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J. Res. 113: 253-258, 2000 Journal of Plant Research 0 by The Botanical Society of JaDan 2000

Phylogenetic Analysis of the and its Relationship to 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 are trees of swamps species in two genera of Sonneratiaceae, seven species in and seacoasts generally, and the inland 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 (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 ’ 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 bretaceae) as outgroups. the trees on the basis that all of the above families have been placed in the same order, , 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 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 (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 Sonneratiaceae Chen 990603 China. Dongzaigang, Hainan AF163701 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, Guangxi, China AF201693 Lagerstroemia indica Lythraceae Shi 9963007 Cult., Lijiang, Yunnan, China AF163696 Lagerstroemia speciosa Lythraceae Shi 990477 Cult., Guangzhou, Guangdong, China AF201689 Lagerstroemia tomentosa Lythraceae Shi 99070620 Cult., Kunming, Yunnan, China AF201688 Lawsonia inermis Lythraceae Qiu 99070207 Cult., Guangzhou, Guangdong, China AF201690 Woodfordia fruticosa Lythraceae Tang 99070504 Cult., Guilin, Guangxi, China AF201692 Combretum alfredii Cornbretaceae Tang 99037504 China. Lipu, Guangxi Afl60471 Rhodomyrtus tomentosa Myrtaceae Shi 9704773 China. Heishiding, Guangdong AF105085 ______中国科技论文在线 www.paper.edu.cn

Phylogenetic Analysis of Sonneratiaceae 255 ______中国科技论文在线 www.paper.edu.cn

256 S. Shi et a/.

Rhodomyrtus tomeniosa* placement of the two genera in the Sonneratiaceae (Mel- ICuphea hookeriana chior 1964, Hutchinson 1973, KO 1983, Cronquist 1988). 71 Takhtajan (1987,1997), however, separated them into two IWoodfordia fruticosa families, the Sonneratiaceae and the Duabangaceae in his new systems. At the same time, Thorne (1992), Dahlgren -00 and Thorne (1984) and Angiosperm Phylogeny Group (1998) Lawsonia inermis included the Sonneratiaceae into the family Lythraceae. Sonneraiia apeiala(1) Based on phylogenetic analyses of the ITS sequences, flSonneratia apetala(2) paraphyly of the traditional Lythraceae was shown with the genus Lagerstroemia nested within the Sonneratiaceae. onneratia ovata The Sonneratiaceae occur within the Lythraceae with high c: Sonneraiia hainanensis bootstrap value support (96%) in the MP tree (Fig. 1). The Sonneratia alba two traditional genera constituting Sonneratiaceae are in different, well-supported clades. The inland genus Duaban- Sonneratia caseolaris(1) ga (Sonneratiaceae) is sister to the clade of Lagerstroemia Sonneraiia caseolaris(2) (Lythraceae) (82%). The mangrove genus Sonneratia (100%) formed the other monophyletic group. The clade of the Sonneratia paracaseolaris genus Duabanga and Lagerstroemia is sister to the clade of Lagerstroemia tomeniosa Sonneratia. The other genera of Lythraceae sampled in this 6Lagerstroemia indica study are shown to be basal to the clades of Lagerstroemia and Duabanga, and Sonneratia. The first clade is Lawsonia, ILagerstroemia speciosa the second is Heimia and the third comprises Cuphea and Duabanga grandifora(1) Woodfordia. The ITS sequence data presented here there- 100 “i c fore do not support recognition of the Sonneratiaceae as a Duabanga grandiyora(2) distinct family, but instead, support the opinion that it should Combretum alfedii* be included within the family Lythraceae. The sequence Fig.l. The single most parsimonious tree of Lythraceae and divergences between Sonneratia and Duabanga (17.70%- Sonneratiaceae based on ITS regions (length=938, CI = 21.10%) are higher than those between Duabanga and Lager- 0.693, RI=0.691, Hl=0.369, RC=0.479). Gaps were stroemia (16.99%-22.13%), and between Sonneratia and treated as missing data. Numbers represent the bootstrap Lagerstroemia (14.97%-16.08%), which indicates that the supports (O/O) for the clades. Asterisk (*) indicates out- genera Sonneratia and Duabanga are phylogenetically iso- group. lated from each other. Sonneratia also differs from Duaban- ga in its ecological preferences and adaptive structures of divergences between Sonneratia and Duabanga range from the pneumatophores and knee . However, the two 17.70% to 21.10%. But the sequence divergences between genera are distinguished from the other genera of Lagerstroemia (Lythraceae) and Sonneratia, and Duabanga Lythraceae by flower, pollen and anatomical characteristics range from 16.99% to 22.13%, and 14.97% to 16.08% respec- too (Jayaweera 1967, KO 1983, A. Graham et a/. 1990). The tively, lower than that between the two genera Sonneratia two should be treated as distinct, but related, genera in the and Duabanga. In the genus Sonneratia the highest diver- Lythraceae. These results support the opinions of Bentham gence is 7.11% between s. caseolaris and s. alba, while the and Hooker (1867), Koorders and Valeton (1894), Thorne lowest value is 2.11% between S. hainanensis and S. alba. (1992), Dahlgren and Thorne (1984) and Angiosperm The sequence divergences among all genera of Lythraceae Phylogeny Group (1998). and Sonneratiaceae sampled in this study range from 16.08% The monophyly of Sonneratia is strongly supported with a to 35.58%. The lowest divergence is between Duabanga 100% bootstrap value (Fig. I), located terminally within the grandiflora and Lagerstroemia tomentosa, and the highest is Lythraceae clade. Sonneratia has been classified into two between Sonneratia apetala and Cuphea hookeriana. sections, Sonneratia and Pseudosonneratia, based on the The phylogenetic positions of the two genera of Sonner- presence or absence of (KO 1993). Section Sonner- atiaceae and the relationships with Lythraceae have been atia, consisting of S. alba, S. caseolaris and S. paracaseolaris, discussed in detail since last century by many authors. has flowers with petals; Section Pseudosonneratia, compris- Lindley (1836) placed Sonneratia in the tribe Myrteae ing S. apetala, S. ovata, S. hainanensis and S. griffithii, has (Myrtaceae) and Duabanga in the Lagerstroemieae flowers without . As noted in the phylogenetic tree (Lythraceae). Miquel (1855) placed Sonneratia in the tribe (Fig. I), paraphyletic relationships are shown among the Sonneratieae (Myrtaceae) and Duabanga in Lythreae species of KO’stwo sections. A close relationship is shown (Lythraceae). Bentham and Hooker (1867) included both between Sonneratia caseolaris and S. paracaseolaris by the genera in the Lythreae (Lythraceae) and this was adopted by very low sequence divergence value (2.43%). Their clade, Koorders and Valeton (1894). Koehne (1881), however, ex- however, is supported with a 72% bootstrap value. Sonner- cluded both genera from his monograph on the Lythraceae. atia apetala, S. ovata, S. alba and S. hainanensis form another However, many authors agreed with Engler and Gilg’s (1924) clade in the genus with 75% bootstrap value support. Their ______中国科技论文在线 www.paper.edu.cn

Phylogenetic Analysis of Sonneratiaceae 257 clade is separated into two branches: 1) S. apetala is close Duck, N.C. and Jackes, B.R. 1987. A systematic revision of to S. ovata (sequence divergence of 4.13%) with 61% boot- the mangrove genus Sonneratia (Sonneratiaceae) in strap value support; and 2) s. aba and s. hainanensis have Australasia. Blumea 32: 277-302 a very close relationship (sequence divergence of 22.11%) Engler, A. and Gilg, E.F. 1924. Sonneratiaceae. Syllabus- with a very high bootstrap value support (97%). Those der Pflanzenfamilien, ed. 9 and 10: 299. results indicate that species formerly placed in two different Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791. sections (Sect. Sonneratia and Sect. Pseudosonneratia) of Graham, A., Graham, S.A. Nowicke, J., Patel, V. and Lee, S. Sonneratia occur within the same clade. KO's taxonomic 1990. Palynology and systematics of the Lythraceae. classification (1985,1993) is therefore not supported by the 111. Genera Physocalymma through Woodfordia, adden- molecular analysis of the ITS regions. study has This da, and conclusions. Amer. J. Bot. 77: 159-177. provided data for reconstructing the phylogeny of the Son- Graham, S.A. 1992. New chromosome counts in neratiaceae and determining its relationship with the related Lythraceae-systematic and evolutionary implications. family Lythraceae. The phylogenetic analyses of the ITS Acta Bot. Mex. 17: 45-51. sequences indicate that the Sonneratiaceae are not a Graham, S.A. Crisci, J. and Hoch, P.C. 1993. Cladistic distinct family, but should be included within the Lythraceae. analysis of the Lythraceae sensu lato based on mor- High sequence divergences, however, which range from phological characters. Bot. J. Linn. SOC.113: 1-33. 16.07%-33.58%, are shown among the genera of the Graham, S.A., Thorne, R.F. and Reveal, J.L. 1998. Valida- Lythraceae and Sonneratiaceae. It is therefore necessary tion of subfamily names in Lythraceae. Taxon 47: to obtain more sequence data from different genes, espe- 435-436. cially genes with a slower rate of evolution, to determine Hutchinson, J. 1973. The Families of Flowering Plants. exact relationships. ed. 3. Clarendon, Oxford. Jayaweera, D. 1967. The genus Duabanga. J. Arnold Arbor. 48: 89-100 We wish to thank Professor Chen Shoucai of the Chinese Kimura, M. 1980. A simple method for estimating evolu- Academy of Tropical Agricultural Science, Tang Shaoqing of tionary rates of base substitutions through comparative Guangxi Normal University, Huang Shiman of Hainan Univer- studies of nucleotide sequences. J. Molec. Evol. 16: sity and Huang Shineng of the Guangdong Institute of 111-120. Tropical Forest Science for kindly providing tissues and KO, W.C. 1983. Sonneratiaceae. In Flora Reipublicae two anonymous reviewers for their valuable comments. 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