees: their monophyly. Therefore, we prefer to follow the Minor Commercial Timber. Plant Resources of treatment of Henderson and Wyatt-Smith (1956) and to South-East Asia No. 5(2), pp. 339−345. Prosea treat the two taxa as distinct species of C. wallichianum Foundation, Bogor, Indonesia. and C. incrassatum. Corner, E.J.H. 1988. Wayside Tress of Malaya. Vol. 1 (3rd ed.). The Malayan Nature Society, Kuala Lumpur, 349 pp. CONCLUSIONS Doyle, J.J. & Doyle, J.L. 1987. A rapid DNA isolation The trnL-trnF sequences were informative and were procedure for small quantities of fresh leaf material. able to solve the taxonomic problems within the family Phytochemistry Bulletin, 19: 11−15. Guttiferae. Based on the molecular phylogeny, we agree Doyle, J.J. & Doyle, J.L. 1990. Isolation of plant DNA from with Stevens (1993) and Turner (2000) to reinstate the fresh tissue. Focus, 12: 13−15. genus Kayea and place Mesua lepidota, M. kunstleri, M. Felsentein, J. 1985. Confidence limit on phylogenies: racemosa and M. corneri within Kayea. The status of an approach using the bootstrap. Evolution, 39: Mesua ferrea, however, remains unchanged. We suggest 783−791. that the taxonomic rank of Calophyllum wallichianum var. Fitch, W.M. 1971. Toward defining the course of wallichianum and C. wallichianum var. incrassatum be evolution: minimum change for specific tree changed from variety to species as C. wallichianum and topology. Systematic Zoology, 20: 406−416. C. incrassatum. Nevertheless, we feel further analyses Gustafsson, M.H.G., Bittrich, V. & Stevens, F. 2002. of additional molecular makers from different genes or Phylogeny of Clusiaceae based on rbcL sequences. genomes are necessary to give a clearer picture of the International Journal of Plant Science, 163: evolution of the family Guttiferae. The inclusion of other 1045−1054. genera from Guttiferae and taxa from Hypericaceae (a Henderson, M.R. & Wyatt-Smith, J. 1956. Callophyllum closely related family) in future studies will be able to Linn. The Gardens Bulletin Singapore, 15: 285−376. shed light on the evolution within and between these two Jones, S.W. 1980. Morphology and Major Taxonomy 150 Radhiah ZAKARIA, Choong CHEE YEN and Ibrahim FARIDAH-HANUM

of Garcinia (Guttiferae). Ph. D. Dissertation, coding regions of chloroplast DNA. Plant Molecular University of Leicester, London. Biolology, 17: 1105−1109. Keng, H. 1969. Orders and Families of Malayan Seed Thompson, J.D., Higgins, D.G. & Gibson, T.J. 1994. Plants. University of Malaya Press. Kuala Lumpur, CLUSTAL W: improving the sensitivity of 429 pp. progressive multiple sequence alignment through Keng, H. 1978. Theaceae. In: Tree Flora of Malaya. Vol. 3. sequence weighting, position specific gap penalties A Manual for Forester (ed. Ng, F.S.P.), pp. 275−296. and weight matrix choice. Nucleic Acids Research, Longman, Singapore. 22: 4673−4680. Kochummen, K. M. 1997. Tree Flora of Pasoh Forest. Turner, I.M. 1995. A catalogue of the vascular plants of Malayan Forest Records No. 44. Forest Research Malaya. National Park Board. Singapore Botanic Institute Malaysia, Kuala Lumpur, 462 pp. Garden. The Gardens Bulletin Singapore, 47: 1−265. Kostermans, A.J.G.H. 1961. A monograph of the Asiatic Turner, I.M. 2000. The taxonomy of Malaysian vascular and Pacific species of Mammea L. (Guttiferae). plants: new taxa (1996−2000) and endemic genera. Pengum. Lemb. Pusat Penjel. Kehut. Indonesia, No. Folia Malaysiana, 2: 41−82. 72, 63 pp. Bogor. Vestal, P.A. 1937. The significant of comparative anatomy Kostermans, A.J.G.H. 1969. Kayea Wall. and Mesua L. in establishing the relationship of the Hypericaceae (Guttiferae). Reinwardtia, 7: 426−431. to the Guttiferae and their allies. The Philippine Linnaeus, C. 1753. Species Plantarum (1st ed.). 111pp. Journal of Science, 64: 199−256. Melchior, H. 1964. Guttiferae. In A. Engler. Syllabus Wallich, N. 1832. Plantae Asiaticae Rariores, 3: 5, t. 10. der Pflanzenfamilien (12nd ed.). Vol. 2, 170−173. Whitmore, T.C. 1973. Guttiferae. In T. C. Whitmore, Gebrüder Borntraeger, Berlin. ed. Tree Flora of Malaya. Vol. 2, 162−236. Malayan Nazre, M. 2000. Ecology and Systematics Studies of Forest Record No. 26. Longman, Hong Kong. Garcinia L. (Guttiferae) in Pasoh Forest Reserve, Negeri Sembilan, Malaysia. MSc. Thesis. Faculty Received 26th Aug. 2004 of Science and Technology. Universiti Kebangsaan Accepted 09th June 2006 Malaysia, 191 pp. Planchon, J.E. & Triana, J. 1862. Memoire sur la famille des Guttiferes. Ann. Sci. Nat. Bot. ser 4, 16: 263−308. Ridley, H.N. 1910. New or rare Malayan plants. J. Straits Branch R. A. Soc. No. 54. ser. V. Ridley, H.N. 1922. Flora of Malay Peninsula. Vol. 1. Lovell Reeve, London, 912 pp. Saitou, N. & Nei, M. 1987. The Neighbor-Joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406−425. Stevens, P.F. 1980. A revision of the old world species of Calophyllum (Guttiferae). Journal of the Arnold Arboretum, 61: 117−424. Stevens, P.F. 1993. A new species and new combination in Clusiaceae−Calophylloideae from New Guinea. Telopea, 5: 359-361. Stevens, P.F. 2005. Angiosperm phylogeny website. Version 6. http://www.mobot.org/ MOBOT/ research/Apweb/. Swofford, D.L. 2000. PAUP＊: Phylogenetic Analysis Using Parsimony (＊and other methods). Version 4. Sinauer, Sunderland, USA. Tarbelet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991. Universal primers for amplification of three non-