Abstract: Dipterocarpoideae is a species-rich subfamily of Dipterocarpaceae. Dipterocarpaceae in Thailand is represented by 65 species in 8 genera. In recent years molecular taxonomist constructed a phylogenetic tree of dipterocarps found in South-East Asia. However, many species of dipterocarps found in Thailand are not included on that tree. In an effort to complete the tree, the phylogenetic relationships among 23 species belonging to 7 genera of dipterocarps found in Thailand were studies using nucleotide sequences of two intergenic spacer region between trnL-trnF and atpB-rbcL in chloroplast DNA. With Neobalanocarpus heimii as an out group, the molecular tree was consistent with the morphological classification and it was clearly resolved two major groups; Vavate-dipterocarpi and Imbricate-Shorea. The analysis showed that each member genera have similar characteristics and base chromosome number of 11 and 7, respectively. The first group consisted of genus Diptercarpus, Anisoptera, Cotylelobium and Vatica. Shorea and Hopea were in the second group.
INTRODUCTION
A large family of massive trees, Dipterocarpaceae is a dominant and important timber species found in tropical forest. The family include over 500 species in 17 genera and is divided into three subfamilies: Monotoideae, Pakarimoideae and Dipterocarpoideae (Aston, 1982). The subfamily Monotoideae comprises over 30 species in three genera and is confined in Africa, whereas 1 species is in Pakarimoideae and is distributed in South America and Guyana. The Asian dipterocarps, Dipterocarpoideae, includes 470 species in 13 genera. The Dipterocarpoideae subfamily is subdivided into two tribe: Dipterocarpeae and Shoreae.
In Thailand, The first recording of Dipterocarpaceae consisted of 10 genera, 46 species and 7 varieties (Craib, 1925). More recently records however, show Dipterocarpaceae in Thailand represented by 65 species in 8 genera: Anisoptera (3), Nebalanocarpus (1), Cotylelobium (1), Dipterocarpus (16), Hopea (14), Parashorea (1), Shorea (22) and Vatica (7) (Pherngklia and Niyomdhum, 1999). The majority of Dipterocarpaceae in Thailand belong to the evergreen species which is scattered all over the country: in gallery forests along the hill stream, in the low-lying land and on hill slopes. Only five deciduous species of D. obtusifolius, D. tuberculatus, D. intricatus, S. obtusa and S. siamensis are distributed at high elevation (Smitinand, 1969).
Dipterocarpaceae is a divers family tree and a species, especially rich in genus of Shorea, Hopea, Dipterocarpus and Vatica. In general, identification is based on morphology, wood anatomy, palinology and fossil record (Aston, 1980). However, identification of this family is not an easy task, because some characteristics vary with age of the tree and it’s habitat (Symington, 1974). Rajaseger et al. (1997) reported that polymorphism of DNA as molecular marker, are suitable for discriminating closely related genotypes. The advantage of DNA-based markers is that they are not influenced by the environment or by the development stage of the plant. Currently, molecular techniques such as RAPD (Rath et al., 1998), RFLP (Tsumura et al., 1996) and nucleotide sequences (Kajita, et al., 1998; Kamiya et al., 1998; Dayanandan et al., 1999) are alternative methods that are used to investigate the relationships between dipterocarps. Beside these, base sequence data base can be used to identify in species level.
In order to obtain a refined phylogeny among species of Dipterocarpaceae in Thailand, phylogenetic analyses based on the nucleotide sequences of intergenic spacer (IGS) region of trnL-trnF and atpB-rbcL of chloroplast DNA were performed.
MATERIALS AND METHODS
Taxon sampling: Leaf samples were obtained from 5 national parks of Thailand. They are Centennial Botanic Gaden, Peninsula Botanic Garden, Pa Klang Ao, Sakaerat Environmental Research Station and Queen Sirikit Botanic Garden. Samples were collected from 23 dipterocarp tree species representing 7 genera during the rainy season of 2002 and 2003. The collected leaf were dried in silica gel while in the field and transport to the laboratory for DNA extraction. The trnL-trnF nucleotide sequences from 10 species that also distributed in Thailand (Cotylelobium lanceolatum, Dipterocarpus baudii, D. kerrii, Hopea pierrei, H. odorata, Neobalanocarpus heimii, Shorea faguetiana, S. guiso, S. roxburghii and Vatica odorata) were retrived from the Genbank database. The nucleotide sequences of atpB-rbcL from 2 species of D. baudii and D. kerrii were a gift from Dr. Choong and Dr. Wickenswari.*** The information on each species is show in Table 1.
Dna extraction, trnl-trnf and atpbrbcl spacer region amplification and sequencing : Total genomic DNA was extracted from leaf samples using the methods of Doyle and Doyle (1987). The trnL-trnF intergenic spacer was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers e and f (Taberlet et al., 1991). Oligonucleotids of atpB (Fofana et al., 1997) and rbcL (Demesure et al., 1995) were developed for amplifying the atpB-rbcL spacers. Amplification reactions contained 1 μL of 10 mM each of dATP, dCTP, dTTP, dGTP, 1 μL of 20 σmol/μL of each primer, 2.5 units of Taq DNA polymerase and 4 μL of 25 mmol/L MgCl2 in a total volume of 50 μL. Thermal cycling for trnL-trnF IGS region was performed in a thermalcycler at 95°C for 5 min followed by 30 cycles of 95°C for 30 sec, 50°C for 30 sec and 72°C for 1 min. The reactions were completed by a 7 min extension at 72°C and held on 4°C. PCR thermal cycling conditions for atpB-rbcL intergenic region were initial denature at 94°C for 3 min, 30 cycles of 40 sec at 94°C, 40 sec at 59°C and 2 min at 72°C, followed by extension at 72°C for 7 min and then a soak at 4°C. The sequencing reactions were performed by using ABI PRISMTM big dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer). Sequencing primers were the same as those for gene amplification. Reactions were then eletrophoresed on an ABI 377 automate sequencer (Perkin-Elmer Applied Biosystems, Inc.).
| Table 1: | Dipterocarpaceae species selected for trnL-trnF and atpB-rbcL sequencing and their sources |
Study site*, CBG = Centennial Botanic Garden, Chachaengsao
/ Sakaew province, Thailand, PBG = Peninsular Botanic Garden (Thung Khai),
Trang province, Thailand, PKA = Pa Klang Ao, Prachuab Kiri Khan province,
Thailand, SERS = Sakaerat Environmental Research Station, Nakonrachasrima
province, Thailand, QSBG = Queen Sirikit Botanic Garden, Chiang Mai province,
Thailand, **Accession Number of DNA Data Bank of GenBank, ***Form Dr. Choong,
C.Y. and Dr. Wickenswari, R. School of Environmental and Natural Resource
Sciences, Faculty of Science and Technology, University Kebangsaan, Malaysia,
43600 UKM Bangi, Selangor |
|
Alignment and phylogenetic analysis: Multiple alignment of the sequences were performed by Clustal W (Thompson, et al., 1994). Alignments were visually adjusted. Sequence data from two nucleotide region were combined in a cladistic analysis using PAUP v. 3.1.1 (Swofford,1993). Phylogenetic analyses were carried out by maximum parsimony and Neighbor-joining (NJ) method. Parsimony trees were constructed using heuristic search TBR branch swapping, stepwise addition of 10 random replicates, an unconstrained number of maximum trees and retention of multiple most parsimonious trees and all characters trees were unweighted. Both strict and 50% majority-rule consensus trees were roots at Neobalanocarpus heimii. NJ analyses were conducted using HKY85 distance.
RESULTS
The combined sequences of the trnL-trnF and atpB-rbcL intergenic spacer region of 23 species were analyzed. The final alignments included 1339 sites. There was a total of 273 informative characters in equally weight of maximum parsimony. The analysis generated 124 mostly equal parsimonious trees, each with a length of 752 steps with Consistency Index (CI) of 0.871 and Rescaled Consistency (RC) index of 0.736 (after excluding uninformative characters).
| Fig. 1: | The strict consensus tree of 124 most parsimonious trees of
combined non-coding region: trnL-trnF and atpB-rbcL of Dipterocarpaceae.
The number above the branches indicate the percentage of bootstrap values
(higher than 50) from 1000 replicates. The total length is 752. Consistency
index is and retention index are 0.871 and 0.736, respectively. This tree
is rooted with N. heimii |
| Fig. 2: | Neighbor-joining tree obtained from combined sequence of two
non-coding region: trnL-trnF and atpB-rbcL of Dipterocarpaceae.
Numbers above the branches indicate bootstrap values from 1000 replicates.
N. heimii is using as an outgroup |
The strict consensus tree of 124 mostly equal parsimonious trees is given in Fig. 1. All branches were well resolved and showed 2 major groups of Valvate-Dipterocarpi and Imbricate-Shorea. The first major clade contained 4 genera of Anisoptera, Cotylelobium, Vatica and Dipterocarpus with a bootstrap value of 100%. This major clade split into 2 subclades Dipterocarpus and Valvate both of with bootstrap value of 100%. Four taxa within subclade Valvate were well resolved with bootstrap supported over 60%. The second subclade of Dipterocarpus formed monophyletic clade sister to valvate subclade. However, the relationships among this subclade remain unresolved. The second major clade, a paraphyletic clade of tribe Shorea (Hopea and Shorea), was well supported with bootstrap value over 60%. Species of Shorea (S. thorelii, S. guiso, S. siamensis and S. faguetiana) formed a clade sister to the clade of S. roxburghii and S. henryana. Hopea clade (H. odorata and H. pierrei) formed a monophyletic clade sister to the clade of Shorea.
Neighbor-Joining analysis were conducted on HKY85 distance matrix. A tree was shown in Fig. 2. All branches showed a bootstrap consensus value of over 50%. Although topology of the NJ tree was not fully consistent with the parsimony trees, all branches were mostly agree with the parsimony trees except Dipterocarpus clade.
DISCUSSION
In this study, phylogenetic relationships within 23 dipterocarp species based on trnL-trnF and atpB-rbcL sequences are mostly in agreement with present taxonomic treatment (Aston, 1980; 1982). Using morphology, pollen analysis and fossil records, (Aston, 1982) classified two tribes in Dipterocarpoideae.The first tribe Dipterocarpeae or Valvate-Dipterocarpi group consist of Anisoptera, Cotylelobium, Dipterocarpus, Stemonoporus, Upuna, Vatica, Vateriopsis and Vatica. The genera of this tribe have valvate sepals in fruit, solitary vessels, scattered resin canals and basic chromosome number x = 11. Present results showed that the genera Dipterocarpus is monophyletic with of the Anisoptera-Cotylelobium-Vatica clade with a bootstrap value of 100% (Fig. 1 and 2). The genera Anisoptera, Cotylelobium and Vatica clustered together (bootstrap probability was 100% in Fig. 1) consistent with the morphological classification of Aston (1982). Our tree showed D. kerrii was closely related with D. baudii as the same result of (Kamiya et al., 1998) and Kajita et al., (1998). The second tribe or Imbricate-Shorea group consist of Balanocarpus, Hopea, Parashorea and Shorea. The genera of this group have imbricate sepals in fruit, grouped vessels, resin canals in tangential bands and basic chromosome number x = 7. Therefore the species rich genera of Hopea and Shorea, can be distinguished by a single characteristic of a number of long fruit calyxes. RFLP analysis (Tsumura et al., 1996) and combined sequences analysis of matK, trnL-trnF IGS region and trnL intron (Kajita et al., 1998) the genus Hopea is sister to the genus Shorea. This is also confirmed by Kamiya et al. (1998). In this study Hopea is also closely related genera forming a sister grouping to Shorea.
Shorea is the largest genus of Dipterocarpaceae, out of 194 species, 163 species which occur in Malesia. Phylogenetic tree of Kamiya et al. (1998) showed S. roxburghii was closely related with S. saimensis and S. obtusa fromed a clade with the Malayan species. Beside these they also suggested that the deciduous species of the genus Shorea in Thailand may evolve in at least independent lineages. In this study, the Imbricate-Shorea group consisted of 3 subclade: they are 2 clade of Shorea and 1 clade of Hopea. Our tree, therefore, indicated that the genus Shorea is paraphyletic. However, our trees were different from the tree of Kamiya et al., (1998) in the case of S. siamensis was not closely affinity with S. roxburghii. Our tree showed S. roxburghii was closely related with S. henryana and S. siamensis fromed a clade with S. faguetiana, S. guiso and S. thorelii.
In the future, the study on Dipterocarpacea is an easy task. Because molecular techniques are an universal tools to access all the problems and get the new knowledge.
ACKNOWLEDGEMENTS
Mahasarakham University is thanks for providing a Postgraduate Scholarship to the first authors. Thanks are extended to the staff of The Royal Thai forest Department for assisting with our field work, P. Taveechai for providing dipterocarps leave collection, Dr. Choong, C.Y. and Dr. Wickneswari, R. for kindly donating the 2 Dipterocarp atpB-rbcL sequences and their useful suggestions in the data analysis. The critical reading of the manuscript by D. Paul, Mahasarakham University, is highly appreciated.