_______________________________________________________________________________www.paper.edu.cn J. Plant Res. 113: 253-258, 2000 Journal of Plant Research 0 by The Botanical Society of JaDan 2000 Phylogenetic Analysis of the Sonneratiaceae and its Relationship to Lythraceae Based on ITS Sequences of nrDNA Suhua Shi’*, Yelin Huang’, Fengxiao Tan’, Xingjin He’ and David E. Boufford’ School of Life Sciences, Zhongshan University, Guangzhou 510275, China Harvard University Herbaria, 22 Divinity Avenue, Cambridge, MA 02138-2020,U.S. A. Phylogenetic analyses were conducted using seven Asia. Species of Sonneratia are trees of mangrove swamps species in two genera of Sonneratiaceae, seven species in and seacoasts generally, and the inland genus Duabanga is five genera of the related Lythraceae and 2 outgroups an evergreen component of the rainforest belt (Backer et a/. based on DNA sequences of the internal transcribed spacer 1954). Approximate five to 11 species are recognized in the (ITS) and the 5.8s coding region of the nuclear ribosomal family worldwide, seven of which occur from south central to DNA to determine the proper systematic placement of the southeast China (Backer and Steenis 1951, Duck and Jackes Sonneratiaceae. Paraphyly of the traditional Lythraceae 1987, KO 1983). The mangrove genus Sonneratia occurs was shown with the genus Lagerstmemia nested within the mainly on Hainan Island in China and consists of five native Sonneratiaceae. The Sonneratiaceae occurred within the species, S. a/& Smith, S. caseolaris (L.) Engler, S, hainanensis Lythraceae with high bootstrap value support (96%). The W.C. KO, E.Y. Chen & W.Y. Chen, S. ovata Backer, S. par- two traditional genera constituting Sonneratiaceae were in acaseolaris W.C. KO, and one exotic species introduced from different well-supported clades. Duabanga (Sonner- Bengal, S. apetala Buch.-Ham. (KO 1985, Chen et a/. 1994). atiaceae) is sister to the clade of Lagerstroemia The inland genus Duabanga consists of two to three species, (Lythraceae) (82%). The mangrove genus Sonneratia Duabanga grandiflora (Roxb. ex DC.) Walpers, D. moluccana (1ooo/0) formed the other monophyletic group. It was locat- Blume and D, tay/orii Jayaweera (Jayaweera 1967, KO 1983). ed terminally within the Lythraceae clade and comprised The species of Sonneratia are major components of tall, two clades: one consisting of S. apetala, S. alba, S. ovata, closed-canopy, mangrove forests where they become the and S. hainanensis; the other including S. caseolaris and S. dominant species in the upper layers (Lin 1993) and grow paracaseolaris. The results indicated that species previ- faster than most of their associated species. Because of ously placed in two dierent sections (Sect. Sonneratia and the plants’ adaptation to a peculiar and important ecological Sect. Pseudosonneratia) of Sonneratia occurred within the habitat and their physiognomy, the family, including both same clade, and the taxonomic classification was not mangrove and inland genera, has been studied mor- supported by the molecular analysis of the ITS region phologically (Backer et a/. 1954, KO 1985), anatomically sequences. Based on the phylogenetic analyses of the ITS (Reinders-Gouwentak 1954), ecologically (Lin 1987,1993), regions, the Sonneratiaceae were shown to be nested within cytologically (Chen et a/. 1994, Muller and Hou-Liu 1966, S. the family Lythraceae. Therefore, the sequence data Graham et a/. 1993, Wang eta/.1998), palynologically (Muller presented here do not support the recognition of the Son- 1969, A. Graham et a/. 1990), cladistically (S. Graham 1993), neratiaceae as a distinct family, but instead support the and molecularly (Conti et a/. 1997, Sytsma et a/. 1999). The inclusion of Sonneratiaceae in the Lythraceae proposed by two genera have been placed in various other families by other authors. different authors (Lindley 1836, Miquel 1855, Bentham and Hooker 1867, Koehne 1881, Niedenzu 1898) before Engler and Key words: Sonneratiaceae - Lythraceae - Phylogeny Gilg (1924) proposed the family Sonneratiaceae to include - ITS regions - Ribosomal DNA the two genera, which has been adapted by many other authors (Melchior 1964, Hutchinson 1973, Cronquist 1988). In some recent classification systems, the Sonneratiaceae The Sonneratiaceae comprise only two small genera, have been included in the Lythraceae (Thorne 1992, Dahl- Sonneratia, which extends from tropical eastern Africa and gren and Thorne 1984, Angiosperm Phylogeny Group, APG, adjacent islands to Queensland of Australia, Micronesia and 1998) or separated into two families, Sonneratiaceae and Melanesia, and Duabanga, which is confined to southeastern Duabangaceae (Takhtajan 1997). The relationships among the species of Sonneratia are somewhat uncertain. They * Corresponding author. Tel: +86 20-8411-3677; Fax: +86 20- have been studied by Duck and Jackes (1987) and by KO 84113652; e-mail: [email protected] (1983,1985,1993) who described some new species and _______________________________________________________________________________中国科技论文在线 www.paper.edu.cn 254 S. Shi et a/. proposed a sectional level system of classification. sequencing kit (US70770, Amersham) and alpha 35S-dATP The objectives of our study were to (1) reconstruct the as a radioactive tracer and compared for all taxa to ensure phylogeny of the Sonneratiaceae based on sequences of the accuracy. The DNA sequences were assembled and the ITS regions of the nrDNA; (2) discuss the relationships boundaries between the coding and spacer regions were between the Sonneratiaceae and Lythraceae; and (3) eval- determined by comparison with the sequences of carrot uate and discuss previous taxonomic treatments of the (Yokota et a/. 1989). The assembled sequences were family. aligned using CLUSTAL X (Thompson et a/. 1997). Sequences generated in this study were submitted to Gen- Materials and Methods Bank. Parsimony analyses were conducted using PAUP 4.0 (Swofford 1999). Parsimony analysis was performed using a This study included sequences from ten taxa of Sonner- branch-and-bound search. Bootstrap analyses were car- atiaceae plus seven species of Lythraceae and two taxa ried out with 1000 replicates using TBR branch-swapping of from related families (Lecythidaceae and Myrtaceae) as the heuristic search (Felsentein 1985). The Kimura (1980) outgroups (Tablei). Seven species in two genera of Son- two-parameter model (Table 2) was used for correcting neratiaceae (following KO1993) are represented in addition to possible multiple hits of nucleotide substitutions. All seven species in five genera of Lythraceae. Rhodomyrtus phylogenetic trees were rooted using Rhodomyrtus tomentosa (Aiton) Hassk. (Myrtaceae) and Combretum alfredii tomentosa (Myrtaceae) and Combreturn alfredii (Com- Hance (Combretaceae) were selected as outgroups to root bretaceae) as outgroups. the trees on the basis that all of the above families have been placed in the same order, Myrtales, in numerous Results and Discussion taxonomic treatments (Takhtajan 1997, Cronquist 1988, Angiosperm Phylogeny Group 1998). The total length of the ITSI, 5.8S, and ITS2 regions of the Total genomic DNAs were extracted from fresh and silica- Sonneratiaceae range from 601 to 629 bases with an ITS1 of dried leaves using the CTAB procedure (Doyle and Doyle 235-240 bases, a 5.8s of 165-167 bases and an ITS2 of 210- 1987), followed by purification with glass milk (made by our 223 bases. The aligned ITS sequences formed in a matrix laboratory). The ITS region of the nrDNA was amplified of 658 nucleotide sites. Of the 658 sites, 301 sites are following Wen and Zimmer (1996). The PCR products of ITS variable and 229 sites are phylogenetically informative. The were purified with centrifugal filter units (UFC3LTKNB, maximum parsimony analysis, treating gaps as missing data, Millipore). Sequencing was done using the Automated generated only a single most parsimonious tree with a tree Sequencer 377 (Applied Biosystems, CA) at China Agricul- length of 938, a consistency index (Cl) of 0.6930, a retention tural University. DNA strands of partial samples were se- index (RI) of 0.6916 and a rescaled consistency index (RC) of quenced manually using the Sequenase Version 2.0 DNA 0.4793 (Fig. 1). The Kimura (1980) two-parameter sequence Table 1. Accessions of Sonneratiaceae, Lythraceae and outgroups sampled in this study. Classification based on Takhtajan (1980). Voucher specimens are at SYS. Species Family Voucher Collection Source GenBank Acc. No Duabanga grandiflora (1) Sonneratiaceae Huang 990407 Cult., Liangfengjiang, Guangxi, China AF163695 Duabanga grandiflora (2) Sonneratiaceae Huang 9942707 Cult., Jianfengling, Hainan, China AF208695 Sonneratia apetala (1) Sonneratiaceae Qiu 974372 Cult., Futian, Guangdong, China AF163697 Sonneratia apetala (2) Sonneratiaceae Chang 977 7907 China. Dongzaigang, Hainan AF208697 Sonneratia caseolaris (1) Sonneratiaceae Qiu 974304 Cult., Futian, Guangdong, China AF163698 Sonneratia caseolaris (2) Sonneratiaceae Huang 990435 Cult., Futian, Guangdong, China AF208696 Sonneratia paracaseolaris Sonneratiaceae Chang 977797 7 China. Dongzaigang, Hainan AF163699 Sonneratia hainanensis Sonneratiaceae Chang 977 7908 China. Dongzaigang, Hainan AFl63700 Sonneratia alba Sonneratiaceae Chen 990603 China. Dongzaigang, Hainan AF163701 Sonneratia ovata Sonneratiaceae Chang 977 7972 China. Dongzaigang, Hainan AF163702 Cuphea hookeriana Lythraceae Tang 99070507 Cult., Guilin, Guangxi, China AF201691 Heimia myrtifolia Lythraceae Tang 99070502 Cult., Guilin,