J. Jpn. Bot. 86: 15–25 (2011) Origin of Hawaiian Endemic Species of Canavalia (Fabaceae) from Sea-Dispersed Species Revealed by Chloroplast and Nuclear DNA Sequences a a,† b Mohammad VATANPARAST , Koji TAKAYAMA , Mario S. SOUSA , Yoichi c a, TATEISHI and Tadashi KAJITA * aDepartment of Biology, Graduate School of Science, Chiba University, 1-33, Yayoi, Inage, Chiba, 263-8522 JAPAN; bDepartamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-367, 04510 México, D. F., MÉXICO; cFaculty of Education, University of the Ryukyus, 1, Senbaru, Nishihara, Okinawa, 903-0129 JAPAN; †Present address: Department of Plant Systematics and Evolution, Institute of Botany, University of Vienna. Rennweg 14, A-1030 Wien, AUSTRIA *Corresponding author: [email protected] (Accepted on July 22, 2010) To reveal the origin of the Hawaiian endemic Canavalia species, phylogenetic analyses of chloroplast DNA (cpDNA) and internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA) sequences were performed. Phylogenetic analyses of 6 cpDNA regions (6386 bp) and of nrDNA ITS (708 bp) for all 6 species of the Hawaiian endemic subgenus Maunaloa together with samples from the other 3 subgenera of Canavalia suggested that subgenus Maunaloa is monophyletic and more closely related to subgenus Canavalia than to other subgenera. Phylogenetic analyses of multiple haplotypes of the nrDNA ITS suggested that the Hawaiian endemic species of Canavalia originated from a sea-dispersed species of subgenus Canavalia, possibly Canavalia rosea (Sw.) DC., which is a pantropical species whose seeds are spread by sea drift. A single origin for subgenus Maunaloa might be also suggested. Key words: Canavalia, chloroplast DNA, Hawaiian Islands, nrDNA ITS, phylogeny, seed dispersal. Approximately 90% of native plant the nearest continents, high dispersibility would species of the Hawaiian Islands are endemic be one of the key factors for the ancestors of (Baldwin and Wagner 2010). This represents endemic species to achieve successful migration an extraordinary example of high endemism in to the Hawaiian Islands. According to the most oceanic islands and has attracted the interest of recent review on studies of Hawaiian endemic many botanists. Many studies have focused on plant species, Baldwin and Wagner (2010) have the origin, speciation, and adaptive radiation suggested that the temperate and boreal regions of the endemic species in the Hawaiian Islands of North America are important sources of (reviewed in Baldwin and Wagner 2010). As the Hawaiian flora. This proposal is well supported Hawaiian Islands lie several thousand km from by molecular phylogenetic evidence (Vargas et —15— 16 植物研究雑誌 第 86 巻 第 1 号 2011 年 2 月 Fig. 1. Distribution range of Canavalia rosea and subgenus Maunaloa (drawn based on Sauer 1964). Plant creeping on beach (top left) and seed (top right) of C. rosea. al. 1998, Lindqvist and Albert 2002). Among the crop species. Species of subgenus Maunaloa species originating from North America, only are endemic to the Hawaiian Islands. Sauer a few examples (Gossypium (Malvaceae) and (1964) studied the morphological differences Jacquemontia (Convolvulaceae)) have seeds and similarities among Canavalia species and that are dispersed by the sea. In the case of the reported that the most primitive subgenus is other species, the seeds are dispersed by birds or probably Wenderothia, and subgenera Catodonia wind. Although sea dispersal is one of the most and Canavalia probably originated from this important modes of seed dispersal for shaping subgenus. He also proposed that subgenus the littoral flora of oceanic islands, detailed Maunaloa would be originated from subgenus studies on Hawaiian plants are lacking. Canavalia because of the presence of a The genus Canavalia Adans. is a good model pantropical species, Canavalia rosea (Sw.) DC., for investigations of the origin of Hawaiian in the subgenus. endemic species from sea dispersed species. Canavalia rosea is a typical member of the The genus Canavalia is distributed in the tropics plant group known as the “pantropical plants and subtropics all over the world. According with sea-drifted seeds” (Takayama et al. 2008). to the latest taxonomic revision, this genus is The members of this plant group are distributed further divided into four subgenera, namely, in littoral areas of the tropics and subtropics all Catodonia (seven species), Wenderothia (16 over the world and their main mode of seed species), Canavalia (23 species), and Maunaloa dispersal is sea dispersal (Fig. 1). In addition (six species) (Sauer 1964, St. John 1970, Schrire to C. rosea, some species of the subgenus 2005). Species of subgenera Catodonia and Canavalia also use sea dispersal to spread Wenderothia are distributed mostly in the New their seeds. Canavalia cathartica Thouars is World. Species of subgenus Canavalia are found distributed over Indo-West Pacific regions, C. in both the Old and New World, including some lineata (Thunb.) DC. is found in South East February 2011 Journal of Japanese Botany Vol. 86 No.1 17 Asia, and C. sericea A. Gray is distributed in the Materials and Methods South Pacific. The other species of Canavalia Taxon sampling whose seeds are subjected to sea dispersal is C. A total of 18 species of Canavalia were bonariensis Lindley of the subgenus Catodonia. used in this study. Data of four species were Other species of the genus Canavalia do not only for nrDNA ITS analyses and obtained have sea-drifted seeds and their main mode of from GenBank (Table 1). Species from all seed dispersal is mechanical (with the seeds 4 subgenera according to the subgeneric being thrown by dehiscent pods) or gravity delimitation of Sauer (1964) were included (Sauer 1964). Given the distribution ranges of (Table 1). Multiple accessions were sampled for the species and their modes of seed dispersal, five taxa,C. rosea (18), C. lineata (2), C. sericea Sauer (1964) and Carlquist (1966) suggested (2), C. hawaiiensis Degener & al. (2) and C. that the endemic species of the Hawaiian galeata Gaudich. (2). Dioclea reflexaHook. f., a Islands might have originated from species that species from the genus Dioclea was used as an reached the Hawaiian Islands by sea dispersal. outgroup, based on the molecular phylogenetic A good candidate species would plausibly be tree of Varela et. al. (2004). Voucher specimens the pantropical species, Canavalia rosea, as it of the newly collected samples in this study were has native distribution in Hawaii according to a deposited in the herbarium of University of the herbarium specimen (Rock s.n., ca. 1910, BISH) Ryukyus (RYU). Some samples were collected and has the widest distribution range among sea- from specimens preserved in the herbaria of dispersed species in the genus. However, this Jardim Botânico do Rio de Janeiro (JBRJ), hypothesis has never been tested using modern Universidad National Autonoma de Mexico molecular phylogenetic approaches. (MEXU), and Bishop Museum (BISH). To test the hypothesis about the origin of Hawaiian endemic species of Canavalia, we DNA extraction, PCR and sequencing employed both nuclear and chloroplast DNA Genomic DNA was extracted from dried (cpDNA) markers. These markers have been leaves or seeds using the method of Doyle successfully used to study the origin of Hawaiian and Doyle (1987). The concentration of crude endemic species in other plant groups (reviewed DNA extract was measured with a GeneQuant in Baldwin and Wagner 2010). Although a 100 electrophotometer (GE Healthcare, Life recent phylogenetic study on subtribe Diocleinae Sciences). For cpDNA sequences, after an initial based on nuclear ribosomal DNA (nrDNA) screening of 15 cpDNA candidate regions (Shaw ITS sequences (Varela et al. 2004) showed that et al. 2007), six regions including intergenic subgenus Canavalia is a sister to the subgenus spacers (IGS) and introns were chosen. The Catodonia, the taxon sampling of the study cpDNA regions and PCR primers used in this was not sufficient to reveal the phylogenetic study were for atpB-rbcL IGS (Hodges and relationships between the four subgenera and the Arnold 1994), ndhD-ndhE (Xu et al. 2000), origin of Hawaiian endemic species. We studied trnH-psbA IGS (Hamilton 1999), rps16 intron all species of the subgenus Maunaloa together (Oxelman et al. 1997), trnD-trnT (Demesure with multiple samples of C. rosea collected from et al. 1995) and trnK, which includes the its wide distribution range. Other species that matK gene (Hu et al. 2000, Lavin et al. 2000, have sea-drifted seeds and representative species Wojciechowski et al. 2004). Polymerase chain of subgenera Wenderothia and Catodonia were reactions (PCR) were performed in reaction also included in the study. volumes of 10–25 µL containing 1.25 units ExTaq (TaKaRa), and 0.2 mM dNTPs., 10× PCR buffer contains 1.5 mM MgCl2, 0.5–1 µM 18 植物研究雑誌 第 86 巻 第 1 号 2011 年 2 月 Table 1. List of Canavalia samples used in this study Oceanic DNA nrDNA Subgenus Taxon region Locality Voucher specimen sample No. ITS** Canavalia C. rosea (Sw.) DC. Indian Ocean South Africa Umdloti T. Kajita 010509 98 cloning Tanzania Dar es salaam T. Kajita 021202 184 cloning Sri Lanka Wattala, Negambo T. Kajita 000729 31S cloning Indonesia Sumatra T. Kajita 000812 500 direct Australia Headland Harbour Y. Tateishi 001123 1 direct West Pacific Marquesas Taipivai, Nuku Hiva T. Kajita & Y. Tateishi 000713 31M direct Tonga Sopu T. Kajita & al. 021024 171 direct East Pacific Mexico Sinaloa T. Kajita & al. 1998-965 965 direct Panama Veracruz T. Kajita & Y. Tateishi 011104 202 direct Ecuador Isla Jambel T. Kajita 990720 1205 direct West Atlantic Panama Cuango, Colon T. Kajita & Y. Tateishi 011031 109 direct Panama Pina, Colon T. Kajita & Y. Tateishi 011030 24 cloning Costa Rica Puerto Viejo T. Kajita 990821 346 cloning Mexico Coatzcoalcos T. Kajita & al. 981029 70M cloning Brazil Gaibu Pernanbuco T. Kajita & al. 991129 70B cloning East Atlantic Senegal Joal-Fadiout T. Kajita 001203 107, 108 cloning Ghana Busua beach Y.