Botanical Studies (2007) 48: 227-232. PLANT BIOSYSTEMATICS Paraphyly and phylogenetic relationships in Lasianthus (Rubiaceae) inferred from chloroplast rps16 data Long-Qian XIAO and Hua ZHU* Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, 650223, Yunnan, P.R. China (Received November 21, 2005; Accepted August 25, 2006) ABSTRACT. A phylogenetic analysis of Lasianthus and some representatives of tribes in subfamily Rubioideae based on the chloroplast rps16 data indicates that Lasianthus as currently circumscribed is paraphyletic, because Saprosma crassipes, representative of the species with two locules per ovary developing into two pyrenes per drupe with a thin wall, and Litosanthes biflora, the species in the monotypic genus Litosanthes, are nested within the highly supported Lasianthus clade. The present delimitation of the tribe Lasiantheae, which includes Saldinia and Trichostachys, is supported by our results. Finally, our results are inconclusive for evaluating the monophyly of the infrageneric classification of Lasianthus. Keyword: Lasianthus; Lasiantheae; Litosanthes; Paraphyly; rps16 intron. INTRODUCTION a thick wall. He also restored Saldinia as a separate genus, and merged part of the species with 2-locular ovaries Lasianthus Jack is a large pantropical genus in developing into 2-pyrene drupes in Lasianthus. Rubiaceae comprising more than 180 species. Of these, The tribal position of Lasianthus has also been c. 160 species occur in tropical Asia, with one extending controversial. Traditionally, Lasianthus was placed in to Australia, c. 20 in tropical Africa and three in tropical the tribe Psychotrieae based on aestivation of the corolla America. The members of Lasianthus are exclusively lobes and the position, attachment, and types of its ovules confined to primary rainforests throughout their (Hooker, 1880; Schumann, 1891). Petit (1964) proposed geographic ranges. The distribution pattern of Lasianthus new circumscriptions for Psychotrieae and Morindeae appears to be important for understanding biogeography and transferred Lasianthus to Morindeae based on its and speciation in tropical rainforests (Zhu, 2002). seeds, which have soft oily endosperm and large embryos. Some regional taxonomic revisions have been made However, molecular data based on a few samples (Bremer, for Lasianthus [e.g., Verdcourt (1976); Denys (1981) 1996; Andersson and Rova, 1999; Piesschaert et al., 1999; for Africa, Wong (1989) for the Malay Peninsula; Deb Bremer and Manen, 2000) indicated that Lasianthus and Gangopadpyay (1991) for India, Zhu (2001) for appeared to be related to Pauridiantha, Perama, Thailand, and Zhu (1994, 1998, 2002) for Eastern Asia]. Trichostachys, and Saldinia. Bremer and Manen (2000) However, the delimitation of Lasianthus has always placed Lasianthus, along with Saldinia and Trichostachys, been controversial and remains unsettled. Jack (1823) in the tribe Lasiantheae. In addition, the only available originally described Lasianthus as a 4-locular ovary comprehensive infrageneric classification of Lasianthus bearing a single basally erected ovule per locule, and a was Hooker’ classification (1880) based mainly on drupe with four pyrenes. Blume (1826) enlarged Jack’ quantitative characters, such as the size of stipules, the s original circumscription to include species with 4-9 occurrence of bracts, and peduncles. Hooker divided locular ovaries and drupes with 4-9 pyrenes. Wight (1846) Lasianthus into four sections: Bracteatae, Nudiflorae, and Korthals (1851) added some species with 2-locular Stipulares, and Pedunculatae. ovaries, developing into 2-pyrene drupes. Later these The identity of the Asian monotypic genus Litosanthes, species were transferred to Saprosma (Schumann, 1891; L. biflorus, has also been controversial. Litosanthes is Boerlage, 1899). In addition, the Madagascar genus characterised by its imbricate corolla, forked stipules, and Saldinia, with 2-locular ovaries and drupes with 1-pyrene, pedunculate inflorescences. Some Asian Lasianthus with was once placed under Lasianthus as a subgenus, (Baillon, pedunculate inflorescences were transferred to Litosanthes 1880). Furthermore, Bremekamp (1957) proposed a new (Deb and Ganopadhyay, 1989; 1991) and recently classification of Lasianthus as species with two or more returned to a section of Lasianthus (Gangopadhyay and locules per ovary and two or more pyrenes per drupe with Chakrabarty, 1992). In the Flora of China, however, * Corresponding author: E-mail: [email protected]; Litosanthes biflorus is treated as a monotypic genus (Lo, Telephone: +86-871-5110721; Fax: +86-871-5160916. 1999). 228 Botanical Studies, Vol. 48, 2007 Here we present a ribosomal protein S16 (rps16) intron (DNASTAR package) and then adjusted manually. phylogenetic analysis with 11 Lasianthus from the tropical Deletions were coded as missing data. Africa, America, and Asia, and 28 representatives from the Maximum parsimony (MP) analysis was performed recognized tribes in the subfamily Rubioideae based on using PAUP 4.0b10 (Swofford, 2001) treating gaps as the classification proposed by Bremer and Manen (2000). missing data using heuristic search options with 1,000 The rps16 intron was chosen because the marker has random replications of stepwise data addition and TBR proven useful for inferring phylogenetic relationships at swapping and Multrees on no tree limit with all characters generic or higher levels (e.g. Andersson and Rova, 1999; weighted equally and unordered. Bootstrap analysis Bremer and Manen, 2000; Nie et al., 2005). Additionally, (Felsenstein, 1985) was performed with 1,000 replicates to many published Rubioideae rps16 sequences are available evaluate internal support. for our studies. Pairwise comparisons of the 17 chloroplast introns shared between tobacco (Nicotiana tabacum L.) RESULTS and rice (Oryza sativa L.) indicate that the rps16 intron is one of the most divergent, with 67% sequence similarity All the newly acquired sequences were submitted to (Downie et al., 1996). The following questions are to be GenBank (Table 1). The total length of 1,191 nucleotides addressed in particular: (1) Is the current circumscription of the rps16 intron sequences in the data matrix, including of the genus Lasianthus monophyletic? (2) What are the 41 species, was determined, and 327 were parsimony- relationships of Lasianthus with other Lasiantheae genera? informative (27.5%). A parsimony analysis of the (3) What are the infrageneric relationships of Lasianthus rps16 intron data matrix resulted in 970 equally most from tropical America, Africa and Asia? (4) And, finally, is parsimonious trees, each with 819 steps, CI = 0.6119, this phylogeny consistent with Hooker’s classification? and RI = 0.807. The strict consensus tree is shown in Figure 1. Saprosma crassipes, Litosanthes biflorus, and all MATERIALS AND METHODS sequenced Lasianthus species formed a strongly supported (BP = 92) monophyletic group. This mostly Lasianthus Eleven species representing Lasianthus from tropical clade was resolved as sister to a highly supported (BP = Africa, tropical America, and tropical Asia, and 28 98) clade containing Saldinia and Trichostachys. However, species representing all recognized tribes in the subfamily the support for this sister-group relationships was weak Rubioideae (except Spermacoceae and Theligoneae) (BP = 58). The Lasiantheae clade was in turn resolved (Bremer and Manen, 2000) were sampled as ingroups. as sister to the tribe Perameae, represented by Perama The outgroups were designated as Ixora amplexicaulis hirsuta. The highly supported (BP = 100) Lasiantheae- (Ixoroideae) and Cinchona pubescens (Cinchonoideae) Perameae clade was resolved with strong support (BP based on the identification of the monophyly of subfamily = 100) as sister to a highly supported (BP = 98) large Rubioideae (Bremer et al., 1995). The materials collected clade containing Saprosma ternatum and the remaining in this study were identified by the second author, Dr sequenced Rubioideae taxa, formally classified into Zhu, H., a specialist on Lasianthus. All sequences have eleven tribes (Morindeae, Gaertnereae, Schradereae, been deposited in GenBank. (For accession numbers Psychotrieae, Craterispermeae, Anthospermeae, for the rps16 intron sequences and vouchers/references Paederieae, Argostemmeae, Danaideae, Coussarieae, information see Table 1.) Urophylleae, and Ophiorhizeae). The two studied species Total genomic DNA was extracted from silica-dried or of the genus Saprosma, S. crassipes and S. ternatum, fresh leaves using a modified CTAB procedure (Doyle and did not form a clade. Finally, the African Lasianthus Doyle, 1987). The primers rpsF and rpsR2 described by batangensis and the Neotropical L. lanceolatus formed a Oxelman et al. (1997) were used for amplifying the rps16 monophyletic group while the sequenced Asian Lasianthus intron from the genomic DNA. PCR reaction volumes did not group together as a clade. (30 ul) contained 1.5 U of Ampli Taq DNA polymerase (Perkin-Elmer 9600). Reactions were incubated at 95°C DISCUSSION for 3 min, then cycled 35 times (95°C for 1 min, 55°C for 1 min, 72°C for 1.5 min), followed by a final extension for Delimitation of Lasianthus 10 min at 72°C. Double-stranded products were purified Lasianthus as presently delimited is not monophyletic, using the E.Z.N.A. Cycle-Pure Kit (Omegabio-tek, USA). unless Litosanthus and Saprosma crassipes are included. Sequencing reactions were performed using PRISM Dye In other words, the species with two