Phylogeny and Biogeography of Tsuga (Pinaceae) Inferred from Nuclear Ribosomal ITS and Chloroplast DNA Sequence Data

Hemlock, Tsuga (Pinaceae), has a disjunct distribution in North America and Asia. To examine the biogeographic history of Tsuga, phylogenetic relationships among multiple accessions of all nine species were inferred using chloroplast DNA sequences and multiple cloned sequences of the nuclear ribosomal ITS region. Analysis of chloroplast and ITS sequences resolve a clade that includes the two western North American species, T. heterophylla and T. mertensiana, and a clade of Asian species within which one of the eastern North American species, T. caroliniana, is nested. The other eastern North American species, T. canadensis, is sister to the Asian clade. Tsuga chinensis from Taiwan did not group with T. chinensis from mainland China, and T. sieboldii from Ullung Island did not group with T. sieboldii from Japan suggesting that the taxonomic status of these distinct populations should be reevaluated. The Himalayan species, T. dumosa, was in conflicting positions in the chloroplast and ITS trees, suggesting that it may be of hybrid origin. Likelihood-based biogeographic inference with divergence time estimates infers an Eocene basal crown group diversification and an initial widespread circumpolar distribution with subsequent vicariance and extinction events leading to the current disjunct distribution. Keywords—ancestral area reconstruction, internal transcribed spacer, phylogenetic incongruence, trnK–matK intron, trnL–F region, rpl16 intron. Hemlock, Tsuga (Endlicher) Carrière (Pinaceae), is an important conifer of subalpine and lowland forest communities in North America and Asia (Wang 1961; Hirokawa 1972, 1976; Burns and Honkala 1990). Hemlocks are slow-growing, long-lived, shade-tolerant trees that cannot tolerate persistent drought. While certain species, especially T. heterophylla in western North America, are a good source of lumber and fiber, Tsuga is generally considered to be more valuable for its ecological functions and aesthetic value. Hemlocks provide important resources for wildlife, including deer (Mladenoff and Stearns 1993), birds (Benkman 1993), and salamanders (Brooks 2001). They are also horticulturally important with more than 250 named varieties and cultivars of T. canadensis alone (Swartley 1984; Cope 1992). Up to 25 extant species of Tsuga have been described, with eight to 13 species accepted by various authors (Fitschen 1929; Flous 1936; Gaussen 1966; Cheng 1983; Silba 1986; Farjon 1990; Page 1990; Fu et al. 1999). We follow Farjon (1990) who recognized nine species, three of which occur in China and the Himalayas, two in Japan, two in western North America, and two in eastern North America (Fig. 1). A comprehensive phylogenetic analysis of Tsuga has not been undertaken. Matsumoto et al. (1995) used eight morphological characters, and Taylor (1972) used phytochemical chromatography to infer Tsuga phylogeneny. Analyses of the nuclear ribosomal internal transcribed spacer (ITS) region agreed that the western North American species are sister to the remaining species, and that the two eastern North American species, T. caroliniana and T. canadensis, were not sister to each other, but that T. caroliniana is located in a clade that includes all of the Asian species (LePage and Bayer 1997; Vining 1999; LePage 2003a). The remaining relationships within Tsuga were not well resolved. The objectives of this study were to reconstruct the phylogeny of Tsuga using nuclear internal transcribed spacer (ITS) and chloroplast DNA (cpDNA) sequence data, and to examine the biogeographic history of the genus. ITS is biparentally inherited, and cpDNA is paternally inherited in the Pinaceae (Mogenson 1996), which allows us to examine potential discordance in ITS and cpDNA phylogenies and to infer possible instances of hybridization. We used the trnK– matK intron, rpl16 intron, and trnT–F cpDNA regions, as these have been used successfully for phylogenetic studies of other groups of closely related plants (e.g. Gernandt et al. 2003; Bell and Donoghue 2005; Chaw et al. 2005; Ran et al. 2006; Liston et al. 2007). The ITS region is also commonly used for phylogenetic analyses of closely related plant taxa, including in the Pinaceae (e.g. Gernandt and Liston 1999; Liston et al. 1999; Gernandt et al. 2001; Wei and Wang 2004). In the Pinaceae, the ITS region is longer than in all other vascular plants, and the ITS1 region has distinctive subrepeats (Liston et al. 1996; Marrocco et al. 1996; Maggini et al. 1998; Gernandt and Liston 1999; Vining 1999; Gernandt et al. 2001; Wei et al. 2003; Campbell et al. 2005, Kan et al. 2007). In addition, ITS copies within a species do not always coalesce (Gernandt and Liston 1999; Wei and Wang 2004; Campbell et al. 2005). To accurately infer phylogenetic relationships among closely related taxa in the Pinaceae using ITS, it is helpful to examine and account for patterns of divergence among copies within and between species. MATERIALS AND METHODS Sampling Design and Outgroup Selection—We obtained 2–10 samples of each Tsuga species and one sample of each outgroup (Appendix 1). Species of Nothotsuga, Abies, Keteleeria, and Pseudolarix were included for rooting purposes because these genera are thought to be most closely related to Tsuga, with Nothotsuga sister to Tsuga (Hart 1987; Price et al. Systematic Botany (2008), 33(3): pp. 478–489 © Copyright 2008 by the American Society of Plant Taxonomists