PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES 31Malays. Appl. Biol. (2007) 36(1): 31–40

* To whom correspondence should be addressed.Present address: Faculty of Forestry, Universiti PutraMalaysia, 43400 Serdang, Selangor, Malaysia.

PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATEDGARCINIA SPECIES WITH SOME WILD RELATIVES

M. NAZRE*, M.M. CLYDE and A. LATIFF

School of Environmental & Natural Resource Sciences,Faculty of Science & Technology,

Universiti Kebangsaan Malaysia 43600 BangiE-mail: nazre@putra.upm.edu.my

ABSTRACT

Garcinia is known for their edible fruit and a number of species from the wild have been selected and cultivated notjust for their fruits but also for other uses such as vegetables and traditional medicine. Phylogenetic relationships offive locally cultivated and 12 wild species of Garcinia were determined using the sequence of the internal transcribedspacer region (ITS). Sequence analysis generated by both Parsimony and Bayesian inference resulted in almost identicaltree topology containing five main monophyletic clades. The monophyly of these clades are congruent with previoustaxonomic classification although a few clades require more representative samples to give clearer picture. Garciniacowa (kandis) and G. atroviridis (asam gelugor) clustered within sect. Brindonia, G. hombroniana (beruas) and G.mangostana (mangosteen) fall within clade of sect. Garcinia and G. prainiana (kecupu) exhibit as basal clade of sect.Xanthochymus.

ABSTRACT

Garcinia telah dikenali kerana buahnya yang boleh dimakan dan terdapat beberapa spesies liar yang telah dipilihdan dijadikan tanaman bukan sahaja kerana buahnya bahkan juga kerana kegunaannya sebagai sayuran dan ubatantradisi. Pertalian filogenetik sebanyak lima spesies yang ditanam dan 12 spesies liar telah ditentukan denganmenggunakan jujukan kawasan yang ditrankripsi dalaman (ITS). Analisis jujukan yang dijana daripada kaedahParsimoni dan inferens Bayes menghasilkan pohon filogeni yang agak serupa topologinya dengan lima klad utama.Monofili klad ini adalah sejajar dengan pengelasan taksonomi yang telah dilakukan walaupun terdapat sebilangankecil klad yang memerlukan lebih banyak wakil spesies bagi memperjelaskan keadaan. G. cowa (kandis) dan G.atroviridis (asam gelugor) terkumpul di dalam seksyen Brindonia, G. hombroniana (beruas) dan G. mangostana(manggis) tergolong dalam klad seksyen Garcinia dan G. prainiana (kecupu) menunjukkan sebagai klad dasar iaitudi dalam seksyen Xanthochymus.

Key words: Phylogenetic relationships, Garcinia species, ITS

INTRODUCTION

Garcinia is a pan-tropical genus that belongs tothe family Guttiferae (Clusiaceae) distributedmostly in the South East Asian region (Stevens,2001). Garcinia species are small to medium-sizedtrees up to 50 m tall and a few are shrubs. Mostof the species produce edible fruits and the mostfamous species is G. mangostana or mangosteen(or locally known as ‘manggis’). In Peninsular

Malaysia there are 49 Garcinia species out of 350species estimated worldwide (Whitmore, 1973;Stevens, 2001). The most common cultivatedspecies are G. atroviridis (asam gelugor), G. cowa(kandis), G. hombroniana (beruas), G. prainiana(kecupu) and G. mangostana (manggis). Apartfrom mangosteen, only a few species arecultivated, either for fruits, vegetables, traditionalmedicines or other domestic uses such as forlandscaping. Sometimes the fruits are preserved orused as added ingredients in local dishes, forexample G. atroviridis, G. cowa and G.hombroniana. In traditional medicine, Burkill(1935) has recorded that Garcinia was used as

32 PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES

after childbirth medication, for menstrualproblems, dysentery and fever. Pharmacologicalinvestigations have shown that some Garciniaspecies contain properties that have potential astreatment for HIV (Rukachaisirikul et al. 2003)or cancer (Nabandith et al., 2004). Previously,there were two significant taxonomic studiesconducted inferring the relationships andphylogeny of Garcinia. The first was by Jones(1980) who established the phylogeny of sectionsin Garcinia using cladistic theory (without anyanalysis) from her taxonomic revision on sectionsin Garcinia. In her treatment, she proposed toclassify Garcinia into 14 sections based onmorphological and pollen characters. Herclassification was reviewed and tested by Rismita(2000) using molecular methods, in particularsequencing data of internal transcribed spacer(ITS) region. Most of Rismita’s (2000) specieswere of Indonesian and Australian origins and herresults (classifications) were in congruence withJones’s (1980). Rismista (2000) however, proposedto elevate some of Jones’s sections to subgenera.Nonetheless, since both works from Jones andRismita remained unpublished, taxonomically,they are invalid to use.

Phylogenetic study of some species in Garciniawas conducted by Yapwatannaphun et al. (2004)using the same regions (ITS sequences) used as inRismita (2000), mainly to investigate therelationship of the mangosteen (G. mangostana)with the wild relatives. The study found that,apart from G. malaccensis, three other wildspecies, i.e. G. hombroniana, G. rostrata and G.celebica were closely related to mangosteen. TheYapwatannaphun et al. (2004) study was basicallyan attempt to test the theory on the origin ofmangosteen based on Richards (1990), whoproposed that mangosteen arose fromhybridisation events of two wild relatives, G.malaccensis (female) and G. hombroniana (male).

The objective of this study is to investigate thephylogenetic relationship between some of thewild species and locally cultivated Garciniaspecies. The results are expected to yield basicinformation that may be useful, especially forfuture genetic manipulation or tree improvementof the cultivated species. Moreover, the data willadd to existing knowledge on the relationships ofwild Garcinia species because there is a hugepotential for wild Garcinia to be exploited as fruittrees since most of the Garcinia fruits are edible.As with the previous studies, the internaltranscribed spacer (ITS) region in the nuclearribosomal DNA has been chosen because it hasproven to contain enough genetic information andis an important locus for phylogenetic studies inangiosperms (Baldwin et al., 1995) and especially

in Garcinia (Rismita, 2000; Yapwatannaphun,2004).

MATERIALS AND METHOD

Plant MaterialsFresh leaves were collected from five locally

cultivated Garcinia and 12 wild species which weremostly located in Pasoh Forest Reserve, NegeriSembilan (Table 1). The identification for wildGarcinia species was based on the works of Nazre(2000). Additionally, two sister taxa in the samefamily as Garcinia (family Guttiferae), namelyMammea brevipes and Mesua racemosa wereselected as outgroup species. The outgroup specieswere selected based on the cladistic analysis ofGustaffson et al. (2002) and availability of thespecies in Peninsular Malaysia. Table 1summarised the species, locality collected and thevoucher numbers of the specimens used in thisstudy.

DNA IsolationDNA was extracted following the method of

Doyle and Doyle (1990) with minor modificationsas in Kamiya et al. (1998) using CTAB(Cetyltriammonium bromide) extraction buffer.The quality of DNA was determined using 1%agarose gel electrophoresis and its concentrationby comparing DNA with λHind III marker.

PCR Amplification and DNA SequencingThe ITS amplification was performed in the

reaction mixture (25 μl) containing 1.0 μl (10 ng)template DNA, 10x PCR buffer (with 15mMMgCl2), 2.0 mM dNTP mixture (200 μM each ofdATP, dTTP, dGTP and dCTP) and 0.2 μl ofAmpliTaq® DNA polymerase (Boehringer®). Allsamples were amplified using the primers of“ITS4” and “ITS5” (5 pmol each) (White et al.1990) and subjected to 30 cycles of amplification(1 min of denaturation at 94ºC, 1 min ofannealing at 48ºC and 2 min of extension at72ºC). Amplification products were purified byGENECLEAN III KIT (BIO 101) following thesupplier’s instructions.

1 μl (10-100 ng) of double-stranded PCRproduct was then sequenced by dideoxy chainterminator method using the ABIPRISMTM BigDye Terminator Cycle Sequencing ReadyReaction Kit (Applied Biosystem Inc., USA). Thereaction mixture contained the same primers asfor the PCR (1 μl of 3.2 pmol each), 4 μl ofterminator ready reaction mixture and 4 μl doubledistilled water. Cycle sequencing was done bothfor forward and reverse directions subjected to 25cycles of denaturation at 96ºC, annealing at 50ºC

PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES 33

and extension at 60ºC. The sequence reading wasperformed using ABIPRISMTM 310 GeneticAnalyser, and the raw sequence data was analyzedwith Sequence AnalysisTM software. Electro-phoregram data was checked manually usingSequence NavigatorTM and compared for bothforward and reverse readings.

Sequence AnalysisAll sequence data were aligned by CLUSTAL

X (Thompson et al., 1997), corrected and refinedmanually using BIOEDIT, compared with othersequences in the GenBank by BLAST programme(Altschul et al., 1997) to determine the boundariesand confirm the correct sequences obtained.

The combined data set of ITS1, ITS2 and 5.8Swas then analysed using the optimality criterionof maximum parsimony method to get thephylogenetic relationship using PAUP Ver. 4.0b10(Swofford, 2002). Parsimony analyses werecarried out by applying the unordered Fitch(1970) parsimony with Branch and Boundalgorithm with the ACCTRAN, STEEPESTDESCENT and COLLAPSE command in effect.Insertions and deletions introduced in thealignment were treated as gaps and were notincluded in the phylogenetic analysis. Pair-wisedistances within the in-groups and between andthe out-group sequences were also performedusing PAUP, and to test the significance of themonophyletic groups in the phylogenetic tree,

statistical analysis by bootstrapping method(Felsenstein, 1985) with 1000 replications wascarried out.

Additionally, an alternative method forestimating phylogenetic relationship usingBayesian analyses, were performed. Bayesiananalyses were carried out using MrBayes 3.00(Ronquist and Huelsenbeck, 2003) to generateposterior probability distribution using MarkovChain Monte Carlo (MCMC) methods. Priorprobabilities were set as default setting and thedata was run for 10 million generation with fourMCMC chains running simultaneously. Thephylogenetic trees produced from BayesianInference were summarised by calculating the 50%majority-rule consensus tree after excluding the‘burn-in’ trees. The burn-in trees were estimatedby plotting (plot command) the ‘lnlikelihood’against number of generations. The posteriorprobabilities produced are used as an estimationof the degree of robustness for the cladesproduced.

RESULTS

Characterisation of ITS SequencesThe ITS region ranged from 617 to 624 bp in

17 species of Garcinia but the length was longerfor the two out-group taxa with 631 and 640 bprespectively (Table 2). For all taxa studied, the

Table 1. Accession and GenBank numbers of taxa used. Species with asterisks indicate the outgroup species

Species

1. Garcinia atroviridis Griff. ex T. Anders.2. Garcinia bancana (Miq.) Miq.3. Garcinia cowa Roxb.4. Garcinia hombroniana Pierre5. Garcinia mangostana L.6. Garcinia nigrolineata Planch. ex T. Anders7. Garcinia parvifolia (Miq.) Miq.8. Garcinia prainiana King9. Garcinia scortechinii King10. Garcinia urophylla Scort. ex King11. Garcinia globulosa Ridl.12. Garcinia nervosa Miq.13. Garcinia griffithii T. Anders.14. Garcinia forbesii King15. Garcinia rostrata (Hask.) Miq.16. Garcinia penangiana Hook f.17. Garcinia opaca King var. minor18. Mammea brevipes (Craib) Kosterm.*19. Mesua racemosa (Planch. & Triana) Kosterm.*

GenBankAccessionNumber

AF367211AF367212AF367213AF367214AF367215AF367216AF367217AF367218AF367219AF367220AF367221AF367222AF367223AF367224AF367225AF367226AF367227AF367228AF367229

VoucherNo.

GA01MN252GC01GH01GM01MN 90MN 109GP01MN 258SK01MN 329MN 97MN 71MN 259MN 100MN 68MN 79AZ 6337ARK 011

Locality Collected

UKM Germplasm Collection, Selangor.Pasoh Forest Reserve, Negeri Sembilan.UKM Germplasm Collection, Selangor.UKM Germplasm Collection, Selangor.UKM Germplasm Collection, Selangor.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.UKM Germplasm Collection, Selangor.Pasoh Forest Reserve, Negeri Sembilan.Fraser Hills, Pahang.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.Pasoh Forest Reserve, Negeri Sembilan.Langkawi Island, Kedah.Pasoh Forest Reserve, Negeri Sembilan.

34 PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES

aligned ITS 1 region (258-261 bp in the in-groupand 251-257 in the out-group) were longer thanITS 2 (199-205 bp in the in-group and 221–224in the outgroup). Sequence lengths in the aligned5.8S region were shorter in the in-group anduniform in size for the out-groups. When all thesequences were aligned, the total aligned lengthwas 702 bp.

Amongst the in-groups and out-groups, theITS region was observed as a GC-rich region. Inthe in-groups, the values of G+C contents werevaried, 47.49%–53.28% in ITS1, 47.50%–54.00%in ITS2 and 52.20%–54.66% in 5.8S. However, theGC contents within out-groups taxa were slightlyhigher in ITS1 (53.78%–63.42%) and ITS2(52.09%–63.39%), respectively, but much lower in5.8S (36.58%–46.21%).

The alignment of all sequences resulted in amatrix of 702 characters. Of these, 9 characters atthe end of the ITS2 sequence region were excludedfrom the analysis because of alignmentambiguities. A total of 175 (24.7%) characterswere phylogenetically informative and 416(58.7%) characters were identical.

The sequence divergence between the taxaranged from 0.16% to 14.79% (Table 3). In theingroup, the smallest divergence was foundbetween G. cowa and G. nigrolineata (0.16%),while the largest was between G. rostrata and G.nervosa (14.9%).

Phylogenetic AnalysesMaximum Parsimony (MP) analysis of the

ITS dataset generated two maximallyparsimonious trees with one of two mostparsimonious tree presented here (Fig. 1). Each ofthe trees required 516 evolutionary steps or treelength with the consistency index (CI) = 0.71,retention index (RI) = 0.72 and rescaledconsistency index (RI) = 0.51.

The Bayesian inference (BI) analysis of theITS sequences produced a fifty percent majorityrule consensus tree (Fig. 2) that was almostidentical in topology as the MP analysis. The onlydifference is the position of G. grifithii and G.

forbesii clades which clustered with G. bancana,whereas in the MP tree, clade of G. bancana isclustered with G. cowa, G. nigrolineata, G.parvifolia and G. globulosa.

Generally, both MP and BI tree topologiescould be divided into five main clades, A - E.Clade A is a highly supported clade with 93%bootstrap value (BV) and 100% posteriorprobabilities (PP), where most of the speciessampled are grouped together. Using Jones’s(1980) taxonomic classification, clade Acorresponds to sect. Brindonia. Within clade A,the cultivated species of G. atroviridis exhibited asthe most distant relatives from the rest of thespecies. Another cultivated species, G. cowa, alsofell within clade A and closely related to G.nigrolineata where it formed a strongly supported(97% BV, 100% PP) monophyletic clade. The restof the wild species in clade A also exhibited amonophyletic relationship with high supportvalues, namely G. griffithii and G. forbesii (98%BV, 100% PP), and G. parvifolia and G. globulosa(98% BV, 100% PP). However, in MP analysis, thesupport values for clade of G. cowa, G.nigrolineata, G. parvifolia and G. globulosa isweakly supported (<60% BV) and within BIanalysis, support values for the monophyly ofclade G. griffithii, G. forbesii and G. bancana isweakly supported (< 60% PP).

Clade B is a highly supported group with twomonophyletic sub-clades; firstly, a moderatesupported group (62% BV) from MP but stronglysupported from BI, the clade of G. hombronianaand G. opaca. Secondly, a strong supported clade(91% BV, 100% PP) of G. mangostana and G.penangiana from both MP and BI analyses. CladeB is congruent with Rismita’s (2000) findingwhich confirmed section Garcinia sensu Jones(1980).

Within clade C, both analyses of MP and BIshow that Clade C is sister to clade B with strongsupport values (100% BV, 100% PP). AlthoughClade C is only represented by two species, G.scortechinii and G. urophylla, it supported Jones(1980) classification of sect. Hebradendron.

TABLE 2. Sequence characteristic of ITS region in Garcinia species and two out-groups

ITS 1 5.8S ITS2 Total Sequence

Length Range [within in-group] (bp) 258–261 159–161 199–205 617–624

Length Range [within out-group] (bp) 251–257 159 221–224 631–640

Aligned Length [including out-group] (bp) 299 161 242 702

G+C content (within in-group) 0.4749–0.5328 0.5220–0.5466 0.4750–0.5400 0.4936–0.5267

G+C content (within out-group) 0.5378–0.6342 0.3658–0.4621 0.5209–0.6339 0.5404–0.6172

PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES 35

36 PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES

Fig. 1. One of two most parsimonious trees from Maximum Parsimony analysis of ITS region with Mammea brevipesand Mesua racemosa as outgroup species. The numbers above branches indicate branch lengths, while the numbersbelow branches indicate bootstrap values (shown for value above 65% only). Asterisks denotes cultivated Garciniaspecies.

PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES 37

Fig. 2. Fifty percent Majority Rule consensus tree resulting from the Bayesian analysis of ITS region with Mammeabrevipes and Mesua racemosa as outgroup species. Numbers above branches indicate posterior probabilities (shownfor values above 60% only).Sectional classification is based on Jones (1980) taxonomic revisional studies and asteriskdenotes cultivated Garcinia species.

The last two clades of phylogenetic tree fromMP and BI analyses show that clades D and E arenot highly supported (<60% BV, 65% PP)compared to clades A-C. However, themonophyly of G. prainiana and G. nervosa (cladeD) confirms sect. Xanthochymus sensu Jones(1980) while G. rostrata belongs to sect.Discostigma (Jones, 1980).

DISCUSSION

A total of five Garcinia species from cultivationand 12 species from the wild were successfullysequenced for the ITS region. The result is inaccordance with previous studies (Rismita, 2000;Yapwatannaphun, 2004) showing that ITS regionprovided enough variation to infer phylogenetic

38 PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES

information for Garcinia. The length of ITSsequence in this study nearly similar with thefinding of Yapwatannaphun et al. (2004) (616 to621 bp) but slightly longer than that reported byRismita (2000) (around 595 bp). Compared withother angiosperm, the length of ITS region forGarcinia found in this study was longer than thosereported in the genus Pinus (Liston et al., 1998),and the families Compositae (Baldwin, 1992) andBrassicaceae (Crespo et al., 2000). Longer lengthof ITS1 region compared to ITS2 region isconsidered as general phenomenon found inangiosperm for instance in Glycine (Kollipara etal., 1997), Compositae (Baldwin, 1992) andFouquieriaceae (Schulties & Baldwin, 1999). Asexpected both the ITS1 and ITS2 regions showmore variability than the 5.8S coding region,which is known to be a conserved region, and theresult similarly found in Yapwatannaphun (2004)and Rismita (2000).

Evidently, the monophyly of Garcinia cladesin this study follow the classification of sectionalclassifications of Jones (1980). The five sections,namely Brindonia (Clade A), Garcinia (Clade B),Hebradendron (Clade C), Xanthochymus (CladeD) and Discostigma (Clade E) are the onlysections that existed in Peninsular Malaysia from14 sections proposed by Jones (1980).

In clade A or sect. Brindonia, the position ofG. atroviridis is reasonably distant compared toother taxa. Similar topology was obtained byYapwattanaphun et al. (2004) on the position ofG. atroviridis as basal taxa with other speciesnamely G. cowa, G. schomburgkiana and G.porrecta. Garcinia atroviridis is a common speciesin lowland and hill forest and is planted by villagefolks especially for the fruits that are used inseasoning or sour relish (Burkill, 1935).Morphologically, Jones (1980) found that thestamens of G. atroviridis were unusual comparedwith other members in sect. Brindonia, arrangedin a ring as opposed to central column. However,Jones (1980) decided to included G. atroviridis intosect. Brindonia because it shared the characters ofthe anther (2-thecous), pollen (4-colporate) andfruits (fleshy). Another cultivated species in cladeA, Garcinia cowa is commonly cultivated innorthern Peninsular Malaysia for their fruits andedible leaves (Burkill, 1935; Whitmore 1973). Theidentity and taxonomic position of G. cowa meritfurther studies because of the morphologicalsimilarities to G. nigrolineata. In term ofmorphological characters, Whitmore (1973) alsofound that sterile specimens of both G. cowa andG. nigrolineata were difficult to distinguish but G.cowa flower has shorter stalk compared to G.nigrolineata. Molecular analyses also supportedthe closeness of these two species with only 0.16%

differences from their sequence divergence(Table 3) and exhibited a strongly supportedmonophyletic clade in the phylogenetic trees withonly one evolutionary change separating betweenG. cowa and G. nigrolineata (Figure 1).

In section Garcinia (clade B), G. hombronianawas found to be clustered with G. opaca butgenetically, both species are quite distant. Thesequence divergence between G. hombroniana andG. opaca at 2.9% (Table 3) while parsimony treeshows that 11 changes were separating them(Figure 1). Morphologically, G. opaca is a smalltree or shrub while G. hombroniana is a mediumto big-sized tree. The fruit of the former a flask-shaped with thin wall, while the latter is globose-shaped with thick wall. Garcinia hombroniana isnot commonly cultivated but some villagersplanted for fruits, vegetables, traditional medicinesor as ornamental trees. Yapwattanaphun et al.(2004) reported that G. hombroniana has a closerelationship with G. rostrata, G. speciosa and G.sizygiifolia. However, the identification of thesethree species is very doubtful. For instance, G.rostrata described by Yapwattanaphun et al.(2004) as having similar fruits as G. hombronianabut smaller in size does not match morphologicaldescriptions of G. rostrata described by mosttaxonomists (e.g. Ridley, 1922; Corner, 1952;Whitmore, 1973). Fruits of G. rostrata aredistinctly different from G. hombroniana especiallythe stigma is disc-like shape while G. hombronianahas elongated stigma with 4-6 lobed (Whitmore,1973; Jones, 1980). Description on G. speciosa,species found in northern Thailand and Burmaprovided by Yapwatnnaphun et al. (2003) was notsufficient but based on taxonomic literature (i.e.Wallich, 1832; Hooker, 1875), G. speciosa isperhaps a synonym to G. hombroniana. As for G.sizygiifolia, no description was provided byYapwattanaphun et al. (2004) making it difficultto asses the identification of this species. However,from morphological and taxonomic classification(e.g. Vesque, 1893; Jones, 1980; Whitmore, 1973),G. sizygiifolia is not related to sect. Garcinia (cladeB) but belongs to sect. Brindonia (Clade A).

Another sub-clade in clade B, the clade ofmangosteen (G. mangostana) is found clusteredwith G. penangiana, a common and widelydistributed species in Peninsular Malaysia. Thisresult adds more information on the wild relativesfor mangosteen because previous studies(Richards, 1990; Rismita, 2000; Yapwattanaphunet al. 2004 & 2005) found only G. hombroniana,G. malaccensis and G. speciosa as closest relatives.Based on the morphological characters,mangosteen is thought to be a hybrid speciesbetween G. hombroniana and G. malaccensis(Richards, 1990) but the parental species for

PHYLOGENETIC RELATIONSHIPS OF LOCALLY CULTIVATED GARCINIA SPECIES 39

mangosteen is still uncertain. Future studies,especially using molecular data on the origin ofmangosteen should incorporate G. penangiana andother members of sect. Garcinia sensu Jones(1980) to verify parental status of mangosteen.

Clade C (sect. Hebradendron) contains twowild species, G. scortechinii and G. urophylla. Bothspecies have a big potential for cultivation as thewhole fruits (including the wall) are edible withsweet and sour tastes. Sect. Xanthochymus (CladeD) contain G. prainiana and G. nervosa, bothspecies differ from the rest of the species in thisstudy having stout leaves nervation and roughleaves. Garcinia prainiana is commonly planted inthe villages in the east coast Peninsular for theirfruits (Whitmore, 1973) but not been sold in themarket. The monophyly of this clade is notstrongly supported and to verify the status of thisclade, further study should incorporaterepresentative species from sect. Xanthochymus.

The basal clade for the phylogenetic trees inthis study is clade E or sect. Discostigma. Eventhough not strongly supported, the phylogenetictree in this study suggests that this is the ancientgroup for Garcinia, in contrast with Jones’s (1980)finding that sect. Garcinia is the most primitivewhile sect. Discostigma is more advanced group.Because of low level of support, it is however tooearly to conclude the evolution of Garcinia beforestudying other representative species of the othersections especially on the African and Americanspecies. Because of that, further study should becarried out to infer the infrageneric evolution inthe genus Garcinia.

CONCLUSION

This study shows that molecular data hasprovided significance results to determine thephylogeny and relationship of some cultivatedGarcinia species with its wild relatives. In Garcinia,the ITS regions show that sectional taxonomicclassification by Jones (1980) is in congruencewith molecular data by exhibiting monophyleticgroups for most of the clades (sections) found inPeninsular Malaysia. For a certain cultivatedspecies like G. cowa, G. hombroniana and G.mangostana, new information generated on therelationship with wild species have been given butfor other cultivated species such as G. atroviridisand G. prainiana, more wild species which arerelated to them should be sequenced to asses theirstatus. Moreover, the inclusion of morerepresentative species will give a clearer picture onthe monophyly of sectional classification andevolution of Garcinia.

ACKNOWLEDGEMENTS

The authors would like to thank Haji AhmedZainudin Ibrahim, Sani Miran and ShamsulKhamis for their field assistance; Dr. KoichiKamiya (Ehime University, Japan) Mr B. K. Songand Dr. C.Y. Choong for their advice on technicalaspects and data analysis. This work wassupported by IRPA grant 09-02-02-0009 toUniversiti Kebangsaan Malaysia from theMinistry of Science, Technology andEnvironment, Malaysia.

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