Molecular phylogenetics and character evolution of morphologically diverse groups, Dendrobium section Dendrobium and allies

Tomoko Takamiya 2 Pheravut Wongsawad 1 Apirada Sathapattayanon 0 Natsuko Tajima 1 Shunichiro Suzuki 2 Saki Kitamura 2 Nao Shioda 2 Takashi Handa 4 Susumu Kitanaka 2 Hiroshi Iijima 2 Tomohisa Yukawa 3 Associate Editor: Chelsea D. Specht 0 Faculty of Science, Srinakharinwirot University , Bangkok 10110, Thailand 1 Institute of Agriculture and Forestry, University of Tsukuba , Tsukuba, Ibaraki 305-8572, Japan 2 School of Pharmacy, Nihon University , Funabashi, Chiba 274-8555, Japan 3 Tsukuba Botanical Garden, National Museum of Nature and Science , Tsukuba, Ibaraki 305-0005, Japan 4 School of Agriculture, Meiji University , Kawasaki, Kanagawa 214-8571, Japan It is always difficult to construct coherent classification systems for plant lineages having diverse morphological characters. The genus Dendrobium, one of the largest genera in the Orchidaceae, includes 1100 species, and enormous morphological diversification has hindered the establishment of consistent classification systems covering all major groups of this genus. Given the particular importance of species in Dendrobium section Dendrobium and allied groups as floriculture and crude drug genetic resources, there is an urgent need to establish a stable classification system. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we analysed the macromolecular characters of the group. Phylogenetic analyses of 210 taxa of Dendrobium were conducted on DNA sequences of internal transcribed spacer (ITS) regions of 18S - 26S nuclear ribosomal DNA and the maturase-coding gene (matK) located in an intron of the plastid gene trnK using maximum parsimony and Bayesian methods. The parsimony and Bayesian analyses revealed 13 distinct clades in the group comprising section Dendrobium and its allied groups. Results also showed paraphyly or polyphyly of sections Amblyanthus, Aporum, Breviflores, Calcarifera, Crumenata, Dendrobium, Densiflora, Distichophyllae, Dolichocentrum, Holochrysa, Oxyglossum and Pedilonum. On the other hand, the monophyly of section Stachyobium was well supported. It was found that many of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the result of convergence. As such, many of the sections that have been recognized up to this point were found to not be monophyletic, so recircumscription of sections is required. - Introduction It is always difficult to construct coherent classification systems for plant lineages having diverse morphological characters. Dendrobium Sw. (Orchidaceae) represents such difficult groups and so far has been established in many alternative systems (e.g. Lindley 1830; Bentham and Hooker 1883; Kra nzlin 1910; Schlechter 1912; Brieger 1981; Clements 2003; Wood 2006; Schuiteman 2011). This genus is one of the largest orchid genera, with around 1100 species (Wood 2006). The distribution range extends from Sri Lanka and India throughout tropical Asia and Oceania, north to Japan, east to Tahiti and south to New Zealand. Enormous diversification of the vegetative organs in accordance with habitat shifts and lack of accessory structures of the pollinarium, a cardinal character in orchid classification, has hindered establishment of consistent classification systems covering all major groups of this genus. Previous studies of systematics based on the morphological characteristics of the group were reviewed by Wood (2006). Given the limits to what can be understood of affinities using morphological characters, Yukawa et al. (1993) analysed the molecular phylogenetics of the subtribe Dendrobiinae (Lindley 1830), which includes the genus Dendrobium and putatively related genera Cadetia, Diplocaulobium, Flickingeria, Epigeneium and Pseuderia based on chloroplast DNA restriction site variation. This analysis resulted in presentation of the first probable phylogenetic relationship between members of this genus. Yukawa et al. (1993) demonstrated that Dendrobium is not monophyletic and comprises two major clades (Asian and Australasian clades: sensu Clements 2003). The Asian clade is predominantly diversified west of Webers line, and the Australasian clade, containing genera Cadetia, Diplocaulobium and Flickingeria, is distributed mostly in Australasia and the Pacific Islands. Subsequent studies on representative members of subtribe Dendrobiinae (e.g. Yukawa et al. 1996, 1999, 2000; Yukawa 2001; Clements 2003, 2006; Schuiteman 2011) incorporated additional macromolecular markers and taxa in their analyses. In addition to providing further support for the above-mentioned phylogeny, these studies identified other infrageneric monophyletic groups. Dendrobium section Dendrobium is one of the largest sections in the genus Dendrobium, comprising 60 species (Wood 2006) distributed across almost the entire geographical range of the genus, with the exception of Micronesia and Melanesia. A number of species are considered important as crude drug sources and are highly sought-after genetic resources with potential value in medicine (Takamiya et al. 2011, 2013). Yukawa et al. (1993) demonstrated that section Dendrobium is nested within the Asian clade. Wongsawad et al. (2001, 2005) analysed sequences for the maturase-coding gene (matK) located in the plastid genome and the internal transcribed spacer regions (ITS) of the nuclear ribosomal DNA of 78 Asian clade species including 35 of section Dendrobium (Wongsawad et al. 2001) and 93 Asian clade species including 42 members of section Dendrobium (Wongsawad et al. 2005). Based on these analyses, Wongsawad et al. demonstrated that section Dendrobium is not monophyletic; that its core clade includes species of sections Breviflores, Densiflora, Holochrysa and Stuposa; and that sections Amblyanthus, Breviflores, Densiflora, Formosae, Holochrysa, Oxyglossum and Pedilonum are not monophyletic. These relationships were confirmed by Xiang et al. (2013). However, given that these studies did not include several species of section Dendrobium and only included a small number of species in sections Aporum, Calcarifera, Calyptrochilus, Crumenata, Distichophyllae, Oxyglossum, Pedilonum, Platycaulon, Stachyobium and Stuposa, which are likely to be closely related to section Dendrobium, our understanding of the relationships between section Dendrobium and other groups within the Asian clade remains incomplete. In this study, we conducted comprehensive phylogenetic analyses of representative species in the Asian clade using the ITS and matK regions to clarify the relationships and the taxonomic position of section Dendrobium. Plant materials The samples for analysis consisted of 210 Asian clade species (214 samples), including 56 species belonging to section Dendrobium. As an outgroup, we chose 10 species of the Australasian clade, based on the results of Yukawa (2001). Plant materials were collected from the living collection of Tsukuba Botanical Garden, National Museum of Nature an (...truncated)