Bull. Natl. Mus. Nat. Sci., Ser. B, 39(3), pp. 137–145, August 22, 2013

Molecular Identification Resolves Taxonomic Confusion in speciosum Complex ()

Tomohisa Yukawa1,* Akihiko Kinoshita1 and Nobuyuki Tanaka2

1 Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan 2 Kochi Prefectural Makino Botanical Garden, Godaisan, Kochi 781–8125, Japan * E-mail: [email protected]

(Received 28 May 2013; accepted 26 June 2013)

Abstract Molecular identification using nuclear ribosomal ITS sequence data revealed four genetic units in Grammatophyllum speciosum complex (Orchidaceae). Investigations of morpho- logical characters support the status of each unit and a provisional taxonomic revision of the complex is proposed. The following species are recognised in the complex: G. speciosum Blume, G. wallisii Rchb.f., G. kinabaluense Ames & C.Schweinf., G. pantherinum Rchb.f., and G. cominsii Rolfe.

Key words : DNA barcoding, Grammatophyllum speciosum, ITS, molecular identification, Orchidaceae, .

Introduction due to its rarity, inaccessibility at canopy habi- tats, irregular and rare flowering nature (Seiden- Grammatophyllum speciosum Blume, com- faden, 1983; Yukawa, unpublished), wide distri- monly called the Giant Orchid, is granted the bution throughout Southeast Asia further to New title of the largest orchid of the world. The vege- Guinea and the Solomon Islands, and unmanage- tative shoot grows to a length of 3.5 m, and the able size of the for collection. flowering stem grows to a length of 3 m. The Interpretations of species boundaries based on total weight of the entire plant was estimated to morphological characters alone frequently gener- be over more than 1000 kg based on a calculation ate misleading results when only a limited num- of a Bornean plant (Lamb, 2011). ber of samples are used because discontinuity of Although noticeable variations in reproductive a character can become continuous if sampling characters exist among individuals, Grammato- intensity is increased. Under these circum- phyllum speciosum has been treated as a single stances, macromolecular (especially DNA) entity in many floristic and taxonomic accounts. markers discriminating closely related species Meanwhile, several entities assignable to this are powerful tools to uncover genetic units corre- species complex have been described so far. sponding to species. To provide a framework for Interpretations on the status of these names are a taxonomic revision of G. speciosum complex, variable and inconsistent. However, a taxonomic we used the internal transcribed spacer (ITS) revision of the G. speciosum complex has not regions of the 18S-26S nuclear ribosomal DNA been attempted. Such situations have been because the regions have been shown to be an caused by limited opportunities for investigations excellent barcoding marker at the species level in 138 Tomohisa Yukawa et al.

Table 1. Materials used for DNA sequencing and a summary of the results Sample code Locality Accession Genotype Identification 1 unknown TBG 78805 A Grammatophyllum speciosum 2 unknown TNS 8500844 A Grammatophyllum speciosum 3 unknown TBG 153268 A Grammatophyllum speciosum 4 Philippines TBG 161752 B Grammatophyllum wallisii 5 Malaysia, Sabah TBG 161754 C Grammatophyllum kinabaluense 6 Malaysia, Sarawak TBG 161753 D Grammatophyllum pantherinum 7 Solomon Islands MBK 20101534 D Grammatophyllum pantherinum Outgroup unknown TBG 118874 various groups of seed (e.g., China Plant number of gaps. All insertions/deletions were BOL Group, 2011), including subtribe Cyrtopo- coded as missing data.