J. AMER. SOC. HORT. SCI. 129(3):368–373. 2004. Phylogenetic Relationship of Mangosteen (Garcinia mangostana) and Several Wild Relatives (Garcinia spp.) Revealed by ITS Sequence Data Chinawat Yapwattanaphun and Suranant Subhadrabandhu1 Department of Horticulture, Faculty Agriculture, Kasetsart University, Bangkok, 10900, Thailand Chitose Honsho and Keizo Yonemori2 Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan ADDITIONAL INDEX WORDS. Garcinia, taxonomy, polyploidy, hybridism, internal transcribed spacer ABSTRACT. The phylogenetic relationships among 17 Garcinia species including G. mangostana (mangosteen) were analyzed by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Both parsimonious and neighbor joining (NJ) analyses revealed that G. mangostana is closely related to G. malac- censis believed to be a progenitor of mangosteen. Another suspected progenitor of mangosteen, G. hombroniana, was more distant from G. mangostana than G. malaccensis phylogenetically. Garcinia hombroniana formed a cluster with G. rostrata, G, speciosa and G. sizygiifolia, and this cluster was connected with a cluster of G. mangostana and G. malaccensis. The ITS sequence analysis showed that G. atroviridis, G. cowa, G. dulcis, G. malaccensis, G. mangostana, G. rostrata and G. vilersiana have nucleotide additivity (two different nucleotides at the same nucleotide position) at several sites in the ITS region. The occurrence of these species might be related to hybridization with ancestors, but the genomic compositions, even chromosome numbers, of these species are still unknown. Mangosteen (Garcinia mangostana L.) is a well-known tasty Sequence analysis of internal transcribed spacer (ITS) region tropical fruit with attractive fruit shape and color. It is reported to of nuclear ribosomal DNA (nrDNA) between the small subunit have originated in southeast Asia and is famed as the ‘queen of (18S) and the large subunit (26S) of nrDNA has been used as a tropical fruitʼ. Many visitors to southeast Asia enjoy this sweet source for analysis of phylogenetic relationships within genera and juicy fruit. In addition to its use as a dessert, the dried fruit and among closely related genera in many angiosperms (Baldwin rind, which contains tannin and xanthones, is also used for treat- et al., 1995). It frequently has been used to resolve phylogenetic ment of infl ammations, diarrhea, and dysentery in native medicine relationships among many plant taxa, such as Coffea spp. (Lash- (Yapwattanaphun et al., 2002). Mangosteen is one of the most ermes et al., 1997), Glycine spp. (Kollipara et al., 1997), Allium important economic fruit crops in Thailand, and the demand of spp. (Dubouzet and Shinoda, 1998), Asarum (Kelly, 1998), Ar- mangosteen fruit usually exceeds the supply (Richard, 1990a; temisia (Kornkven et al., 1998), Loasaceae (Moody et al., 2001), Yaacob and Tindall, 1995). and Mangifera (Yonemori et al., 2002). In this study we used a Mangosteen reproduction is very interesting because only fe- sequence analysis of ITS region of nrDNA between the small male trees have been found. Since mangosteen is apomictic, all subunit and the large subunit of nrDNA to describe the relationship mangosteen trees are thought to be clones with the same genotype. among Garcinia species to provide fundamental information for Richard (1990b) reported that G. mangostana is allopolyploid and the breeder, because it will be important for genetic improvement derived from the hybridization of G. hombroniana and G. malac- of mangosteen in the future. censis. He reported that 13 important morphological characteristics of G. mangostana are intermediate between G. hombroniana and Materials and Methods G. malaccensis. However, his hypothesis has not been confi rmed by analytical methods using molecular techniques. The origin of PLANT MATERIAL AND DNA EXTRACTION. Seventeen species mangosteen is still unclear, although it is a very important fruit of Garcinia and some accessions were collected from Thailand, crop in southeast Asia. Indonesia, Malaysia and Australia (Table 1). Mammea siamensis Mangosteen belongs to the genus Garcinia, and many Garcinia and Calophyllum inophyllum were selected as outgroup taxa. species are distributed in southeast Asia. In the tropical forest These two species belonged to the same family as Garcinia, the in Thailand, there are 22 reported Garcinia species (Smitinand, family Guttiferae, and M. siamensis was selected because it was 1980). Several of the species are important for commercial use easy to obtain in Thailand and C. inophyllum because it was such as timber, resin, or various natural products, in addition to morphologically close to Garcinia. the production of edible fruit. However, information on genetic For DNA extraction, 5 g of leaf tissue were ground into a diversity and phylogenetic relationships of these species to G. powder with liquid nitrogen in a mortar. The cetyltrimethyl am- mangostana (mangosteen) is very limited. (Yaacob and Tindall, monium bromide (CTAB) method reported by Doyle and Doyle 1995; Yapwattanaphun et al., 2002). (1987) was used for DNA isolation with a slight modifi cation as described by Schnell et al. (1995). The isolated DNA was purifi ed Received for publication 19 June 2003. Accepted for publication 18 Nov. 2003. by precipitating with a polyethylene glycol (PEG) solution (Helig This work was supported, in part, by The Royal Golden Jubilee PhD program of et al., 1992). Then, DNA was quantifi ed by visual comparison the Thailand Research Fund. 1Deceased 16 July 2003. with a DNA standard under UV light following electrophoresis 2Corresponding author; e-mail [email protected]. on a 1% agarose gel and stained with 50 ppm of ethidiumbromide 368 J. AMER. SOC. HORT. SCI. 129(3):368–373. 2004. 9464-Genet 368 3/13/04, 12:16:15 PM for 30 min. The DNA concentration was adjusted with Tris-EDTA ing the aligned sequence with previously published sequences (TE) buffer (pH8.0) to ≈50 ng·µL–1. (Baldwin 1992; Kollipara et al., 1997; Lashermes et al., 1997). AMPLIFICATION AND SEQUENCING OF ITS REGION. The entire ITS The 5.8S coding sequence was also considered for phylogenetic region, including the 5.8S nrDNA coding region, was amplifi ed analysis, although only a few variations were found among the by PCR using primers ITS-4 and ITS-5 (White et al., 1990). species examined. Reaction mixtures (100 µL) contained 10 mM Tris-HCl (pH 8.3), PHYLOGENETIC ANALYSIS. The aligned sequence data matrix 50 mM KCl, 1.5 mM MgCl2, 5% glycerol, 0.2 mM each dNTP, 25 was analyzed by a PAUP 4.0b10 program (Swofford, 2002) by pmol of each primer, 20 to 40 ng genomic DNA and 5 units of treating the gap as fi fth base. Unordered parsimony analysis with TaKaRa rTaq DNA polymerase (TaKaRa Shuzo Co. Ltd., Shiga, accelerated transformation (ACCTRAN) optimization was per- Japan). Amplifi cation was carried out in a TaKaRa PCR Thermal formed by a heuristic search procedure with simple for stepwise- Cycler MP (TP3000; TaKaRa Shuzo Co., Ltd., Shiga, Japan) for addition options, options of tree-bisection-reconnection (TBR) 5 min at 98 °C, followed by 35 cycles of 1 min at 97 °C, 30 s at branch swapping, and saving the multiple trees (MulTrees). The 57 °C and 1.5 min at 72 °C, with a fi nal run of 7 min at 72 °C. most parsimonious trees were also found by a heuristic search The amplifi ed products were then purifi ed through a spin column procedure. In addition, bootstrap analysis with 250 replications (SUPREC-02; TaKaRa Shuzo Co. Ltd., Shiga, Japan) for direct was conducted to assess the degree of support for each branch with sequencing. consensus tree option of retaining group with frequency >50%. Forward and reverse sequencing reactions were performed In the analysis, two outgroup taxa were used to root the tree. using a Big Dye Terminator ver.3 Cycle Sequencing Ready Reac- The phylogenetic reconstruction was also performed by neigh- tion Kit (Applied Biosystems, Perkin Elmer, Foster City, Calif.) bor-joining (NJ) method (Saitou and Nei 1987) with PHYLIP using ITS-2 and ITS-5, and ITS-3 and ITS-4 (White et al., 1990) (Felsenstein, 1993) after the distance matrices were calculated as sequencing primers for ITS1 and ITS2 regions, respectively. by the Kimura 2-parameter model of Dnadist in PHYLIP v.3.57 Sequencing reactions were done according to the standard pro- (Felsenstein, 1993). In the analysis, the polymorphic site from tocol provided by Perkin Elmer with the kit. Sequence analysis additivity having a nucleotide and a gap was treated as a gap at was performed with an automated DNA sequencer (ABI PRISM this site, since we could not defi ne the site for this kind of additiv- 310 Genetic Analyzer; Applied Biosystems, Perkin Elmer, Foster ity in PHYLIP. NJ tree was drawn by a Tree Draw Deck hacked City, Calif.) after the extra fl uorescent ddNTPs were removed with into Hypercard (Gilbert, 1990) from Drawgram and Drawtree Centri-Sep Spin Columns (Applied Biosystems, Perkin Elmer, programs of the PHYLIP. Foster City, Calif.). SEQUENCE ALIGNMENT. Sequence data of the ITS1, 5.8S, and Results ITS2 regions were fi rst aligned by using GENETYX-MAC 7.3 (Software Development Co. Ltd., Tokyo, Japan), and then the ITS SEQUENCING. The entire ITS region in Garcinia species alignment was checked and slight modifi cations made manually. ranged from 616 to 621 bp (Table 2). The length of ITS1 region The boundaries of ITS1 and ITS2 were determined by compar- was 254 to 257 bp and of ITS2 was 199 to 202 bp. There is no Table 1. List of Garcinia species and two outgroup taxa with their collected place. Species Collection place Accession no. G. acuminata Chantaburi , Thailand AB110796 G. atroviridis Nakon Srithammarat, Thailand AB110797 G. costata Ubon Ratchathani, Thailand AB110798 G. cowa Chantaburi , Thailand AB110799 G. dulcis accession 1 Ubon Ratchathani, Thailand AB110800 G. dulcis accession 2 Bangkok, Thailand AB110801 G.