J. Jpn. Bot. 92(1): 34–43 (2017) Harashuteria, a New Genus of Leguminosae (Fabaceae) Subfam. Papilionoideae Tribe Phaseoleae a a b, Kazuaki OHASHI , Koji NATA and Hiroyoshi OHASHI * aSchool of Pharmacy, Iwate Medical University, Yahaba, Iwate, 028-3694 JAPAN; bHerbarium TUS, Botanical Garden, Tohoku University, Sendai, 980-0862 JAPAN *Corresponding author: [email protected] (Accepted on November 25, 2016) A new genus, Harashuteria K. Ohashi & H. Ohashi, is proposed as a member of the tribe Phaseoleae of Leguminosae (Fabaceae) based on Shuteria hirsuta Baker by comparative morphological observation and molecular phylogenetic analysis of Shuteria and its related genera. Molecular phylogenetic analysis was performed using cpDNA (trnK/matK, trnL–trnF and rpl2 intron) markers. Our molecular phylogeny shows that Shuteria hirsuta is sister to Cologania and is distinct from Shuteria vestita or Amphicarpaea, although the species has been attributed to these genera. A new combination, Harashuteria hirsuta (Baker) K. Ohashi & H. Ohashi is proposed. Key words: Amphicarpaea, Cologania, Fabaceae, Glycininae, Harashuteria, Hiroshi Hara, new genus, Phaseoleae, Shuteria, Shuteria hirsuta. The genus Shuteria Wight & Arn. was protologue. Kurz (1877) recognized Shuteria established on the basis of S. vestita Wight & hirsuta as a member of Pueraria, because he Arn. including 4–5 species in Asia (Schrire described Pueraria anabaptista Kurz citing 2005). The genus belongs to the subtribe S. hirsuta as its synonym (hence Pueraria Glycininae in the tribe Phaseoleae and is anabaptista is superfluous). Amphicarpaea closely allied to Amphicarpaea, Cologania, lineata Chun & T. C. Chen is adopted as a and Dumasia especially in the flower structures correct species by Sa and Gilbert (2010) in the (Lackey 1981). Among Shuteria, S. hirsuta recent treatment of Fabaceae for Flora of China, Baker (Fig. 1) is a divergent species from others but is treated by Ohashi and Ohashi (2016) as a of the genus in having a spurred standard, synonym of Shuteria hirsuta. monadelphous stamens and linear pods with Recently, however, Shuteria is consistently septa between the seeds. The species has so separated from Amphicarpaea, Cologania, far been placed in a controversial taxonomic Pueraria and other members of the subtribe position among Amphicarpaea, Pueraria and Glycininae in molecular phylogenetic analyses Shuteria. Baker (1876) described the species (Doyle and Doyle 1993, Lee and Hymowitz based on two syntypes those were referred to 2001, Stefanović et al. 2009, Egan et al. 2016). Amphicarpaea ferruginea Benth. as indicated Shuteria belongs to a Kennediinae-Desmodieae on the original label of the specimens (Fig. clade, while the other genera to a Glycininae 1), although the name was not cited in the clade within the Core-Phaseoleae clade. In these —34— February 2017 The Journal of Japanese Botany Vol. 92 No. 1 35 Fig. 1. Lectotype of Shuteria hirsuta Baker (©The Board of Trustees of the Royal Botanic Gardens, Kew). 36 植物研究雑誌 第 92 巻 第 1 号 2017 年 2 月 Table 1. Voucher information and GenBank accession numbers on data newly sequenced for this paper Taxon Locality Voucher Shuteria hirsuta (1) Cultivated in Setsunan University, Osaka Hiroko Murata (TUS) (Origin: Myanmar) (2) Nepal. Koshi Zone, Sankhuwasabha Distr. S. Noshiro & al. 9810179 (TUS) (3) Thailand. N. Chiang Mai H. Koyama & al. T-48821. (TUS) Shuteria vestita (2) Nepal. Between Thare and Dhuche H. Tabata & al. 8631 (TUS) Dumasia truncata Japan. Miyagi Pref., Higashi-Matsushima-shi, H. Kasai 3873 (TUS) Miyatojima Toxicopueraria peduncularis China. Yunnan: Li-jiang, Wen-bi-feng Murata & al. 662 (TUS) GenBank accession number Taxon trnK/matK trnL intron trnF–trnL spacer rpl2 Shuteria hirsuta (1) LC197941 LC197931 LC197935 LC196161 (2) LC197938 — — — (3) LC197939 — — — Shuteria vestita (2) LC197942 LC197932 LC197936 LC196162 Dumasia truncata LC197940 LC197930 LC197934 LC195833 Toxicopueraria peduncularis LC197943 LC197933 LC197937 LC196160 (1), (2), and (3) correspond to the individuals of Shuteria hirsuta or S. vestita in phylogenetic tree in Figs. 1 and 2. Sequence data of S. vestita (1) are obtained from GenBank. Missing data are indicated by a dash (–). molecular analyses, however, Shuteria hirsuta collected from fresh leaves of a cultivated plant has not been examined, and only S. vestita was in The Botanical Garden, Setsunan University. used as the representative of the genus. Voucher specimen of the plant is deposited in This study aims to solve the controversial TUS. Samples listed in Table 1 including other taxonomic position of Shuteria hirsuta, because S. hirsuta individuals were collected from it has such clear morphological differences herbarium specimen kept in TUS. Already between other species of Shuteria as mentioned published DNA sequences of S. vestita and other above. We present here molecular evidence that species were obtained from GenBank (listed in Shuteria hirsuta is distinctly separated from S. Table 2). vestita hitherto examined and is closely related to Cologania. Shuteria hirsuta and Cologania, DNA extraction, amplification and sequencing however, differ in the flower and legume Genomic DNA was extracted from leaf morphology, the chromosome numbers and their tissue using the modified CTAB method of distribution. In consequence, we here describe Doyle and Doyle (1987). Three chloroplast a new distinct genus Harashuteria K. Ohashi & markers were amplified: trnK intron including H. Ohashi to accommodate the single species, S. matK (trnK/matK), trnL–trnF (trnL intron and hirsuta Baker. trnF–trnL spacer) and rpl2 intron. The PCR primers used for trnK/matK were trnK1L and Materials and Methods trnK2R (Hu et al. 2000). The primers for trnL– Materials trnF (trnL intron and trnF–trnL spacer) were One of the samples of Shuteria hirsuta was primer ‘c’ and primer ‘f’ (Taberlet et al. 1991). February 2017 The Journal of Japanese Botany Vol. 92 No. 1 37 Table 2. Sequence data already published and obtained from GenBank GenBank accession number Taxon trnF–trnL spacer/ trnK/matK rpl2 trnL intron Amphicarpaea bracteata EU717399 EU717317 EU717364 Apios americana EU717426 EU717312 EU717392 Bituminaria bituminosa EU717398 EU717349 EU717362 Bolusafra bituminosa EU717413 EU717309 EU717362 Cajanus cajan EU717414 EU717310 EU717361 Campylotropis macrocarpa EU717418 EU717298 EU717384 Clitoria ternatea EU717286 EU717355 EU717528 Cologania angustifolia GQ246140 – – Cologania lemmonii EU717405 EU717319 EU717371 Cologania pallida JQ619980 – – Cologania tenuis JQ619979 – – Desmodium barbatum EU717420 EU717290 EU717386 Erythrina sousae EU717411 EU717313 EU717377 Galactia striata EU717356 EU717428 EU717394 Glycine max AF142700 EU717321 EU717363 Hardenbergia violacea EU717425 EU717331 EU717391 Kennedia nigricans EU717424 EU717335 EU717390 Kummerowia stipulacea EU717417 EU717299 EU717383 Lablab purpureus EU717408 EU717339 EU717374 Macroptilium atropurpureum EU717409 EU717340 EU717375 Macrotyloma uniflorum EU717410 EU717341 EU717376 Neonotonia wightii U717402 EU717323 EU717368 Ophrestia radicosa EU717430 EU717359 EU717396 Pachyrhizus erosus EU717401 EU717324 EU717367 Pseudovigna argentea EU717403 EU717325 EU717369 Psophocarpus tetragonolobus EU717412 EU717343 EU717378 Pueraria phaseoloides EU717404 EU717327 EU717370 Shuteria vestita (1) EU717423 EU717328 EU717389 Teramnus uncinatus EU717400 EU717330 EU717365 (1) corresponds to the individuals of Shuteria vestita in phylogenetic tree in Figs. 1 and 2. Missing data are indicated by a dash (–). For amplification of rpl2 intron, rpl2 intron f products were run on an Applied Biosystems (5ʹ-GCT CTA GCT AAT TGT CCA CCC TT-3ʹ) 3130xl Genetic Analyzer. and rpl2 intron r (5ʹ-AAA ATG GGA AAT GCC CTA CCT T-3ʹ) were used. In case of failure Phylogenetic analyses of amplification, internal primers were used to Sequence alignment was initially performed amplfy the region in two or more fragments. using MUSCLE (Edgar 2004) in Mega version PCR was performed using KOD Fx neo 7.0 (Kumar et al. 2016) and manually adjusted. (TOYOBO, Osaka, Japan) in 20 μL reactions Phylogenetic analyses were carried out using with the following PCR program: 94°C for 5 Bayesian inference (BI), maximum likelihood min; 40 cycles of 98°C for 10 s 52°C 30 s 72°C (ML), and maximum parsimony (MP) 90 s; 72°C 5 min. PCR products were purified approaches. by polyethylene glycol (PEG) precipitation or An appropriate model of sequence evolution ethanol precipitation with NH4OAc and EDTA. for the combined dataset was estimated using the The sequencing reactions were performed program Kakusan4 (Tanabe 2007). The dataset using the BigDye Terminator, v3.1, Cycle was divided into data partitions (rpl2 intron, trnL Sequencing Kit (Applied Biosystems, Foster intron, trnF–trnL spacer, 5ʹtrnK, 3ʹtrnK, and City, California, U.S.A.), and the reaction matK as protein coding sequence). Models of 38 植物研究雑誌 第 92 巻 第 1 号 2017 年 2 月 Fig. 2. The 50% majority-rule consensus tree (Bayesisn analysis) of Shuteria with Paseoleae species based on the cpDNA dataset (trnK/matK, trnL–trnF and rpl2 intron). Support values on the branches are presented as follows: BI-PP/ML- BS/MP-BS. NS indicates node not supported. sequence evolution for each of the partitions and discarded as burn-in from the final tree set that for all combined datasets were determined. was used to determine the posterior probability Bayesian inference (BI) analyses were distribution. conducted using MrBayes version 3.2 Maximum likelihood (ML; Felsenstein 1981) (Ronquist et