Bot. Bull. Acad. Sin. (2003) 44: 217-222

Peng et al. — Phylogenetic position of Dipentodon sinicus

Phylogenetic position of Dipentodon sinicus: evidence from DNA sequences of chloroplast rbcL, nuclear ribosomal 18S, and mitochondria matR genes

Yalin Peng1, Zhiduan Chen2, Xun Gong3, Yang Zhong4, and Suhua Shi1,*

1Key Laboratory of Gene Engineering of The Ministry of Education, Zhongshan University, Guangzhou 510275, The People's Republic of China

2 Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100873, The People's Republic of China

3 Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, The People's Republic of China

4 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, The People's Republic of China

(Received June 25, 2002; Accepted February 24, 2003)

Abstract. Phylogenetic position of the monotypic genus Dipentodon has long been controversial. We investigated its position with 125 accessions representing 50 genera and 40 families of eudicots in the APG system. Four data sets—including the chloroplast gene rbcL, the nuclear 18S ribosomal DNA, and the mitochondrial gene matR, as well as the combined matrix—were used in the study with the maximum parsimony (MP) and Bayesian inference (BI) analyses. The phylogenetic trees based on individual genes and the combined data suggested that Dipentodon is sister to Tapiscia (Tapisciaceae) and that Dipentodon could be placed in euroside II of the APG system. The clade of Dipentodon and Tapiscia is closest to Malvales and Sapindales. Such finding does not support the previously suggested close relationship between Dipentodon and various other groups, including Celastraceae, Samydaceae, Flacourtiaceae, Hamamelidaceae, and Santalales.

Keywords: Bayesian inference; Chloroplast rbcL; Dipentodon sinicus; Maximum parsimony; Mitochondrial matR; Molecular phylogeny; Nuclear ribosomal 18S.


The genus Dipentodon Dunn consists of a single species D. sinicus Dunn, native to southern China and adjacent Burma and northeastern India (Merrill, 1941; Fischer, 1941; Li, 1986; Thorne, 1992; Bhattacharya and Johri, 1998). Its systematic position has been controversial since it was established and placed in the family Celastraceae in 1911 by S. T. Dunn. For example, Sprague (1925) moved it into the family Samydaceae based on the same variation range of floral base numbers in Dipentodon and Samydaceae. Many authors put the genus into the family Flacourtiaceae (including Samydaceae) (Fischer, 1941; Loesener, 1942; Metcalfe and Chalk, 1950; Lobreau, 1969). Record (1938) considered that Dipentodon is close to Hamamelidaceae based on the wood anatomic characters. Merrill (1941) proposed an independent family Dipentodontaceae Merr. and placed the family in Rosales between Hamamelidaceae and Rosaceae (Merrill, 1941;

Hutchinson, 1959, 1973; Schultze-Motel, 1964; Dahlgren, 1980; Cronquist, 1981; Takhtajan, 1987, 1997). However, Cronquist (1981) put the Dipentodontaceae into the order Santalales based on similar characters of the gynoecial structure. In the update Angiosperm Phylogeny Group (APG) classification scheme of flowering plants, the phylogenetic position of Dipentodontaceae is still uncertain (Angiosperm Phylogeny Group, 1998; APG II, 2003).

Recently, DNA sequences of the chloroplast, nuclear ribosomal, and mitochondrial genes have been widely used in plant phylogenetics, especially in reconstructing angiosperm phylogeny (Hoot et al., 1995; Qiu et al., 1999; Soltis et al., 1998; Kuzoff and Gasser, 2000). More importantly, combining multiple genes from three genomes in plants has proved effective in reducing homoplasy generated by gene-, function-, and genome-specific molecular evolutionary phenomena (Qiu et al., 1999). However, phylogenetic information about Dipentodon is poorly known from molecular data. In the present paper, therefore, we conduct phylogenetic analyses of the rbcL, 18S, and matR sequences to determine the phylogenetic position of Dipentodon and its relationships with related groups.

*Corresponding author. Tel: 86-20-84113677; Fax: 86-20-84113652; E-mail:

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Materials and Methods

In-Group and Out-Groups

According to the APG classification scheme, 45 taxa for rbcL, 42 taxa for matR, and 31 taxa for 18S nrDNA, which represent the families Celastraceae, Samydaceae, Flacourtiaceae, and others from the core eudicots were selected as the in-group. Platanus occidentalis (Platanaceae) and Akebia quinata (Lardizabalanceae) were selected as the out-groups for the phylogenetic analyses. GenBank accessions and the taxa used in this study are listed in Table 1.

DNA Extraction and Sequencing

Total genomic DNA was extracted from fresh and silica-gel-dried leaves using the CTAB procedure (Doyle and Doyle, 1987), and then purified with a DNA purification system (DPS) kit made by our laboratory. Aliquots of the total DNAs were used for sequencing all of the matR gene and part of rbcL and 18S rRNA genes. The PCR products of all samples were purified by using the QIAquick PCR Purification Kit (CN 28104, QIAGEN), and sequenced by using an ABI 377 Genetic Analyzer (Applied Biosystems, CA). All sequences have been deposited in GenBank (for accession numbers see Table 1).

Phylogenetic Analysis

The sequences used in this study were aligned with the Clustal-X program (Thompson et al., 1997) and modified manually. For phylogenetic analyses based on individual genes, the maximum parsimony (MP) method was used with PAUP* 4.0b5 (Swofford, 1999) (the heuristic search option with TBR branch-swapping and simple addition). Characters were assigned equal weights at all nucleotide positions (Fitch, 1971). Gaps were treated as missing data. Bootstrap analyses (Felsenstein, 1985) with 1000 replicates were performed to examine the relative level of support for individual clades on the phylogenetic trees. All phylogenetic trees were rooted using Platanus occidentalis and Akebia quinata as the out-groups.

A combined data set of the chloroplast, nuclear ribosomal, and mitochondrial sequences from 33 taxa was analyzed using the MP method implemented in the PAUP* 4.0b5 and the Baysian inference (BI) method implemented in the MrBayes 2.0 (Huelsenbeck and Ronquist, 2001; Huelsenbeck et al., 2001). Since there was no topological difference between trees of the two out-groups and with either one out-group, P. occidentalis was selected as the out-group for the combined MP and BI analyses. MrBayes uses a MCMC algorithm that runs four Markov chains simultaneously. The Markov chains were started from a

Figure 1. The most parsimonious tree (MPT) based on the combined data set. Numbers above branches represent the bootstrap values (%) for the clades with 1000 replicates. For tree parameters, see Table 2.

Peng et al. — Phylogenetic position of Dipentodon sinicus

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

549 bp; and 4) the combined data set containing 33 taxa was 4,537 bp. The parameters of the most parsimonious trees (MPTs) obtained from the four data sets are presented in Table 2.

The strict consensus trees of the MPTs based on the individual and combined data sets showed a congruent topology. In the combined tree (Figure 1), Dipentodon sinicus is shown to be sister to Tapiscia sinensis (Tapisciaceae), with a relatively high bootstrap support value (95%).

The BI tree based on the combined data set is shown in Figure 2. The sister group relationship between D. sinicus and T. sinensis is strongly supported with a high

random tree and run for 100,000 generations sampling every 50 generations for a total of 2,000 samples each run. The first 100 samples from each run were discarded as burn-in. The gamma distribution (Yang, 1994) and HKY model (Hasegawa et al., 1985) were used in the BI analysis.

Results and Discussion

After sequence alignment, four data sets were formed for phylogenetic analyses: 1) the chloroplast rbcL data set containing 48 taxa was 1,382 bp in length; 2) the nuclear ribosomal 18S data set containing 33 taxa was 1,734 bp; 3) the mitochondrial matR data set containing 44 taxa was

Figure 2. Phylogenetic tree determined by Bayesian Inference from the combined data set. Numbers above branches represent Posterior probabilities (PP). (HKY85 model: Ka=3.12).

Peng et al. — Phylogenetic position of Dipentodon sinicus

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posterior probability (PP = 0.98). The two species are then sister to the clade consisting of three orders: Malvales, Sapindales, and Brassicales with the same posterior probability support (PP = 0.98). The clade, which can be identified as the eurosid II in APG, is also shown to be sister to the clade consisting of the eurosid I clade and the genus Staphylea. Within the eurosid I clade, the sister group relationship between the families Flacourtiaceae and Celastraceae is well supported (PP = 0.97).

Based on the small number of genera sampled, our study suggests that the genus Tapiscia is most closely related to Dipentodon. The genus Tapiscia was established by Oliver (1890) and placed in the family Sapindaceae. However, it has been placed in the family Staphyleaceae by many authors (Diels, 1909; Bean, 1909; Schneider, 1912; Cronquist, 1981) and recognized as a distinct family by Takhtajan (1987). In the APG system, the family Tapisciaceae has not been assigned to any order but at the base of the "eurosid II" (APG, 1998). Simmons et al. (1998) indicated that the data from rbcL and ITS sequences suggested a different ordinal placement for Huertea and Tapscia from other members of Staphyleaceae.

Our study also shows that the clade Dipentodon and Tapiscia and that of Malvales and Sapindales form a sister group. This does not support a close relationship of Dipentodon to any of the families to which it traditionally been considered closely related. Tapisciaceae are also shown to have a distant relationship to Staphyleaceae. However, there are relatively low bootstrap values at some deeper nodes. In general, the results obtained from the three different genes mostly agree with the treatments about those families in APG system (APG, 1998).

Acknowledgments. This study is supported by grants from the National Science Funds for Distinguished Young Scholars (39825104), the Major Research Project of the Chinese Academy of Sciences (KSCXZ-SW-101A), National Natural Science Foundation of China (30070053, 30130030, 30170071), Natural Science Foundation of Guangdong Province (001223), National Ministry of Education Foundation for Key Member Teachers and for Post-doctor program (20010558013) and Qiu Shi Science & Technologies Foundation. We would like to thank Jason Clower of Harvard University for critical reading of the manuscript.

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