New evidence on the origin of mangosteen (Garcinia mangostana L.) based on morphology and ITS...

RESEARCH ARTICLE

New evidence on the origin of mangosteen (Garciniamangostana L.) based on morphology and ITS sequence

M. Nazre

Received: 5 November 2013 / Accepted: 5 February 2014 / Published online: 19 February 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Mangosteen (Garcinia mangostana L.),

known as one of the most desirable tropical fruits of

Southeast Asia, has been considered as an obligate

agamospermous hybrid, thought to have arisen from

two wild species, G. celebica L. (syn. G. hombroniana

Pierre) and G. malaccensis Hook. f. However, this

putative origin was based on a misidentification of

G. malaccensis, which was confused for G. penangi-

ana Pierre. Intensive field studies and molecular

investigations based on internal transcribed spacer

(ITS) sequence data of 22 samples were conducted,

which included six samples of true G. malaccensis.

Morphological observation shows that mangosteen

highly resembles G. malaccensis, particularly in its

vegetative and fruit characters, even sharing similar

taste of ripe fruits. ITS data revealed that mangosteen

shared more than 99 % of its sequence with

G. malaccensis with a few accessions identical with

wild populations in Peninsular Malaysia. Phylogenetic

analysis revealed that clades of mangosteen are

paraphyletic per se, but monophyletic if both

mangosteen and G. malaccensis are grouped together.

This show that mangosteen and G. malaccensis are so

closely related that they should be combined together

as one species. I propose two theories on the origin of

mangosteen, first, that it is a hybrid of different

varieties of G. malaccensis, and second, that it may be

a product of multiple, superior selections from differ-

ent populations of female trees of G. malaccensis

originating in Peninsular Malaysia.

Keywords Garcinia malaccensis � Garcinia

mangostana � Peninsular Malaysia �Wild

relatives

Introduction

Mangosteen (Garcinia mangostana L.), locally known

in South East Asia as ‘‘manggis’’, is one of the most

desirable local fruits, occasionally it is even called

‘‘queen of fruits’’. The species was recognised by

Linnaeus (1753) when L. Garcin’s (1733) description

was made available to him, and, in honour of Garcin,

Linnaeus named the species as Garcinia mangostana

in the family Guttiferae (Clusiaceae). In recent years,

mangosteen has been planted in many other tropical

areas, such as Northern Australia, South America and

tropical Africa, but the major producing countries of

mangosteen are still in Southeastern Asia, namely

Thailand, Malaysia, the Philippines and Indonesia

(Cruz 2001).

M. Nazre

Laboratory of Plant Systematics, Department of Biology,

Graduate School of Science, Chiba University, 1-33

Yayoi-chu, Inage-ku, Chiba 263-8522, Japan

M. Nazre (&)

Herbarium Faculty of Forestry, Universiti Putra Malaysia

(UPM), 43400 Serdang, Selangor, Malaysia

e-mail: [email protected]; [email protected]

123

Genet Resour Crop Evol (2014) 61:1147–1158

DOI 10.1007/s10722-014-0097-2

Theories on the origin of mangosteen are rather

interesting. Richards (1990b) notes the most crucial

controversy, whether mangosteens could be found in

the wild. Historically, the only published report on the

existence of wild mangosteen was by Corner (1940),

but this was refuted by Whitmore (1973) who

re-identified specimens collected by Corner from

the state of Terengganu, Malaysia, as Garcinia

malaccensis Hook. f. In the absence of documented

wild populations, an analysis of earlier reports and

cytological information (Krishnaswamy and Raman

1949; Tixier 1953; Ha et al. 1988; Richards 1990a),

led Richards (1990b) to suggest that apomictic

mangosteen arose from hybridization between two

wild species, G. celebica L. (syn. G. hombroniana

Pierre, see Nazre (2010)) as the male parent, and

G. malaccensis as the female. His claim was consis-

tent with the work of Ridley (1922) and Whitmore

(1973), who also notes that mangosteen morpholog-

ically resembles two wild species in Peninsular

Malaysia, G. malaccensis and G. celebica. While

the identity of G. celebica or seashore mangosteen is

widely recognised, information about G. malaccensis

is not fully known, apart from information on herbar-

ium specimens, and the identity of G. malaccensis had

not thoroughly been checked by previous authors,

which suffered from a classic case of species

misidentification. For example, Ha et al. (1988)

misidentified G. penangiana Pierre from Pasoh Forest

Reserve, Malaysia as G. malaccensis for their cyto-

logical studies, and, subsequently, this information

was used by Richards (1990b) who claimed that

mangosteen was probably allopolyploid with respect

to its related taxon, G. hombroniana (= G. celebica),

and the misidentified G. penangiana. More recent

morphological, anatomical and molecular analyses

showed that G. malaccensis in Pasoh Forest Reserve

should be treated as G. penangiana (Nazre 2000,

2006; Nazre et al. 2007, 2009). Similar misapplication

of G. malaccensis for G. penangiana could also be

found in the works of Saw et al. (1991), Kochummen

(1997), Thomas (1997) and Abdullah et al. (2012).

And, with regard to the exact ploidy level for

mangosteen, numbers are uncertain, with reported

somatic chromosomal counts ranging from 56 to 96

(Krishnaswamy and Raman 1949; Tixier 1953; Kaur

et al. 1978; Ha et al. 1988; Soepadmo 1989).

Since it was widely accepted that mangosteen is a

hybrid, although attempts to backcross with

mangosteen and G. celebica/G. malaccensis have not

succeeded, Richards (1990b) speculated that mango-

steen might be an offspring of a single event, but he

also suggested that there was a possibility that the

cross might have occurred on more than one occasion.

Yapwattanaphun et al. (2004) agreed with the single

hybrid ancestor theory based on amplified fragment

length polymorphism (AFLP) data, and they sug-

gested that any genetic variation in mangosteen

resulted from somatic mutation. However, Ramage

et al. (2004), who investigated 37 accessions of

mangosteen with a randomly amplified DNA finger-

printing (RAF) technique, disagreed with the single-

origin theory and suggested two alternatives. The first

possibility was that mangosteen might have arisen

from independent hybridization events, and multiple

hybrids were selected for cultivation. And the second

view postulated that related backcrossing events

occurred between a male and a closely related female

tree of the respective species.

Although mangosteen has been considered an

obligate agamospermous apomictic, as proposed by

Richards (1990b), molecular data have proven that

there is considerable genetic variation among its

cultivars (Ramage et al. 2004; Sando et al. 2005;

Sobir et al. 2011) and stable phenotypic variation has

also been confirmed in Malaysia (Osman and Milan

2006) and Indonesia (Mansyah et al. 2010). Today in

Peninsular Malaysia, there are two types of mango-

steen that are being planted, one with normal globose

fruits and another with odd ovoid-shaped fruits,

known as masta (Osman and Milan 2006; Raziah

et al. 2007). Phenotypic differences could result from

environmental differences, geographic adaptation, or

cultural practices such as pruning techniques and the

shading of trees. Alternatively, they may have a

genetic basis via natural mutation and, if the data on

cytology are correct, chromosomal instability may

also occur and directly contribute to morphological

variation (Ramage et al. 2004). However, there is also

the possibility that mangosteen could be a facultative

apomict, in which sexual reproduction sometimes

occurs. Male individuals of mangosteen have been

reported by Burkill (1935), Idris and Rukayah (1987)

and Osman and Milan (2006). The rarity of males may

be related to the cultural practice of cultivators in

Peninsular Malaysia (and Southeast Asia in general)

who chop down any male fruit trees because they

believe they are of no benefit. A male tree reported by

1148 Genet Resour Crop Evol (2014) 61:1147–1158

123

Idris and Rukayah (1987) suffered the same fate, as

documented by a research team from the National

University of Malaysia (UKM) that visited the village

where it had been seen and found that it had been

chopped down because the villager felt it was not

worth keeping. Earlier, Burkill (1935) had also noted

that villagers in Indochina had practised the same

action.

Taxonomically, mangosteen is grouped within

Garcinia L. section Garcinia, suggesting that all

members of section Garcinia should be closely related

to mangosteen (Jones 1980; Nazre 2006; Sweeney

2008). However, Sando et al. (2005), Sobir et al.

(2011) and Abdullah et al. (2012), all showed that G.

celebica is so genetically distant as to be an unlikely

parent for mangosteen. Abdullah et al. (2012) further

noted that another wild species, G. opaca King (syn. of

G. diospyrifolia Pierre), is the closest relative of

mangosteen without realising that her data actually

demonstrated that G. penangiana (because of misi-

dentification as G. malaccensis) was the closest

relative of mangosteen. Earlier, Nazre et al. (2007)

also found that among the cultivated Garcinia of

Peninsular Malaysia, mangosteen was most closely

related to G. penangiana. Based on samples from

Thailand, Malaysia and cultivated trees in Java,

Yapwattanaphun et al. (2004) concluded that of the

17 species of Garcinia they studied, mangosteen was

more closely related to G. malaccensis than to G.

celebica. Similar results were obtained by Nazre

(2006), Sweeney (2008) and Sobir et al. (2011), where

phylogenetic trees showed that G. malaccensis is a

sister taxon to mangosteen.

It was clearly shown that two sister taxa,

G. malaccensis and G. penangiana, are the closest

species to mangosteen rather than G. celebica, but

there are no comprehensive studies to date that include

these sister taxa. This is understandable because

samples of G. malaccensis are not easily obtained,

considering its limited geographic distribution in

Peninsular Malaysia, Sumatera and Borneo, and

difficulty in identifying it in the field, especially in

the absence of flowers and fruits. As for G. penangi-

ana, it is a common species that can be found from

southern Thailand to Sulawesi and is easily identified,

except that it was long confused with G. malaccensis.

This current study was designed to provide new

evidence on the origin of mangosteen based on

morphological and molecular variation, incorporating

multiple samples of G. malaccensis and G.

penangiana.

Materials and methods

Morphological investigation was done by using her-

barium specimens kept at E, K, KEP, L, BO, SAN,

SAR (for abbreviations see Thiers), and the herbarium

of the Malaysia Agriculture Research and Develop-

ment Institute (MARDI) to compare G. mangostana,

G. malaccensis and G. penangiana, specimens of

different populations for mangosteen, G. malaccensis

and G. penangiana throughout the Malaysian forests

collected by ourselves. Voucher specimens were made

and are kept in the UPM herbarium. Important

characters for species delimitation, as mentioned by

Whitmore (1973), i.e., male flowers, fruits and veg-

etative characters, such as latex differences, were

observed and compared. In order to do that, morpho-

logical characters were examined by careful observa-

tion of specimens with the help of a hand-lens or stereo

microscope. All informations including qualitative

and quantitive measurement were scored in the

PADME database system, developed at Royal Botanic

Garden Edinburgh (RBGE).

For molecular analysis, internal transcribed spacer

(ITS) was chosen for sequencing because nucleotide

sequence variation found within the ITS region have

often been well-suited for comparing related species

(Soltis and Soltis 1998) and for broader applications,

including DNA barcoding (Li et al. 2011). In Garci-

nia, previous studies (Sari 2000; Yapwattanaphun

et al. 2004; Nazre 2006; Nazre et al. 2007; Sweeney

2008; Abdullah et al. 2012) solely based on ITS

variation have reported well-resolved phylogenetic

trees that were able to differentiate among species. My

initial DNA work was carried out in Royal Botanic

Gardens Edinburgh, and further experiments and

analyses were done in the Systematics Lab, Chiba

University, Japan. I used 23 accessions of Garcinia for

molecular analysis (Table 1), including 11 ITS

sequences obtained from GenBank. Garcinia atrovir-

idis Griff. ex T. Anders. in section Brindonia

(Thouars) Choisy was chosen as an outgroup. All

DNA samples were extracted from mature fresh

leaves preserved in silica gel by using the CTAB

(Cetyltriammonium bromide) extraction buffer

following the protocol of Doyle and Doyle (1990).

Genet Resour Crop Evol (2014) 61:1147–1158 1149

123

PCR amplification was performed by using ITS

primers as described by White et al. (1990). Cycle

sequencing was performed with an ABITM Big Dye

terminator Cycle Sequencing system (Applied Biosys-

tems Inc., Foster City, California, USA) and analysed

on the ABI3100 (Applied Biosystems Inc., USA).

Sequences were aligned with CLUSTAL X (Thomp-

son et al. 1997) and refined and checked manually by

using BIOEDIT. Analyses to find the most parsimo-

nious trees were run with PAUP* Ver. 4.0b10 for

Windows (Swofford 2003), using ‘Heuristic’ search

under the Fitch (1971) parsimony criterion where

character-state changes were weighted equally and

unordered. Two rounds of heuristic searches were

performed. The first search involves multiple repli-

cates, each of which has two stages: (1) the initial tree is

obtained by connecting the taxa one at a time by using

stepwise addition and additional taxa are then added in

random order; (2) to search for more parsimonious

trees, branches were swapped by using tree bisection

reconnection (TBR). To evaluate the confidence values

of the clades, 100,000 bootstrap replicates (Felsenstein

1985) were then carried out with simple addition of

sequences and TBR branch-swapping. All trees pro-

duced from the parsimony search were visualised via

TreeView 1.6.6 (Page 1996).

Results

Morphological variation

Morphologically, mangosteen trees are easily distin-

guishable from G. malaccensis or G. penangiana.

Based on the colour of their bark, mangosteen bark is

lighter than the dark grey brown bark of G. malaccensis

or the dark brown of G. penangiana. Leaves of

G. penangiana are a bright reddish colour when dry,

Table 1 List of species and accession used in this study

Species Locality/origin GenBank/DDBJ* no. Author/[collectors-voucher deposited]

1. G. mangostana SAM South America AJ 509214 see Gehrig et al. (2003)

2. G. mangostana PM1 Peninsular Malaysia AF 367215 see Nazre et al. (2007)

3. G. mangostana PM2 Peninsular Malaysia AB 110808 see Yapwattanaphun et al. (2004)

4. G. mangostana PM3 Peninsular Malaysia AB856024* [Shamsul Khamis SKMY10 (UPM)]

5. G. mangostana MASTA Peninsular Malaysia AB856025* [Rosslan 11 (UPM)]

6. G. mangostana JAV Java Island AB 110807 see Yapwattanaphun et al. (2004)

7. G. mangostana TH1 Thailand AB 110809 see Yapwattanaphun et al. (2004)



10. G. mangostana LAO Laos AB856023* [Newman s.n. (E)]

11. G. malaccensis MY1 East coast, Peninsular Malaysia AB856027* [Nazre BB04 (UPM)]



14. G. malaccensis MY4 North East, Peninsular Malaysia AB856026* [Nazre 23 (UPM)]

15. G. malaccensis MY5 South, Peninsular Malaysia AB856030* [Nazre 18 (UPM)]

16. G. malaccensis SUM1 Sumatra, Indonesia AB 110805 see Yapwattanaphun et al. (2004)

17. G. malaccensis SUM2 Sumatra, Indonesia AB 110806 see Yapwattanaphun et al. (2004)

18. G. malaccensis SBH Sabah, Malaysia AB856031* [Nazre SP01 (UPM)]

19. G. penangiana 1 West Peninsular Malaysia AF 367226 see Nazre et al. (2007)

20. G. penangiana 2 East Peninsular Malaysia AB856033* [Nazre BB07 (UPM)]

21. G. diospyrifolia West, Peninsular Malaysia AF 367227 see Nazre et al. (2007)

22. G. celebica Peninsular Malaysia AB856032* [Nur.03 (UPM)]

23. G. atroviridis Peninsular Malaysia AF 367211 see Nazre et al. (2007)

Asterisk denotes sequences that were submitted to DDBJ

Without asterisk sequences refers to GenBank accessions


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compared with mangosteen or G. malaccensis with

yellowish green to brown colour. Intramarginal vein is

present and located close to the leaves margin for

mangosteen and G. malaccensis but is absent on the

leaves of G. penangiana. Glandular lines which only

run in one direction from the midrib to the leaf margin,

across the secondary veins, are fine and widely spaced

for mangosteen and G. malaccensis, but G. penangiana

has very fine and closely spaced glandular lines.

Exudate or latex colour, however, is similar between

mangosteen and G. malaccensis which are both

yellow, but G. penangiana has whitish latex. The

morphological comparison of mangosteen with its

closest relatives are summarised in Table 2, and details

on the two most important characters, i.e., fruits and

male flowers, are noted below.

Male flower

Male mangosteen stamen bundles are 4-angled

surrounding the base of a prominent fungiform

pistillode (Fig. 1a, b). In contrast, male flowers of

G. malaccensis have slightly 4-angled or conical- or

cylindrical-shaped stamen bundles, either tipped by a

smaller pistillode or the pistillode is absent (Fig. 1b, c).

Garcinia penangiana (Fig. 1d) strictly lacks pistill-

odes, and its stamen bundles are somewhat shorter and

cruciform-shaped.

Fruit

The fruit colour for mangosteen and G. malaccensis

is similar, even when the fruits are ripening. When

young and unripe, the fruit is greenish yellow, then

turning reddish-pink and finally purplish black

when ripe (Fig. 2a, b). The fruits of these two

species can only be differentiated by comparing

their stigma bundles; G. malaccensis has coarse

corrugated surfaces compared to smooth surfaces

for mangosteen (Fig. 2b), except for the masta type,

with its distinctly ovoid fruit and protruding stigma

Table 2 Comparison of

important morphological

characters for Garcinia

penangiana, G. malaccensis

and G. mangostana

(mangosteen)

Characters Species

G. penangiana G. malaccensis G. mangostana

(mangosteen)

Male flowers

Petals colour No information Pinkish red Pinkish red

Pistillode Absent i. Absent Fungiform c. 5 mm

ii. Fungiform c. 2 mm

Stamens Cruciform i. Slightly 4-angled 4-angled

ii. Conical-cylindrical

Fruits

Colour (ripe) Reddish pink i. Yellowish red Purple-black

ii. Reddish pink

iii. Purple-black

Shape i. Ovoid i. Ovoid i. Ovoid

ii. Globose ii. Ellipsoid ii. Ellipsoid

iii. Globose iii. Globose

Stigma (bundles) Nodule-like surface Corrugated surface Rather smooth surface

Taste Sour Sweet–sour Sweet–sour

Leaves

Colour (dry

specimens)

Reddish brown Yellowish green to

brown

Yellowish green to

brown

Secondary nerves No intra marginal

veins

With intra-marginal

veins

With intra-marginal

veins

Glandular lines Very fine, closely

spaced

Fine, widely spaced Fine, widely spaced

Exudates White Yellow Yellow


123

bundles with their surfaces closely resembling those

of G. malaccensis but slightly smoother (Fig. 2c).

For G. penangiana, the fruit shape is ovoid or

globose with fewer stamen bundles that are widely

spaced and display a nodule-like surface, easily

differentiated from G. malaccensis or mangosteen

(Fig. 2d).

Sequence analysis

ITS sequence data showed that sequences of mango-

steen and G. malaccensis produced 617 bp of partial

sequence of ITS1 and ITS2, and a complete sequence

of the 5.8S region. The close similarity of ITS

sequences among mangosteen and G. malaccensis

Fig. 1 Variation of male flowers; a and b G. mangostana L.

with 4-angled stamen bundles and dwarf-fungiform pistillodes;

c G. malaccensis Hook. f. with conical-cylindrical stamens

bundles tipped with small pistillodes; d G. malaccensis with

slightly 4-angled stamen bundles tipped with small pistillodes

(left) and without stamen bundles without pistillodes (right);

e G. penangiana Pierre with cruciform-like stamen bundles

without any pistillodes


123

accessions can be seen in the distance matrix gener-

ated from PAUP with values ranging from only 0.16 to

0.33 % (Table 3). Nine substitution sites were

detected from 18 individuals at positions 26, 115,

180, 200, 250, 444, 490, 527 and 616 (Fig. 3). There

are four groups within mangosteens based on nucle-

otide substitutions. The first group, LAO, JAV, TH1,

TH2, TH3, PM2 and PM3, has unique M4 substitu-

tions sites, while the second group with only one

member, SA, has a deletion at M5 and unique M9

substitution sites. The third group (MASTA), how-

ever, shared nucleotide sequences with G. malaccensis

P4, and the last group, PM1, shared similar ITS

sequences with a group of G. malaccensis including

P1, P2 and P3 (Fig. 3).

For G. malaccensis, differences of base substitu-

tions are observed that may reflect differences in

geographic distribution; for instance, the group con-

taining P1, P2, and P3 all were collected from the east

coast of Peninsular Malaysia. Accession P4 (which

shares sequences with the masta form of mangosteen)

has a unique base substitution, M8, is found in

northern part of east coast of Peninsular Malaysia;

P5, collected from southern Peninsular Malaysia,

groups together with SM1 and SM2 of Sumatera

origin (but cultivated in Bogor) has a unique substi-

tution, M2; and Bornean G. malaccensis SBH from

state of Sabah, Malaysia displays the unique substi-

tution site, M7 (Fig. 3).

Phylogenetic analysis of these ITS sequences was

done by incorporating sequenced data from closely

related taxa from section Garcinia, namely G. penan-

giana, G. diospyrifolia Pierre, G. celebica, and

rooted with data from G. atroviridis (Garcinia sect.

Brindonia). The resulting cladogram (Fig. 4) showed

that clades of mangosteen and G. malaccensis formed

a monophyletic group with considerably high boot-

strap values (81 %) in parsimony analysis (Fig. 4).

However, separate clades for mangosteen and

G. malaccensis have low support from bootstrap

values because of the high degree of shared ITS

sequences among these accessions. As expected, the

closest sister taxon with this group is G. penangiana,

not G. diospyrifolia or G. celebica. All mangosteens

with the unique base substitution M4 (LAO, PM2,

PM3, TH1, TH2, JAV) clustered together in clade

Fig. 2 Variation of fruits of G. mangostana L. (mangosteen),

G. malaccensis Hook. f. and G. penangiana Pierre; a indistin-

guishable mangosteen and G. malaccensis fruits; b stigma

bundles with corrugated surfaces for G. malaccensis (left) and

smooth surfaces for G. mangostana (mangosteen); c protruding

and weakly corrugated stigma-bundle surfaces of mangosteen-

masta with a distinct ovoid shape; d widely spaced stigma

bundles in G. penangiana


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Table 3 Distance matrix from Internal Transcribed Spacer (ITS) sequences for G. mangostana (mangosteen) and G. malaccensis

1 2 3 4 5 6 7 8 9 10 11

1. G. mangostana LAO –

2. G. mangostana TH3 0.0000 –

3. G. mangostana JAV 0.0000 0.0000 –

4. G. mangostana TH1 0.0000 0.0000 0.0000 –

5. G. mangostana TH2 0.0000 0.0000 0.0000 0.0000 –

6. G. mangostana PM2 0.0000 0.0000 0.0000 0.0000 0.0000 –

7. G. mangostana PM3 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 –

8. G. mangostana PM1 0.0016 0.0000 0.0016 0.0016 0.0016 0.0016 0.0016 –

9. G. mangostana SAM 0.0033 0.0016 0.0033 0.0033 0.0033 0.0033 0.0033 0.0016 –

10. G. mangostana MASTA 0.0032 0.0016 0.0032 0.0032 0.0032 0.0032 0.0032 0.0016 0.0033 –

11. G. malaccensis MY4 0.0032 0.0016 0.0032 0.0032 0.0032 0.0032 0.0032 0.0016 0.0033 0.0000 –

12. G. malaccensis MY1 0.0016 0.0000 0.0016 0.0016 0.0016 0.0016 0.0016 0.0000 0.0016 0.0016 0.0016

13. G. malaccensis MY2 0.0016 0.0000 0.0016 0.0016 0.0016 0.0016 0.0016 0.0000 0.0016 0.0016 0.0016

14. G. malaccensis MY3 0.0016 0.0000 0.0016 0.0016 0.0016 0.0016 0.0016 0.0000 0.0016 0.0016 0.0016

15. G. malaccensis SUM2 0.0033 0.0016 0.0033 0.0033 0.0033 0.0033 0.0033 0.0016 0.0033 0.0033 0.0033

16. G. malaccensis SUM1 0.0033 0.0016 0.0033 0.0033 0.0033 0.0033 0.0033 0.0016 0.0033 0.0033 0.0033

17. G. malaccensis MY5 0.0032 0.0016 0.0032 0.0032 0.0032 0.0032 0.0032 0.0016 0.0033 0.0032 0.0032

18. G. malaccensis SBH 0.0032 0.0016 0.0032 0.0032 0.0032 0.0032 0.0032 0.0016 0.0033 0.0032 0.0032

12 13 14 15 16 17 18

13. G. malaccensis MY2 0.0000 –

14. G. malaccensis MY3 0.0000 0.0000 –

15. G. malaccensis SUM2 0.0016 0.0016 0.0016 –

16. G. malaccensis SUM1 0.0016 0.0016 0.0016 0.0000 –

17. G. malaccensis MY5 0.0016 0.0016 0.0016 0.0000 0.0000 –

18. G. malaccensis SBH 0.0016 0.0016 0.0016 0.0000 0.0000 0.0000 –

Fig. 3 Substitution sites and indels in internal transcribed spacer (ITS) sequences for G. mangostana L. (mangosteen) and

G. malaccensis Hook. f


123

A. Mangosteens in clade B (TH3) and G (SAM) did

not cluster with others due to the presence of point

mutations in their sequences. Clade C, which shared a

unique M2 base substitution, consists of G. malaccensis

from Sumatera (SM1 and SM2) and southern Peninsular

Malaysia (MY5). Clade D consists of mangosteen-

masta along with G. malaccensis P4, supported by a

64 % bootstrap value because both shared sufficient ITS

sequence homology and the unique substitution site M8.

Garcinia malaccensis MY1, MY2, MY3 and mango-

steen PM1 grouped together as clade E due to 100 %

ITS sequence similarity. This cladogram also shows

Fig. 4 One of most parsimonious phylogenetic tree from a total

of three trees generated from 621 characters of internal

transcribed spacer (ITS) sequences. Number above branch

shows bootstrap values (shown for values [ 50 %). Informative

characters = 26, tree length = 101, consistency index

(CI) = 0.9307, homoplasy index (HI) = 0.0693, retention

index (RI) = 0.8478, rescaled consistency index

(RC) = 0.7891


123

that G. malaccensis from Borneo (Sabah) is quite

different and serves as the basal clade (clade G) for other

mangosteen and G. malaccensis accessions.

Discussion

Garcinia mangostana or mangosteen is morphologi-

cally closer to G. malaccensis than to G. penangiana

based on important characters of the male flowers,

especially petal colour, presence and shape of pistill-

odes, fruit, leaf colours, and glandular line patterns.

Some of these similarities were also observed by

previous taxonomists (Corner 1940; Whitmore 1973).

ITS sequence data also supported the close relation-

ship between mangosteen and G. malaccensis in

agreement with previous studies by Yapwattanaphun

et al. (2004) and Sweeney (2008). Most mango-

steen ITS sequences closely resemble those of

G. malaccensis, with some accessions such as man-

gosteen PM1 and masta displaying ITS sequences

identical to those of certain G. malaccensis accessions

(Fig. 3). It should be noted that morphological vari-

ation can be observed within different populations of

G. malaccensis, and between mangosteen and man-

gosteen-masta, and these are translated into paraphy-

letic clades in the cladogram (Fig. 4).

Mainly based on the absence of male individuals,

Richards (1990b) and Abdullah et al. (2012) stated that

mangosteen is an obligate agamospermous species

that originated from two different species, with

G. malaccensis being one of its parents. If mangosteen

is indeed an obligate agamospermous species, this

would indicate that no sexual reproduction has

occurred since the F1 generation that initially pro-

duced it. Thus, both parental ITS sequences should be

retained throughout the succeeding obligate apomictic

generations, for example, as in Passiflora (Lorenz-

Lemke et al. 2005), and, thus, there should be

considerable heterozygosity present within mango-

steen accessions, reflecting those parental differences.

Nevertheless, the electropherograms of ITS sequences

of mangosteen accessions indicate that only one

individual displays any heterozygosity at all (mango-

steen accession TH3 at position 200; Fig. 1), suggest-

ing that it is highly unlikely that mangosteen is an

obligate apomict originated from two different species

as suggested by Richards (1990b) and Abdullah et al.

(2012).

Based on ITS sequence similarity, the monophyletic

clade that includes both G. mangostana and

G. malaccensis, and close morphological resemblance,

these two species should be grouped together into a

single species that might then be differentiated into

different botanical varieties. The present study sug-

gests that Corner’s (1940) observation of wild mango-

steen is likely valid. By uniting G. mangostana and

G. malaccensis, there are still at least two possibilities

regarding the origin of mangosteen. Firstly, mangosteen

may indeed be a hybrid between different varieties of

G. malaccensis but that, in the generations since initial

hybridization, ITS sequences have undergone a phe-

nomenon known as concerted evolution. Concerted

evolution is a molecular process that homogenizes

different loci within multigene families (Arnheim et al.

1980). It is driven by two molecular processes, gene

conversion and unequal crossing over (Koch et al. 2003)

and is only effective with sexual reproduction. Koch

et al. (2003) outlined three possibilities for the evolution

of ITS in individuals that result from hybridization

between parents with two different ITS sequences: (1)

unidirectional concerted evolution leads to the loss of

one copy and fixation of the second; (2) concerted

evolution results in new ITS sequences that represent a

mixture of the two parental sequences; (3) both parental

ITS sequences are retained in the ‘non-concerted

evolution’ alternative. Mangosteens in Clade D and E

fit the description on possibility 1 of Koch et al. (2003)

where concerted evolution occurs in a unidirectional

way, which results in only one of the parental sequences

being retained. Although there is no information on how

much sexual reproduction is needed for ITS sequences

to homogenize in mangosteen, other findings such as in

Armeria Wild. (Fuertes Aguilar et al. 1999) indicated

that concerted evolution and homogenization to one

parental sequence could occur at a very rapid rate,

within two generations after hybridization.

The only problem with this possibility is the

chances of hybridization between varieties of

G. malaccensis is hindered by the difficulty of finding

male individuals since concerted evolution required

sexual fertilization. From the herbarium specimens

studied, I only found three males out of 20 specimens.

Thomas (1997) also observed a similar rarity of males

in some wild Garcinia species in the lowland forests of

Malaysia and suggested that if a species shows a

female biased sex ratio, it is probably a facultative

agamosperm. If this assumption is a good indication of


123

the presence of agamospermy, then G. malaccensis

may be facultative agamosperm and explains the rarity

of male trees. However, more study will be needed to

confirm the presence or type of agamospermy in G.

malaccensis, and it should be noted that data on

pollination biology in Garcinia is scarce with excep-

tion for mangosteen, G. celebica and G. penangiana

(see Ha et al. 1988; Richards 1990a, b).

If sexual reproduction is rare, a second possibility is

that mangosteen has arose from the products of multiple

human selection events from various wild populations

of G. malaccensis with a strong preference for only

retaining pistillate forms. Morphological and molecular

data have shown that variation exists within populations

of G. malaccensis and conveniently can be distin-

guished by geography into accessions from the east

coast of Peninsular Malaysia (MY1, MY2 and MY3),

the northern part of the east coast of Peninsular Malaysia

(MY4), southern Peninsular Malaysia and Sumatera

(MY5, SUM1 and SUM2), and Borneo (SBH). For two

of the most widely cultivated mangosteens in Peninsular

Malaysia, the common mangosteen originated from

wild populations of G. malaccensis resembling acces-

sions MY1, MY2 and MY3, while masta likely

originated from G. malaccensis MY4 or a close relative.

Taxonomic implications

Based on these findings, clearly G. mangostana and

G. malaccensis form a single interrelated taxon. Based

on priority of publication, if this group is to be treated as

a single species, G. mangostana would take priority

over G. malaccensis and the domesticated mangosteen

could still be recognised at the varietal level. A full-

taxonomic revision of Garcinia sect. Garcinia is now

underway, including proper taxonomic treatments of

the varieties of G. mangostana.

Acknowledgments This paper is an extension of works from

the author’s Ph.D. project supervised by Prof. Dr. Toby

Pennington and Dr. Mark Newman of Royal Botanic Garden

Edinburgh (RBGE), to whom the author is indebted. Numerous

field expeditions and molecular analyses were made possible

through grant RUGS 9364500 from the University Putra Malaysia

(UPM). I would like to thanks to the curator of these herbaria; A,

K, L, P, SING, SAR and UC for the loan materials. Curator and

staff of the following herbaria during my visit: K, BM, BO, KEP,

KINA, MARDI, SAR and SAN. My special thanks to Dr. Tadashi

Kajita of Chiba University for giving permission using his lab

during my stay in Japan, which was supported by the Japanese

Society for Promotion of Science (JSPS). My personal gratitude

also goes to Prof. Emeritus Dr. Abd. Latif Mohamed (UKM) for

his comments on the manuscript, James E. Richardson (RBGE)

for his valuable advice, the Forestry Department of Peninsular

Malaysia (especially to Senior Ranger Salleh Endut for

contribution of his photograph), Dr. Jamili Nais (Sabah Parks),

John Sugau (Sabah Forestry Centre), Rosslan Yaacob, and

colleagues in UPM especially Pn Latifah Zainal Abidin,

Shamsul Khamis and Nur Asyikin Psyquay.

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