Litsea Complex, Lauraceae) Inferred from Sequences of the Chloroplast Gene Matk and Nuclear Ribosomal DNA ITS Regions

Plant Syst. Evol. 246: 19–34 (2004) DOI 10.1007/s00606-003-0113-z Phylogenetic relationships within the ‘core’ Laureae (Litsea complex, Lauraceae) inferred from sequences of the chloroplast gene matK and nuclear ribosomal DNA ITS regions Jie Li1, D. C. Christophel2, J. G. Conran3, and Hsi-Wen Li1 1Laboratory of Plant Phylogenetics and Conservation Biology, Xishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, Kunming, Yunnan, P.R. China 2Department of Biological Science, University of Denver, Iliff, Denver, CO, USA 3Centre for Evolutionary Biology and Biodiversity, Discipline of Environmental Biology, School of Earth and Environmental Sciences, Darling Building DP418, The University of Adelaide, Australia Received April 17, 2003; accepted November 24, 2003 Published online: February 26, 2004 Ó Springer-Verlag 2004 Abstract. A phylogenetic analysis of the ‘core’ Key words: Lauraceae, Laureae, matK, ITS, Laureae (Litsea complex) was conducted using the molecular phylogeny, Litsea, Lindera, classification. chloroplast gene matK and nuclear ribosomal DNA ITS sequences to investigate generic relationships The ‘core Laureae’ or Litsea complex consists and boundaries within the complex. Despite low genetic divergence for matK, rooting of the tree with of ten genera with 500–700 species concen- Sassafras resulted in Iteadaphne as the basal member trated in tropical-subtropical Asia, but with of the complex and five resolved clades: a Neolitsea representatives in Australasia, the Mediterra- clade and then Laurus, Parasassafras, Litsea and nean, North and Central America. The Lindera clades in a large polytomy with unresolved monophyly of the complex is well supported Lindera sections plus Umbellularia. A combined (Chanderbali et al. 2001; Rohwer et al. 1991; analysis of the data (identical to the ITS results) Rohwer 2000; Li 1985, 1995; van der Werff provided a more resolved phylogeny of the Laureae, and Richter 1996) and it is distinguished from with four major lineages: the Laurus, Litsea, Lindera other Lauraceae by possessing basically thyr- and Actinodaphne II clades. These clades also appear soid open, pseudo-racemose inflorescences to reflect the importance of inflorescence structure bearing involucral bracts and which appear and ontogeny within the Laureae, as well as data to be umbellate due to short axes, and mainly from cuticular micromorphology, but there was no support for traditional generic characters such as introrse anthers in the third whorl (Li and 2- versus 4- celled anthers. As a result, genera such as Christophel 2000, Li 2001). Actinodaphne, Litsea, Neolitsea and Lindera were Generic delimitation within the complex polyphyletic in all analyses. Parasassafras was has traditionally been problematic (Koster- related to Sinosassafras by the matK data, but mans 1957, Hutchinson 1964, Richter 1981), distant from it in the ITS and combined analyses. the major point being the significance of 20 J. Li et al.: Molecular phylogeny of the Litsea complex 2-locular versus 4-locular anthers as generic shaped fruiting cupules; sect. Daphinidium descriptors, although modern Lauraceae clas- (Nees) Benth. has evergreen, trinerved leaves sifications, tend to downplay the significance and un-developed terminal buds on shortened of anther cell number in generic delimitation brachyblasts; sect. Polyadenia Nees is ever- (e.g. Rohwer et al. 1991, Rohwer 1993, van der green with penninerved leaves and well-devel- Werff and Richter 1996). Li and Christophel oped terminal buds on shortened (2000) incorporated traditional morphological brachyblasts and sect. Uniumbellatae Tsui characters with leaf cuticles in a phylogenetic has evergreen, trinerved leaves, and the analysis of the complex, but although they also long-pedunculate pseudo-umbel is solitary in concluded that anther cell number did not the axil of a normally developed leaf. have strong phylogenetic value at the generic In previous Lauraceae classifications, the level, they also demonstrated that morpholog- number of anther cells was a critical character ical data were insufficient to resolve relation- in distinguishing the different genera and ships within the Laureae. Kostermans (1957) considered it as the third The genus Litsea was divided into four most important character after inflorescence sections under the classification of Li et al. type and fruiting cupule shape. This view was (1984), with sect. Litsea defined as evergreen followed by Li (1985, 1995), Tsui (1987) and with alternate, penninerved leaves, a racemi- Hyland (1989), although Li (1985, 1995) con- form inflorescence and non-enlarged perianth sidered that the relationship may not be as tubes with absent or reduced perianth lobes. close as originally thought, and that the Sect. Conodaphne (Bl.) Benth. et Hook. f. is similarities between Litsea and Lindera may evergreen with alternate or opposite penni- be the result of parallel evolution. Li (1985) nerved leaves and non- to slightly enlarged and Tsui (1987) also suggested that there is perianth tubes. Sect. Cylicodaphne (Nees) parallel evolution between and within Litsea Hook. f. was considered to be evergreen with and Lindera at the generic and sectional levels, alternate, penninerved leaves, an enlarged making generic delimitation difficult using perianth tube and cup-shaped fruiting cupule. traditional morphological characters. Sect. Tomingodaphne (Bl.) Hook. f. has decid- Because morphological data did not pro- uous, alternate, penninerved leaves and non- vide strong phylogenetic signal, our study enlarged, 6-lobed perianth tubes. involving molecular sequences was undertaken Lindera was similarly divided into eight to find a more robust phylogeny for the sections by Tsui (1987) as follows: sect. Laureae. The matK gene is one of the most Lindera with deciduous, penninerved leaves rapidly evolving coding regions found so far in and well-developed terminal buds on short- the chloroplast genome (Olmstead and Palmer ened brachyblasts; sect. Sphaerocarpae Tsui 1994), and its high rates of synonymous and with deciduous; triplinerved or trinerved especially non-synonymous substitutions mean leaves and well-developed terminal buds on that it is considered appropriate for construct- shortened brachyblasts and sect. Palminervia ing infrafamilial phylogenies (e.g. Johnson and Meissn. with deciduous, lobed, trinerved Soltis 1994, 1995; Steele and Vilgalys 1994; leaves and well-developed terminal buds on Plunkett et al. 1996, 1997; Liang and Hilu shortened brachyblasts. Sect. Aperula (Bl.) 1996; Kron 1997; Gadek et al. 1996; Manos Benth. has evergreen, penninerved leaves, and Steele 1997; Xiang et al. 1998; Denda well-developed terminal buds on shortened et al. 1999), Nevertheless, Rohwer (2000) brachyblasts and long-pedunculate, racemi- found that matK sequence divergence in form inflorescences; Sect. Cupuliformis Tsui Lauraceae was surprisingly low (only 9.7% possesses evergreen, penninerved leaves, fun- informative characters), so a second marker nel-shaped glands on the third anther whorl was incorporated to improve recovery of and enlarged perianth tubes forming cup- phylogenetic signal (Chase et al. 1997). J. Li et al.: Molecular phylogeny of the Litsea complex 21 The ITS spacer region, has been utilised as in Actinodaphne in lateral sub-sessile fascicles’’. widely in phylogenetic studies of many taxa of van der Werff and Richter (1996) also considered flowering plants (e.g. Baldwin et al. 1995) and Sassafras to be close to the Laureae, noting that recent work on Lauraceae by Chanderbali both lack marginal parenchyma and possess similar et al. (2001) found that it provided good signal phloem fibres in the wood, nevertheless, it is resolution and therefore should be suitable for separated from the Laureae by its unique accentu- ated growth ring structure in both secondary xylem the Laureae. However, because ITS is a and phloem. nuclear gene spacer, phylogenetic conclusions Similarly, although Umbellularia is usually using ITS sequences are advised to be corrob- placed within the Laureae (Kostermans 1957, orated with data from other sources, including Rohwer 1993, van der Werff and Richter 1996), plastid genes such as matK (Donoghue and Chanderbali et al. (2001) and Rohwer (2000) Sanderson 1992). suggested that it should be placed in the Cinnamo- The goals of our study using maximum meae variously on the basis of ITS, trnL-F, parsimony analysis of sequence data from psbA-trnH and matK sequences and morphology nuclear ITS (internal transcribed spacer of (bisexual flowers and extrorse anthers in the ribosomal genes) and the chloroplast matK innermost staminal whorl). As a result, we have gene are to: (1) examine the circumscription of included it in our study, but treated it in the the Laureae; (2) test the hypothesis that Litsea, analyses as an outgroup. DNA extraction. Total DNA was extracted Lindera, Actinodaphne and Neolitsea are each using the CTAB method of Doyle and Doyle monophyletic; and (3) investigate major lin- (1987). Approximately 1 cm2 leaf tissue was ground eages in order to try to explain evolution with a minute amount of sterile sand and 1 ml of within the complex. warmed (60 °C) CTAB + BME in a warmed (60 °C) mortar and pestle; an additional 1 ml CTAB was added and mixed, then the slurry was Materials and methods poured into two 1.5 ml tubes and incubated at Ingroup sampling. Taxon sampling included repre- 60 °C in a water bath for 45 minutes. Two volumes sentatives of all previously recognised generic of chloroform: isopropanol (24:1) were added, the segregates

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