NAT. NAT. HIST. BUL L. SIAM Soc. 55(2): 307-322 ， 2007

A STUDY OF () IN THAILAND BASED ON CHLOROPLAST trnL AND h ・nG INTRON SEQUENCES

Yuttaya Yuttaya Yuyen I， Jessada Denduangboripan t?， Piya Chalermglin 3， Quentin Quentin C. B. Cron J(l and Vilaiwan Anusarnsunthorn I

ABSTRACT

Goniothalamus Goniothalamus (Annonacωe) is a large (ca. 120 species) and highly diverse genus in Southeast Southeast Asia. Numerous infrageneric (subgeneric and sectional) c1 assification systems have been been proposed in the past ， mainly on the basis of stamen characteristics and the number of ovules. ovules. To investigate the relationships of the Goniothalamus species occuπing in Th ailand ， we performed a molecular phylogenetic analysis of 20 Thai accessions using comparative

nucleotide nucleotide sequences of chloroplast trnL and trnG introns. Our sequence alignment r，巴 sulted in in a combined data-matrix of 1， 127 basepairs ， though with only 5.9% parsimonious informa- tive tive characters. Maximum-parsimony ，neighbor-joining and maximum- Ii kelihood analyses produced produced similar trees. Our results suggest that there are probably six groups of Goniotha- lamus lamus in Thailand ， but only one of these has high bootstrap support. The six groups reflect similarities similarities in floral morphology but not biogeography. Elements of both Boerlage's and Ban's infrageneric infrageneric c1 assifications are reflected in our analysis ，although our results are not s仕ictly congruent congruent with either of theirs.

Keywords: Keywords: Annonaceae ，Goniothalamus ， molecular phylogeny ，Thailand ， trnL intron ， trnG intron intron

INTRODUCTION

The Genus Goniothalamus (Blume) Hook. f. & Thoms.

Annonaceae 紅 'e a pantropical family of shrubs ， trees and lianas. The family consists of about 130 genera and 2，300 species. The largest number of genera and species 紅 e known from Asia (including Australia and the Pacific) ，with ca. 60 and 1000 ，respectively (MOLS & KESSLER ， 2003). Among the Asian Annonaceae ， Goniothalamus (Blume) Hook. f. & Thoms. is is a large important genus of some 120 species (MAT -SALLEH ， 2001). It is widely distributed

1 Department of Biology ，Faculty of Science ， Chiang Mai University ， Chiang Mai ，Thailand. 2 Department of Biology ，Faculty of Science ， Chulalongkom University ，Bangkok ，Thailand. 3 Th ailand lnstitute of Scientific and Technological Research (Tl STR) ，Bangkok ，Thailand. 4 UBC Botanical Garden and Centre for Re 郎searc 油E Vancouver ，Canada V6T l Z4. Received Received 5 April 2007; accepted 7 November 2007.

307 308 YU 1T AYA YUYEN ET AL in in lowland and submontane tropical forests in SoutheastAsia ， with a center of species diver- sity sity in Indochina and Westem Malesia (SAUNDERS ， 2002; 2003). They 紅 'e shrubs or small trees ，characterised by having outer petals which are slightly or not spreading ， while the inner petals 紅 'e smaller ，clawed and curving over the sexual organs to form a dome-shaped struc- ture ture (mitreform dome) during anthesis. Th e stamens have broad apical connectives and their shapes shapes range from truncate to apiculate. Based on the mo 甲hological characters ， SINCLAIR (1955) classified the family into 6 tribes (Uvarieae ，Unonieae ，Miliuseae ，Mitrephoreae ，Annonieae and Xylopieae). Goniothalamus was placed in the tribe Mitrephoreae by the character of inner petals curving over the sexual organs organs forming a dome-shaped (mitreform) structure ， along with Pseuduvaria ，Neo-uvaria ， O 呼 mitra ， Mitrephora and Popowia. In contrast ，results from RICHARDSON ET AL. (2004) ， who studied the historical biogeography of Annonaceae using rbcL and trnL-F plastid DNA sequences ， suggested that Goniothalamus was totally unr eJ ated to otha rni 住eform genera of Annonaceae. 百 ey found that Goniothalamus was a strongly supported group ， but the sister group group to the genus was unclear. Their molecular data indicated that Goniothalamus is closely related related to Anonidium ，Neostenanthera ，Disepalum ，Asimina ，Annona ，and Rollinia.

History History of Classification

Goniothalamus Goniothalamus is an interesting genus that is in need of study. The genus is poorly understood ， both in terms of basic morphological t飢 onomy and phylogenetic relationships. It It has been subject to several di 妊erent subgeneric ，sectional and subsectional classifications (Table (Table 1). The name Goniothalamus was first proposed by BLUME (1 830) in Flora Javae as a section of the genus Polyalthia to accommodate Polyalthia macrophylla ，a species with an “ angled receptacle". HOOKER & THOMSON (1 855) later elevated this section to a genus in in their Flora Indica. BOERLAGE (1899) incorporated Beccariodendron ，a monospecific New Guinean genus ， into Goniothalamus. The section Beccariodendron of BOERLAGE was founded founded to accommodate Beccariodendron grandiflorus and the multi-ovulate Goniothalamus species. species. Other Goniothalamus with only one or two ovules were then grouped into the section section Eu-Goniothalamus. BAN (1 974) proposed a more hierarchical infrageneric classification based mainly on stamen characters. He divided the genus into two subgenera: subgenus Goniothalamus (apiculate (apiculate stamens) and subgenus Truncatella (truncate stamens). He also suggested that subgenus subgenus Goniothalamus could be divided into two sections: section Goniothalamus (with subsections subsections Goniothalamo か'P us and Pleiospermi) and section Lo ngistigma. The 0 白ersubgenus Truncatella Truncatella was also divided into two sections: section 1ゆ， ndibulistigma (with subsections PO かspermi and In. かndibuliformes) and section Truncatella (with subsections Multiseminales and Pauciseminales). However ，MAT-SALLEH (1 993 ， 2001) in studying Goniothalamus species species from Bomeo ， suggested that an infrageneric classification would be more natural if based based on flora l/ leaf characters and habits. Recently ，further Southeast Asian Goniothalamus collections (especially (especially collections from Peninsular Malaysia and Sumatra) have been critically studied (SAUNDERS ， 2002; 2003) and the usefulness of MAT-SALLEH'S taxonomic characters further emphasised. emphasised. Table Table 1. Infrageneric classifications of 出e genus Goniothalamus proposed by BOERLAGE (1 899) and BAN (1 974).

Authors Infrageneric classification Morphological characters

BOERLAGE (1 899) Section 1. Beccariodendron More th 加 one ovule

Section Section 2. Eu-Goniothalamus One or two ovules 〉 BAN (1 974) Subgenus 1. Truncatella Connective truncate stamen 2 Section Section 1. lnfundibulistigma Style short; stigma funnel-shaped or fusiform ， broad 0 Subsection Subsection 1. Polyspermi Ovarywith 3・ 10 ovules 0 Subsection Subsection 2. lnfundibuliformes Ov 紅 y with 1-2 ovules 。 Section Section 2. Truncatella Style long ，cylindrical or subuliform; stigma minute ， =言 integral integral or slightly bilobed 。 Subsection Subsection 3. Multiseminales Ovary with 3-8 ovules g Subsection Subsection 4. Pauciseminales Ovary with 1-2 ovules Subgenus 2. Goniothalamus Connective apiculate stamen s Section Section 3. Goniothalamus Style distinctive ，cylindrical or subuliform; stigma minute ， 〉 integral integral or bilobed 。至 Subsection Subsection 5. Goniothalamotypus Ovary with 1-2 ovules z 〉 Subsection Subsection 6. Pleiospermi Ov 釘 y with 3-10 ovules Section Section 4. Lo ngistigma Style cone-shaped; stigma filiform and extremely long E 盟

国 主 。主

凶{)。 310 YUTIAYA YUYEN Ef AL ，

Economic Impor 旬nce of Goniothalamus

The genus is not normally recognised as an important forest produc t. However ，some species 釘 'e used as omamental because of their small size and strongly fragrant f1 owers. The species most commonly cultivated are G. griffithii Hook. f. &百lO mson ， G. laoticus laoticus (Finet & Gagnep.) B 釦， G. macrophyllus (Blume) Hook. 五&百 lomson ， G. malayanus Hook. f. & Thomson ，G. repevensis Pierre ex Finet & Gagnep. ， G. sawtehii Fischer ， G. tapis Miq. ， G. tortilipetalus Henderson ， G. umbrosus J. Sinclair and G. undulates Rid l. BURKILL (1935) reported 血at G. macrophyllus ， G. umbrosus ，and G. tapis were widely utilised utilised by village midwives in the Malay Peninsula in their 位'aditional practices. Goniotha- lamus lamus malayanus and G. macrophyllus have been used too in Bomeo for the same pu 中 ose (MAT-SALLEH ， 1993). Javanese mountain dwellers also treated patients su 紅白ngwi 出 fevers with with aromatic roots of G. macrophyllus (BURKILL ， 1935). There have been several recent phytochemical studies on Goniothalamus focusing on their their potential for anti-cancer therapies. Styryl-pyrrone and styryllactone derivatives from Goniothalamus Goniothalamus have been suggested to have anti-cancer activities (LI ET AL. ， 1998; INAYAT- HUSSAIN ETAL. ， 2002; UMAR-TsA 田町AL. ， 2004; TIAN ET AL. ， 2006).

Goniothalamus in Thailand

Thailand Thailand is 佃 important distributional 紅 'ea for Goniothalamus. Although only nine species species were reported in Thailand by CRAIB (1 925) ，a more recent checklist (BVGRAVE ， 1997) 1997) listed 21 species. A recent survey by CHALERMGL 町 (200 1) found 23 species of 官 lai Goniothalamus. Goniothalamus. We suspect that the real number of Goniothalamus species in Thailand is likely likely to be considerably higher. So far ，no comprehensive treatment of Goniothalamus in Th ailand has been undertaken and existing studies of Th ai species are based on mo 中hological characters alone. Th e use of molecular molecular data is of potential value in helping to assess the utility of morphological taxonomic characters ，and to assist in the natural classification of the genus. For 血is reason we conducted a molecular systematic investigation on 白e Th ai species using using chloroplast trnL and trnG intron sequences.τ 'h e trnL intron sequences have been used successfully successfully in other genera of the Annonaceae ， for instance in a molecular phylogeny of Miliusa Miliusa and its allies in the tribe Saccopetaleae (MOLS ET AL. ，2000) ，and phylogenies of some Neotropical Neotropical genera (CHATROU ET AL. ，2002). 百le trnG intron has never previously been used for for phylogenetic studies in the An nonaceae ， but it has occasionally been used in other groups of of plants (PEDERSEN & 1也DENAS ， 2003; SHAW & SMALL ， 2004). In In this study ，we reconstruct a molecular phy logeny of 20 Thai Goniothalamus taxa using the the combined trnL and trnG intron sequences.τ 'h e results are discussed in 血e context of the infrageneric infrageneric classifications ofBOERLAGE (1899) and BAN (1974). A STUDY OF GONIOTHALAMUS (ANNONACEAE) IN THAILAND 311

MATERIALS AND METHODS

In group and Outgroup Taxa

Fresh Fresh leaves of 20 Thai Goniothalamus taxa were collected in the wild and kept dry in in silica gel until used (Fig. 1). Plant specimens were identified to species following CRAIB (1 925) ， SINCLAIR (1 955) ，MAT-SALLEH (1 993) ，佃 d SAUNDERS (2002 ， 2003). Some plants were unidentifiable and had to be informally named (using 血 e collecting locality) pending either either identification with species not currently recorded from 百lailand or description as new species. species. Work on describing these informally named specimens is 泊 progress. Herbarium specimens specimens and spirit materials were deposited at CMU (Chi 叩 g Mai University) Herbarium ， Chiang Mai ，Thailand. We used all 20 Gonioth( 均 mus as ingroup taxa (Table 2) while two other Thai Annon- aceae ，Orophea enteroca 中 a Maingay ex Hook. f.&Thoms. and Artabotrys spinosus Craib were used as outgroups to root the phylogeny. RICHARDSON El' AL. (2004) reported other genera genera more phylogenetically closely related to Goniothalamus ， but Artabotrys is the only genus genus of the tribe Mi 位ephoreae 出at we could collect in Thailand and successfully sequence its its DN A. In fact ，two more outgroups from the tribe Mitrephoreae (Friesodielsia desmoides (Craib) Steenis Steenis (Craib) and Mitrephora kethii Ridley) were also tested but none of the PCR products gave gave a readable sequence. On the other hand ，Orophea enterocarpa was also selected as the outgroup outgroup because it is in tribe Miliuseae (SINCLA 限， 1955) which is clOsely related to 白e tribe tribe Mitrephoreae.

Genomic DNA Extraction ，PCR Am pl 出cation ，and DNA Sequencing

Total Total genomic DNA was extracted from a leaf sample of each plant using DNeasy 骨 Plant Mini kit (QIAGEN GmbH ，Germany) or the CTAB method (DOYLE & DOYLE ， 1987). DNA quantity quantity and quality was examined by agarose gel electrophoresis. Chloroplast trnL intron sequences sequences were amplified with the Polymerase Chain Reaction (PCR) technique using a for- ward primer trnL ・C (5'-CGAAAT CGG TAG ACG CTA CG-3') and a reverse primer trnL-D (5'-GGGGAT AGAGGG ACTTGAAC-3') ，followingTABERLETETAL. (1 991). Amplifica- tions tions were carried out in 50 ・μ1 reaction volume with 0.8μ1 of2.5 unitofTaq DNAPolymerase

(Finnzyme ，Finland) ，5μ1 of 10x optimised Taq Polymerase buffer with 1.5 mM MgC1 2 included included (Finnzyme ，Finland) ，1μ1 of 10 ・mM dNTP ，5μ1 of 10 ・μM each primer ，and 10 ー20 ng of genomic DN A. The PCR cycling profile to amplify trnL 泊tron sequences was modified modified from BRUNEAU ET AL. (2001). The PCR amplification was performed using a GeneAmp@ PCR system 9700 (Applied Biosystems ，Singapore) ， prograntmed for pre-dena- turation turation at 95 0 C for 5 min ， 35 cycles of denaturation at 94 0C 1 min ，annealing 55 0 C 1 min and extension extension 72 0 C2 min ， with final extension step at 72 0C for 7 min. Chloroplast Chloroplast trnG intron sequences were amplified using a forward primer 3'trnG

(ター GTA GCG GGA ATC GAA CCC GCA TC ・3') and a reverse primer 5'trnG2G (タ-GCG GGT ATA GTTTAG TGG TAAAA-3') following SHAW ET AL. (2005). Amplifications were

C紅 ried out in 25 ・μ1 reaction volume with 0.25μ1 of 5 unit of Taq DNA Polymerase (New

England BioLabs ，USA) ，2 .5 μ1 of 10x Taq DNA Polymerase buffer with 1.5 mM MgC1 2 included included (New England BioLabs ，USA) ，2.5μlof2 mM dNTPand 1μlofl0μM each primer (forward (forward and reverse) ，and 1μ1 (1 0- 20 ng) oftotal DN A.百 le PCR CYcling profile to amplify 312 YUTrAYA YUYEN 67' AL.

MYANMA.It

2. G. marcanii 3. G. sp. Narathi wat

sawtehii sp. Aung laeonai undulatus

laoticus 2 G. laoticus 9. G. cheliensis

10. G. repevensis

tortilipetalus umbrosus

sp. Sun yataram sp. Maerim

sp. Chongyen sp. Phuye

tapis 19. G. malayanus

20. G. macrophyllus N t

Figure I. Collecting localities of 20 Thai Goniotha/amus specimens used in this study. The fl ori sti c-region map of Thail and is after Smitinand (2001) . Abbreviations of the fl oristi c-regions are as follows: N = North, NE = Northeast, E = East, S W = Southwest, C = Central, SE = Southeast, PEN = Peninsular. Table Table 2. Collector number and GenBank accession numbers of 20 Goniothalamus samples and two outgroups (Artabotrys spinosus and Orophea Orophea enterocarpa) collected and examined in this study.

No. No. Taxoo Locality (provioce) Collector 00. GeoBaok accessioo 00.

〉 cheliensis G. cheliensis Hu DoiPhu 即1a (Nan) Yuyen 220 EU249788 ，EU249766 ∞ 2. 2. G. elegans Ast Phu Phan (Sakon Nはhon) Yuyen 229 EU249783 ，EU249761 C→ 0 3. 3. G. griffithii Hook. f. &百 1omson Doi Saket (Chiang Mai) Yuyen 208 EU249798 ，EU249776 4 0 4. 4. G. laoticus (Finet & Gagnep.) Ban (sample 1) 阻1ao Yai (N 紘 hon Ratchasima) Yuyen250 EU249800 ，EU249778 守1 c 5. 5. G. macrophyllus (Blume) Hook. f. & Thomson 回1ao Pho Ta Luang Kaew (Ranong) Yuyen 211 EU249789 ，EU249767 o Phu Phu Moo (Mukdahan) Chalermglin 25-4-47 (1) EU249787 ，EU249765 6. 6. G. marcanii Craib oミ &百lO Ba Cho (N 紅 athiwat) Yuyen209 EU249796 ，EU249774 '--l 7. 7. G. malayanus Hook. f. mson E 8. 8. G. repevensis Pierre ex Finet & Gagnep. 回1ao Soi Doaw (Chanthaburi) Yuyen 232 EU2 49794 ，EU249772 9. 9. G. sawtehii Fischer Phanoenthong (Phetchaburi) Yuyen 235 EU249784 ，EU249762 i q 10. 10. G. tapis Miq. Sai B uri (p attani) Yuyen 205 EU249793 ，EU249771 仁司 1. 11. G. tortilipetalus Henderson Thong Pha Phum ，(Kanchanaburi) Yuyen 234 EU249790 ，EU249768 〉 z 12. 12. G. umbrosus J. Sinclair Khlong 官10m (Kr abi) Yuyen 236 EU249797 ，EU249775 o 3. 13. G. undulatus Rid l. Khlong Na Kh a (Ranong) Yuyen 217 EU249786 ，EU249764 z 。〉 14. 14. G. sp. Aunglaeonai (êh~q11'W) Aunglaeonai (Chachoengsao) Yuyen 213 EU249785 ，EU249763 開 〉 15. 15. G. sp. Chongyen (~Ð~L~ 'W) Chong Yen (Kamphaeng Phet) Chalermglin 6ふ47 (1) EU249791 ，EU249769 廿3 16. 16. G. sp. Maerim (弘前剖) Kanchanaburi Yuyen 239 EU249802 ，EU249780 z 17. 17. G. sp. Narathiwat ('W 11ii11~) Mueng (Narathiwat) Yuyen206 EU249795 ，EU249773 g 百10ng Pha Phum (Kanchanaburi) Yuyen 219 EU249792 ，EU249770 〉 18. 18. G. sp. Phuye ('可凶) 「司 19. 19. G. sp. Sunyat 訂am( 明間 111 剖) 百10ng Pha Phum (Kanchanaburi) Yuyen 218 EU249801 ，EU249779 z〉 20. 20. G.laoticus 2 (Finet & Gagnep.) Ban (sample 2) Dongph 油uan (Ub on Ratchathani) Yuyen 228 EU249799 ，EU249777 o Out Out group Artabotrys spinosus Craib Dongphahuan (Ub on Ratchatani) Yuyen 247 EU249782 ，EU249760 Out Out group Orophea enterocarpa Maingay Maingay ex Hook. f. &百lO mson Dongphahuan (Ub on Ratchatani) Yuyen 246 EU249781 ，EU249759

凶-一凶 314 Y U1・ fAYA YUYEN EI' A L.

trnG trnG intr 'O n sequences was m 'O dified from SHAW ET AL. (2005). The PCR amplificati 'O n was perf 'O rmed 'O n Mastercycle~ PCR gradient system (Eppend 'O rf AG ，Hamburg) pr 'O grammed f'O r pre-denaturati 'O n at 80 "C f'O r5 min ，30 cycles 'O f denaturati 'O n at 95 0 C 45 sec ，annealing 48-52 0 C 30 sec and extensi 'O n 72 0 C 1 min; with final extensi 'O n step at 72 0 Cf 'O r5 min. PCR pr 'O ducts were purified using QIAquick PCR p町 ificati 'O n kit (QIAGEN GmbH ， Germany) pri 'O rt 'O cycle sequencing reacti 'O n. Sequencing pr 'O ducts were analysed 'O n an aut 'O mated DNA sequencer ABI Pri sm@ 377 (Applied Bi 'O systems ，USA) 'O r 叩 ABI3730XL sequencer sequencer 'O f Macr 'O gen Inc. (in Se 'O ul ，S 'O uth K 'O rea). B 'O th f'O rward and reverse sequencing reacti 'O ns were perf 'O rmed f'O r sequence c'O nfirmati 'O n 'O f each sample.

Phylogenetic Phylogenetic Analyses

The trnL and trnG intr 'O n sequences were aligned using the alignment pr 'O gram Clus 凶 Xwi 白凶n'O rmanu 剖叫ustments. Sequence b 'O undaries 'O f tr 札 and trnG intr 'O n were determined determined using published sequences 'O f 'O ther genera in the GenBank nucle 'O tide da 旬base. Single Single and c'O mbined trnL and trnG intr 'O n sequence matrices were analysed with a phyl 'O ge- netic netic rec 'O nstructi 'O n pr 'O gram PAUP* versi 'O n 4.0b lO (SWOFFORD ， 1998) running 'O n Macint 'O sh P 'O werb 'O'O k (CPU G3 ・300MHz ，192 Mb RAM) with character states un 'O rdered and equally weighted. weighted. Phyl 'O genetic 住'ees were rec 'O nstructed using a maximum-parsim 'O ny (MP) meth 'O d

as as implemented in the pr 'O gr 創 n. Gaps (indels) were treated as missing data. All sequence characteristics characteristics 'O f the single trnL intr 'O n， trnG in 位'O n， and c'O mbined sequence data ma 凶 ces were 'O btained 企'O mPAUP*. We perf 'O rmed branch-and-b 'O und search in 'O rder t 'O find the m 'O st parsim 'O ni 'O us trees (MPTs). (MPTs). The analyses were c紅 ried 'O ut with 'O ut using a gap ma 凶 x since n 'O di 首erent tree- t'O p'O l'O gy was f'O und 企'O m the initial analysis. A semistrict c'O nsensus tree was calculated 企'O m all all f'O und MPTs. B 'O'O ts 回 P (FE 凶 ENS 官民 1985) and jackknife (LANYON ， 1985) analyses were perf 'O rmed 1，000 replicates using PAUP* set t 'O a heuristic-search 'O pti 'O n and a simple sequence sequence additi 'O n. Descriptive statistic indices reflecting am 'O unts 'O f phyl 'O genetic signals in 血ep 紅 sim 'O ny analyses were given by c 'O nsistency index (CI) ，retenti 'O n index (RI) ，and rescaled rescaled c 'O nsistency (R C) index. A neighb 'O ur-j 'O ining (NJ) tree and a maximum likelih 'O'O d (ML) 紅白 from the 1227-bp c'O mbined sequence data matrix were als 'O rec 'O nstructed using PAUP* pr 'O gram t 'O c'O mpare with with the MP results. Ev 'O luti 'O nary m 'O del and parameter settings f'O r the ML analysis were

sugges 旬 d by a pr 'O gr 創 nM 'O deITest versi 'O n 3.06 (POSADA & CRANDALL ， 1998). TVM+G m 'O del was selected with a substituti 'O n-rate ma 佐ix 'O f 0.5093 ，1. 7681 ，0.3491 ，0.7361 ，阻 d 1. 7681; nucle 'O tide frequenciesA= 0.34250 ，C= 0.15890 ，G =0.19350 ，and T=0 .3 05 1O; and agammadis 凶buti 'O np 紅 ameter (G) = 0.9324. 百le ML analysis was executed with a heuristic search search strategy using a rand 'O m sequence additi 'O n and TBR branch-swapping.

RESULTS

Collection Collection of Goniothalamus Speci 回 in Thailand

Fr 'O m 'O ur field expediti 'O ns (f 'O cusing mainly 'O n 血en 'O rth 'O fthe c'O untry) ，we were able t 'O l'O cate 20 taxa 'O f Goniothalamus inτbailand. F 'O urteen 'O f these were identifiable with kn 'O wn A STUDY OF GONIOTHALAMUS (ANNONACEAE) IN THAILAND 315 species. species. The other six were unknown and had to be named following the collecting localities: G. G. sp. Narathiwat ， G. sp. Aunglaeonai ， G. sp. Sunyataram ， G. sp. Maerim ， G. sp. Chongyen ， and and G. sp. Phuye. Of the 14 identifiable taxa ，five were new records for Thailand. We found five five ofthe nine species reported from Thailand by CRAIB (1925) ，eight ofthe 21 species listed by BYGRAVE (1 997) and 16 ofthe 23 taxa found by CHALERMGLIN (2001).

Sequence Sequence Alignment and Matrix Characteristics

Sequence Sequence characteristics of the trnL and trnG intron data for 20 Thai Goniothalamus and and two outgroups are summarised in Table 3. The aligned trnL and trnG matrices can be obtained obtained from the first author. From the 1，227 ・basepair combined data matrix ， only 73 nucle- otides otides (5.9%) were found to be parsimony-informative ， while 127 nucleotides (10 .4%) were autapomorphic autapomorphic characters. An initial MP analysis of the whole 1，227 ・bp alignment yielded more MPTs than could be held in the computer memory; therefore ， only the 73 parsimony- informative informative characters were used in further analyses. The consistency index (CI) and retention index index (RI) values were 0.8667 and 0.8824 ，respectively ， while the rescaled consistency index (R C) was 0.7674. When alll ， 227 nucleotides ofthe matrix were analysed ， the indices were slightly slightly higher (CI = 0.9492 ，RI = 0.8824 ， and RC = 0.8375).

Table Table 3. Sequence characteristics of the single trnL intron ， trnG intron ， and combined sequence sequence data matrices used in this study.

Parameters Parameters trnLintron trnG intron Combined sequence Length Length range (total) 519-575 bp 602-723 bp 1143-1284 bp Length Length mean (total) 555 bp 635 bp 1190 bp Length Length range (ingroups only) 523-575 bp 602-723 bp 1152-1284 bp Length Length mean (ingroups only) 557 bp 636 bp 1193 bp Length Length range (outgroups only) 519-561 bp 62 牛631 bp 1143-1192 bp Length Length me 加 (outgroups only) 540 bp 627 bp 1168 bp G + C content range 35-39% 33-37% 35-38% G + C content mean 38% 36% 37% Number Number of excluded sites 52bp 80bp 132 bp Number Number of sites a 白er exclusion 540bp 687bp 1227 bp Aligned Aligned length (total) 592 bp 767bp 1359 bp

Sequence Sequence divergence (i ngroups) 0.00- 1. 91% 0.00-3 目52% 0.09-2 .4 39 も Sequence Sequence divergence (in/outgroups) 10 .56- 12.12% 6 .1 0-9.09% 8.55- 10.11 % Number Number of indels (ingroups) 6 72 78 Number Number of indels (total) 8 73 81 Size Size of indels (ingroups) 1-8 bp 1-21 bp 1-21 bp Size Size of indels (ωtal) 1-9 bp 1-21 bp 1-21 bp Number Number of constant sites 459 bp (85%) 568 bp (82 .7%) 1027 bp (8 3. 7%) Number Number of variable sites 81 bp (1 5%) 119 bp (17 .3%) 200 bp (1 6.3%) Number Number of MP-informative sites 51 bp(9 .4%) bp 22 (3. 2%) 73 bp (5.9%) Number Number of autapomorphic sites 30 bp (5.6%) 97 bp (1 4.1 %) 127 bp (1 0.4%) Transition: Transition: transversion ratio 1: 0 3 ・2 4:2 凶目。 Maximum parsimonious Boerlage's B 姐 's Semishictconsensus E 曜血 Qmk Sec t. Stamen/Stvl e.l尉 I!m1 B SubgenJSect. '且冒且・ EEEEEEBBBEEEEEEBBEBEuuuuuueeeuuuuuueeueuGGGGGG00000000000000 :…… ..μ二;伝説tdh- …………..~ Northeast convexlshortlclavate TR .l ln. 74 74 ，広言1_ G. macranii はJNorth 悶 t convex/shortlclavate TR .l ln. 1 convex/shortlclavate TR .l ln. 石壬---士山 RNpm-w jM 叩 lar ・ A2 ~ 1ゐ...戸ニム '0: ・ぬ・ル働 ir...... r 肯l ~ou~west 加 ncate/lon g/ minute TR .l Tr.

且 -四 M ~I-:.~;:.I1 <5 r!~~ 企 "1 ト~Southeast convex /l ong/minute TR .l Tr. 1 ，0 L- G. sp. Aungl 一一一 'A <5 0 undu 伽 s Peninsular convex/shortlclavate TR .l ln. ~L一一一一 G. ~ 今 99235 二..川三・"(1:必必面玄'::::':::":::::::， East 3 CCC------CC・ convexlshortlfusiform TR .l ln. 弓 3 .. laoticus laoticus : 副 convexlshortlfusiform TR /l n. :戚右L_ G. E 刈斗 司 G. cheliensis North convexlshortlcylindrical TR .l ln. r宝石1~ 'A GGGGGG .. G .. ふんji--1 ニ J ・Sou 曲師t truncate/shortlcylindrical TR .l ln. J ・ 「 1 apiculate /l on g/ minute GO .l Go. qコ : ロ;訓示副長 ， Southwest ・且 〉a apiculate /l on g/ cylindrical GO .l n.a. JSJ G. G. umbros 回 目す lPenins 凶釘 'I' I∞ apiculate /l on g/ minute GO .l Go. 「 C4 94 87 且・且 ， ;仁川 iiτM 託 早 町戸 即 叩叫伽明官宙側st apiculate /l on g/ minute GO .l Go. 間 G. G. Sun ly ata n ~ S th w Z G. G. 叩 .Ma 総E由血rim ~ 伽加S t伽h蜘w鴨芭団鮒喧峨 t apiculate /l on g/ minute GO .l Go. 匂 ζJ 』a ・・ truncate/shortlfusiform τ'R.江 n. hl Iω Iω 弓，“・ 乙 山 s託t truncate/shortlfusiform TR .l ln. E 附叩 57 ，<50 G.s? ト刊川.刊加m恥 u戸 回 S叫oω 側州u凶仙白th G γ:に...コ....に:コ...戸三 :"1 説&丘"沿》諒 t云訟 's...... 1 ・ apiculate/shortlfunnel GO .l n.a. 内 1∞ L'i C-・ truncate/shortlcoiled TR .l Tr. 55 ， 51 ←一一 L 一 G. 仰 ω均ya 仰n附 己子引 J Pen 凶nins 叫1 G .…...瓦己瓦..ふん 'phyllus … P，叩 insul 紅 apiculate/shortlclavate GO .l n.a.

Artbo 町IS spinosus Orophea enterocarpa

Fi 伊 re 2. Semistrict consensus 回 e of 3， 273 equally most-parsimonious trees based on 白e 73 ・basep 誼∞mbined trnUtrnG intron sequence matrix of 20 百凶 Goniothalamus Goniothalamus with two Annonaceae outgroups. Numbers above branches (in ita Ii cs) are identical percentages of the branch among aII congruent congruent parsimonious trees. Numbers below branches designate 胸 tstrap and jackkni た support of 1， 000 replicatω ，抑制ively. Collecting regions ， important important florai characteristics ， and Boe r1 age's and Ban's taxonomic assignments were also indicated. Abbreviations of subgenera and sections as follow: Eu ・Go. = se 氾tion Eu-Goniothalamus ， Bec. = section Beccariodendron ， TR. = subgenus Truncatella ，GO. = subgenus Goniothalamus; In. = section In ルndibulistigma ，Tr. = section Tr uncatella ， Go. = section Goniot 加， lamus ， and n.a. = unable ωcl 蹴 ify to section. A STUDY OF GONIOTHALAMUS (ANNONACEAE) IN THAILAND 317

Phylogenetic Phylogenetic Analyses

A branch-and-bound search on the 73-basepair trn L/ trnG intron sequence matrix gave 3，273 equally most-parsimonious trees ， of 227 steps in length (phylograms not shown). A semistrict semistrict consensus tree (Fig. 2) from the entire 3，273 MPTs showed that a1 1 20 Goniotha- lamus lamus were separated from the outgroup species as a single clade with 100% bootstrap and jackknife jackknife supporting values. From the semistrict consensus analysis ，six plausible groups were recognised recognised within the Thai Goniothalamus group. Each group was present in the majority of of MP trees. Groups I-VI were recovered in 74% ， 91 %， 80% ，100% ，100% ，and 100% of trees ，respectively. Likewise ， the neighbour-joining tree and the maximum-likelihood tree of the the 1，227 ・bp combined sequence data matrix of the 20 Thai Goniothalanus taxa (Fig. 3) also revealed revealed the same six subgroups. Two species ， G. repevensis and G. macrophyllus ，were the only only taxa that did not cluster with any of the six groups. Group 1 contains G. elegans ， G. marcanii ，and G. sp. Narathiwat; group 11 has G. sawtehii ， G. G. sp. Aunglaeonai ，and G. undulatus; group III ，G.laoticus 2，G.laoticus ，and G. Cheliensis; group group IV ， G. tortilipetalus ， G. umbrosus ， G. griffithii ， G. sp. Sunyataram ，and G. sp. Maerim; group group V ， G. sp. Chongyen and G. sp. Phuye; and group VI contains G. tapis and G. malayanus. Only three groups (IV ，V ，and VI) have bootstrap support ， but generally low. The exception is is group IV ，which has high support in all three analyses (bootstrap values of 94% ，92% ，and 95% on the MP ，NJ ，and ML trees ，respectively).

DISCUSSION

Goniothalamus Chloroplast trnL/trnG Introns vs. Nuclear ITS Regions

We found that the average lengths of Goniothalamus trnL and trnG introns (557 bp and 636 bp ，respectively) were similar to those of other plants. SHAW ET A L. (2005) investigated phylogenetic phylogenetic utilities of many noncoding chloroplast DNA sequences of phanerogams and reported reported the average length oftheir trnL introns as 499 basepairs (ranging from 697 to 1008 bp) bp) while the average trnG-intron length was 636 basepairs (395 to 602 bp). Moreover ，we also also found that the trnL and trnG introns of Goniothalamus had fairly low GC-content values ， only only 38% and 36% in average ，respectively. Problematically ， the numbers of parsimony-informative characters of Goniothalamus trn L/ trnG introns are remarkably low ， only 9.4 9もand 3.2% ，respectively. The average value was even lower (5.9%) in the combined sequence matrix. Data ofRICHARDSON ETA L. (2004) ， suggested suggested that Goniothalamus is a very young genus ， with an estimated age of only 3.~.8 Ma. Therefore ， the very low informative sites of Goniothalamus chloroplast trn L/ trnG introns found found in this study could support their hypothesis that this genus has a short evolutionary history. history. finding Our finding that Goniothalamus trnL intron sequences had comparatively higher useful- ness ness than the trnG introns is contrary to the study of SHAW ET A L. (2005). They suggested that that the trnG introns of phanerogams were generally more informative than trnL introns. Nevertheless ，PACAK & SZWEYKOWSKA-KuLINSKA (2000) found that liverwort trnG intron sequences sequences provided more nucleotide substitutions than the trnL introns. Moreover ，PEDERSEN & HEDENAS (2003) reported that the trnG intron of an apoca 中 ous moss provided nearly twice as as many as the trnL-trnF sequence. Maximum Likelihood (ML) 凶同市町

口コ

E コ

IVI I ペ C44

〉〈〉

d「 ci 直E 『開 Z 匂』』「 1∞ IVI IIVI rn rn

Orophea enterocarpa Artbotrys Artbotrys spinosus 一ー 0.005 substitutions/site 一一 0.005 substitutions/site

Fig 日記 3. Neighbour-joining (l eft) and maximum likelihood (right) 回 es based on the 1， 223 ・basepair combined sequence ma 甘ix of 20 Thai Goniothalamus taxa with two outgroups based on the combined tmUtrnG in 町on sequence analyses. Th e ML 出 e was reconstructed using TVM +G model (ー Ln = 2983 .4 9826). Six

rec 町 ring subgroupings congruently found on all MP ，NJ ， and ML trees were proposed. Numbers along branches indicate 1000 ・replicate bootstrap suppo 停

泊g-values. No number was assigned to any branch having less th 佃 50% support. A STUDY OF GON/OTHALAMUS (ANNONACEAE) IN THAILAND 319

To solve our problem on the low parsimony-informative level of Goniothalamus chloroplast chloroplast trnL and trnG introns ，we had also tried to implement other chloroplast DNA regions regions and ITS (Intem a1 Transcribed Spacer) regions of nuclear ribosomal genes. Unfo 武u-

nately ，a1 1 sequencing experiments on chloroplast DNA trnS-trnG ，rpoB-trnC ，trnT-trnDF ，佃 d trn fM -trnS regions followed SHAW ET AL. (2005) could not give amplifiable PCR-products from a1 122 plant specimens. We also performed additional experiments on ITS amplifications followed followed the works of MOLLER & CRONK (1997) on Saintpaulia (Gesneriaceae) and MEADE (2000) (2000) on Uvaria (Annonaceae). Only MEADE'S ITS primers could amplify all our 22 plant specimens ，resulting in approximately 900 ・bp PCR products. However ，none of the PCR products products gave readable sequence. Further studies (beyond the scope ofthis project) involving cloning cloning experiments of the PCR products are needed to solve this problem.

Goniothalamus Molecular Phylogeny vs. Biogeography

We suggest that the genus Goniothalamus in Thailand may be divided into six putative groups groups on the basis of 出e phylogenetic analysis. However ， the supporting values for these groups groups (with the exception of group IV) were not high ，due to the gener a1 1y low number of p紅 simony informative characters. In addition ， the positions of G. repevensis and G. macro- phyllus phyllus were unstable in 白e 位'ees 叩 d probably contribute to the low support values of the other other groups. On mo 叩hologic a1 grounds it is su 中rising 白紙 G. repevensis does not group with with G. undulatus with which it shares similar ovule number. Th ere is very little biogeographical patteming evident in 白e clade structure. For instance ， a1 though the two northeastem species ， G. elegans and G. marcanii ，occur 出 sister taxa in group 1， they were allied with G. sp. Narathiwat from the southem border. Likewise ， the eastem taxon-p 泊r of G. laoticus 2 and G. laoticus are allied to the northem species ， G. cheliensis ， to form group III. These two cases may be explained by the low support for 血e group; however ，

出， e same pattem is evident in the very s位ongly 制 supported group IV of five Goniothalamus taxa. taxa. Members of 白is clade come from distant regions of 出e country: G. tortilipetalus ， G. G. sp. Maerim 叩 d G. sp. Suyataram 紅 e found in the southwest; G. gr iJ. 芦thii from 出e north; and G. umbrosus from the pen 泊sular. Widely distributed 紅 eas of members of each group may be due to the effective 仕uit dispersal dispersal of 血e genus. Birds and mammals are the most likely seed vectors ，even though studies studies of seed dispers a1 in the genus are stilllacking. SAUNDERS (2002 ， 2003) suggested that that Goniothalamus has its center of diversity in Indochina and westem M a1 esia. Notably ， YUYEN& Boo 限 ERD (2002) had suggested before that 百lailand (and particularly Pr achuap Kh iri Kh an Pr ovince) has long been a meeting point of M a1 esian ，Indo-Burmese ，and Indo- Chinese Chinese elements.

Taxonomic Im plications of the Thai Goniothalamus Phylogeny

Th ough with low supporting levels in some ， the six groups found in this study to some extent extent could reflect flor a1 morphology including the number of ovules and the shape of 白e stamen stamen connective ，characteristics used in 由einf 同generic classification systems ofBOERLAGE (1899) (1899) and BAN (1 974) respectively. All All taxa in 也e groups 1，11 ，IV ，佃 d VI had only one or two ovules (i. e. section Eu-oni ・ othalamus othalamus (one or two ovules). On the other hand ， the groups 111 and V were the only two 320 YUTrAYA YUYEN ET AL

q ， L mn B C 'm D 1E 12 crn Icrn A

l， nn '2 mm l l2 m F G H J

Figure Figure 4. A ，F lower of Goni Ol lzala ll/ us II/ ar ι，:a l/ ii; B-J rnain type s of pistils showin g ran lhe ge of slyle and st ig rna varialion in th e ge nu s; F，Fllnnel-shap 巴d s li grna: B ，G ，cla val e st igrna ;C ，H ，st ig ma minute ;

fllsifonn 0 ，1， fllsifonn sti g ma; E，J， cy lin dr ical stigma .Plant sw ilh minute sti g mas have long styl 巴s ， all othel tyles styles are shor l. Species : B ，G 川 rcanii ，C， G. griffilliii ，0 ， G. sp Phuye ，E ， G. clieli el/ sis ，F ，G lapi s，G ，G. undulalus ，H ， G. sawlelzii ，1 ， G. laoli cus ，J ， G. repev 凶 Isis. Orawn by Y. Yuyen.

subgroups subgroups that had many ovul 巴s (i .e. section B eccar iod endron). However ，in our tree section Beccariodendron Beccariodendron appears to b巴 a paraphyletic group ， as G. repevensis and the clades 1，II ， and and III are clu stered together. However ， support for thi s grouping (that suggests paraphyly) is is very weak ， so a monophyletic section Becc α riodendron cannot b巴 rul 巴d ou t. Similarly Similarly our six propos 巴d groups can b巴 compared to BAN 'S subgeneric clas sificatio n using using the character of truncate or convex vs. apiculate stamen connectives. The clustering ofth 巴 trun cate or convex taxa ， G. repevensis and clades 1，II ， and III ，is possibly suggestive of of a natural group of truncate spec ies (subgenus Trun cate ll α) ，separat 巴d from the subge nus Goniothalamus Goniothalamus (exemplifi 巴d by group 1V). However ， group Vl of G. tapis (apiculat 巴) and

G. malayanus (truncate) ， have mixed stam 巴n connective characteristics. A relationship study of of these two species is needed in the future 1n 1n addition to the importance of th 巴 stamen connect ive ，BAN (1974) recommended us- ing ing style and stigma features to further divide the two subgen 巴ra of Goniothalamus into four sections. sections. 1t seems that stigma and sty le ch 加.acters may be more comp l ex than detailed by BAN. For in stanc 巴 in th 巴 Tha .i spec ie s we note fiv 巴 types of stigmas (Fig. 4) . ln addition to minute ， fusiform and funne l-shaped stigmas ，there are the clavate stigmas of G. elegans ，G. marcanu ， and G . sp. Narathiwat ， and the cylindrical st igmas of G. repevensis ，G. umbrosus ， and and G. undulatus (see Fig. 4) A lth ough ， our findings and attempts to reconstruct the phylogeny of Goniot h. alamus should should be useful for Annonaceae systematists ，it is important to note that Goniothalamus is a large and complex g巴nus (ca. 120 sp巴cies) and consequently thi s st udy is very preliminary . Less Less than 17% of the genus was sequenc 巴d in our studi 巴s and the addition of other genes and more taxa a.r e clearly needed to further test our results and hypoth 回目 A STUDY OF GONIOTHALAMUS (ANNONACEAE) IN THAILAND 321

ACKNOWLEDGMENTS

We would like to thank Mr. J. F. Maxwell (CMU Herbarium ，Chiang Mai University ， Thailand) for his taxonomic suggestions. The first two authors would like to thank Pongtip Jaidee (Biology Department ，Science Faculty ，Chulalongkom University ，Thailand) and Dorothy Cheung (UBC Botanical Garden ，University of British Columbia ，Canada) for their help in the molecular laboratories. This investigation was carried out while Yuttaya Yuyen was funded by the Royal Golden Jubilee Ph.D. program (TRF grant no. PHD/OI22/2546) of Thailand Research Fund (TRF) which is gratefully acknowledged. The second author was also funded by TRF (MRG-4680001).

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