J. Jpn. Bot. 85: 337–349 (2010)

Molecular Phylogeny of Sect. Amurense (Leguminosae)

a, b a Yasuhiko Endo *, Byoung-Hee Choi , Daisuke Kakinuma , c d e Gregory Kenicer , Xiang-Yun Zhu and Hiroyoshi Ohashi

a,*College of Science, Ibaraki University, Mito, 310-8512 Japan; bDepartment of Biological Sciences, Inha University, Incheon, 402-751 Korea; cRoyal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR UK; dInstitute of Botany, the Chinese Academy of Sciences, Beijing, 100093 China; eHerbarium (TUS), Botanical Garden, Tohoku University, Sendai, 980-0862 Japan *Corresponding author: [email protected]

(Received on May 11, 2010)

Vicia sect. Amurense (tribe , family Leguminosae), consisting of four species, is mainly distributed in western China. The species of the section share a terete style, a globose stigma, and a swollen pollen mesocolpium. However, in recent molecular phylogenetic studies of Vicia, species with a terete style were presumed to be polyphyletic. In order to clarify phylogenetic relationships of the species of sect. Amurense we did a molecular phylogenetic study comparing data from the ITS region of nrDNA and matK of the chloroplast genome. The analysis showed sect. Amurense to be nested in a clade corresponding to sect. Vicilla, which contains mainly East Asian Vicia species. The monophyly of sect. Amurense was not supported. This discrepancy between the phylogenetic relationships of sect. Amurense presumed from the floral and pollen morphological characteristics and those from the molecular phylogenetic analysis indicates needs of further phylogenetic studies on the section.

Key words: East Asia, Leguminosae, matK, molecular phylogeny, nrDNA-ITS, section Amurense, Vicia.

Vicia L. (tribe Fabeae, family Leguminosae) 1976, Endo 1994, Endo and Ohashi 1995, includes approximately 160 species and is 1996a, 1996b, 1997, 2001, Choi et al. 2006). distributed mostly in the temperate regions of the Most of the species can be classified into the Northern Hemisphere (Lock and Maxted 2005). following four stylar groups (Kupicha 1976, Its main center of diversity is the Mediterranean Choi et al. 2006, Endo et al. 2008). The names region, with smaller centers in North and South of the groups in the following parentheses were America (Kupicha 1976). Around 20 species suggested by Choi et al. (2006). are considered endemic to East Asia (Endo (1) Dorsi-ventrally compressed and abaxially and Ohashi 1996b), making the region another tufted hairy stylar group (Dabt-type stylar group). center of diversity for the . (2) Laterally compressed stylar group (Le- The species of Vicia can be classified into type stylar group). several groups according to the differences in (3) Terete stylar group (Te-type stylar group). stylar features (Trautvetter 1875, Radzhi 1971a, (4) Dorsi-ventrally compressed and evenly 1971b, Kupicha 1976, 1981, Gunn and Kluve hairy stylar group (De-type stylar group).

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Based on molecular phylogenetic studies, the phylogenetic tree, presumed by a comparison first and second groups have been considered of their ITS sequences of nrDNA (Choi et al. to be monophyletic (Choi et al. 2006, Endo et 2006, Endo et al. 2008), and matK region of al. 2008). The third group has been considered cpDNA (Steele and Wojciechowski 2003). to be polyphyletic, and the fourth paraphyletic Fabeae has been shown to be monophyletic (Choi et al. 2006, Endo et al. 2008). The features and its sister group has been presumed to be of Dabt-type, Le-type, and Te-type styles were Trifolieae (Wojciechowski et al. 2000, Steele considered to be apomorphies and the De-type and Wojciechowski 2003). In order to clarify style was considered to be a plesiomorphy (Choi the phylogenetic position of the species of sect. et al. 2006, Endo et al. 2008). Amurense, we compare their DNA sequences Most Vicia species endemic to East Asia with those of other Fabeae and Trifolieae have De-type or Te-type styles. Therefore, the species. The species examined and the voucher monophyly of the East Asian species had been specimens of Vicia species sequenced in the in question, but the monophyly of these species present study are listed in Table 1. has been suggested by molecular phylogenetic studies (Choi et al. 2006, Endo et al. 2008). Molecular phylogenetic study The East Asian Te-type stylar species, Vicia ITS (ITS1 and ITS2) regions of nrDNA and amurensis Oetting., the type species of sect. matK of chloroplast gene were examined for Amurense, was nested among the East Asian the present molecular phylogenetic study. The De-type stylar species in the study of Choi et methods for DNA extraction, polymerase chain al. (2006). However, the remaining species of reaction, sequencing, and phylogenetic analysis section Amurense, three Te-type stylar species were same as in our previous studies (Choi et al. in the west part of China, i.e., Vicia dichroantha, 2006, Endo et al. 2008). The ITS sequences of V. nummularia, and V. tibetica (Endo and six Vicia species and matK for 20 Vicia species Ohashi 1996b), have not been examined were sequenced for the first time. under molecular phylogenetic studies, so their phylogenetic positions are still in question. Tracing character changes on the phylogenetic We aimed to clarify the relationships of these tree three species under a molecular phylogenetic Stylar shapes and stylar hair patterns were study. A further Te-type stylar species, V. crocea treated as unordered multistate characters and (Desf.) B. Fedtsch. from the Caucasus, has been optimized on to the Bayesian trees, obtained considered phylogenetically related to the East in the present study, by using MacClade 4.06 Asian species by Kupicha (1976). We try to (Maddison and Maddison 2003). The stylar clarify the phylogenetic position of that species. shapes and stylar hair patterns of the species Furthermore, we added several additional examined were determined from Kupicha (1976, species for the present analysis, which have 1981), Maxted (1992),Endo and Ohashi (1996b), not been examined in molecular phylogenetic Venora et al. (2000), and Endo et al. (2008). studies. Results and Discussion Materials and Methods Sequence characteristics of ITS and matK Species examined regions Vicia is included in tribe Fabeae together The alignment of combined sequences of the with , , , and ITS 1 and 2 regions comprised 504 positions (Lock and Maxted 2005). Lathyrus, Lens, and with 204 variable characters. The proportion Pisum were nested in Vicia on a molecular of G and C of ITS 1 and 2 regions ranged December 2010 Journal of Japanese Botany Vol. 85 No.6 339

Table 1. List of the GenBank accession numbers of ITS1, ITS2, and matK, voucher specimens and collection sites of samples Species ITS1 ITS2 matK Voucher specimen Collection site L. —1) — AF522084 GenBank2) L. davidii Hance HM026369 HM026373 HM026393 Lee 207001 (IUI) Korea, Gangwon Prov., Jeongseon-eop, Mt. Gariwang L. gmelinii Fritsch — — EU025895 GenBank L. japonicus Willd. AF335217 AF335218 — GenBank L. japonicus Willd. 1 — — HM026394 Lee 105002 (IUI) Korea, Gyeonggi Prov., Hwaseong-si, Maehyang- ri L. japonicus Willd. 2 — — HM026395 Lee 205019 (IUI) Korea, Gangwon Prov., Gangneung-si, Jeongdongjin L. lanszwertii Kellogg — — EU025896 GenBank L. latifolius L. AF335219 AF335220 AF522085 GenBank L. palustris L. ssp. pilosus HM026370 HM026374 HM026396 Lee 10622 (IUI) Korea,Gangwon Prov., Goseong-gun, Geojin-eop, (Cham.) Hultén Bongpyeong-ri L. quinquenervius (Miq.) Litv. HM026371 HM026375 HM026397 Lee 10613 (IUI) Korea, Gyeonggi Prov., Pocheon-si, Gwangneung L. sativus L. — — AF522086 GenBank L. tingitanus L. — — AF522087 GenBank L. vaniotii H. Lév. HM026372 HM026376 HM026398 Lee 20723 (IUI) Korea, Gangwon Prov., Jeongseon-gun, Mt. Banryun L. vestitus Nutt. — — AF522088 GenBank Lens culinaris Medik. AF335221 AF335222 AF522089 GenBank Lens ervoides (Brign.) Grande — — AF522090 GenBank Melilotus officinalis (L.) Pall. AB115777 AB115778 — GenBank ssp. suaveolens (Ledeb.) H. Ohashi Pisum sativum L. AB107207 AB107208 AY386961 GenBank Trifolium repens L. AF335223 AF335224 AF522131 GenBank Vavilovia formosa (Steven) Fed. EU543569 (ITS 1, 2) — GenBank Mühl. ex Willd. — — AF522152 GenBank V. americana Mühl. ex Willd. 1 DQ351832 (ITS 1, 2) — GenBank V. americana Mühl. ex Willd. 2 DQ351834 (ITS 1, 2) — GenBank V. amoena Fisch. AF333559 AF333560 — GenBank V. amoena Fisch. — — HM026399 Cho 80810 (IUI) Korea, Chungcheongbuk Prov., Danyang-gun, Mt. Sobaek V. amurensis Oett. AF335207 AF335208 — GenBank V. amurensis Oett. — — HM026400 Endo 3623 (TUS) Japan, Akita Pref. V. angustepinnata Nakai AF335203 AF335204 — GenBank V. articulata Hornem. — — AF522153 GenBank V. benghalensis L. AF335187 AF335188 AF522154 GenBank V. bifolia Nakai AF333561 AF333562 — GenBank V. bithynica (L.) L. AJ130831 (ITS 1, 2) — GenBank V. bungei Ohwi 1 HM026416 HM026436 HM026379 Endo and Iokawa China, Yunnan, between Degen (Shengping) and 548101 (TUS) Benzilan V. bungei Ohwi 2 AF333583 AF335186 — GenBank V. cassubica L. HM026417 HM026437 HM026411 Kenicer 101a Hungary, Matra area (TUS) V. chosenensis Ohwi AF333563 AF333564 — GenBank V. cracca L. AF335189 AF335190 — GenBank V. crocea (Desf.) B. Fedtsch. 1 HM026431 HM026451 HM026405 Maxted et al. Armenia, Shumookh, Goris to Kafan 6843 (E) V. crocea (Desf.) B. Fedtsch. 2 HM026432 HM026452 HM026406 Davis et al. Turkey, Prov. Bolu, Above Devrek 37715 (E) V. dichroantha Diels 1 HM026418 HM026438 HM026380 Endo and Iokawa China, Yunnan, between Baihanchang and Lijiang 564101 (TUS) V. dichroantha Diels 2 HM026419 HM026439 HM026381 Endo and Nemoto China, Sichuan, between Hexi（Xexi）and 588101 (TUS) Pingchuan V. dumetorum L. AB185885 AB185886 — GenBank V. dumetorum L. — — HM026407 Endo 3624 (TUS) Cultivated from seeds obtained from Botanischer Garten, Bot. Museum, Berlin-Dahlem V. eristalioides Maxted AJ010808 AJ010809 — GenBank V. ervilia (L.) Willd. AF335211 AF335212 — GenBank V. faba L. AF335215 AF335216 AY386899 GenBank V. fauriae Franch. & Sav. AF333565 AF333566 — GenBank V. galilaea Plitm. & Zohary AJ131077 (ITS 1, 2) — GenBank V. hirsuta (L.) Gray DQ351827 (ITS 1, 2) — GenBank V. hirsuta (L.) Gray 1 — — AF522157 GenBank V. hirsuta (L.) Gray 2 — — HM026382 no voucher Japan, Ibaraki Pref., Niihari-gun, Yasato-machi 1) No data 2) Data from GenBank. 340 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

Table 1. Continued Species ITS1 ITS2 matK Voucher specimen Collection site Vicia hirticalycina Nakai AF333567 AF333568 — GenBank V. humilis Kunth — — HM026408 Endo and Nemoto Mexico, San Rafael, Dos Arroyos 43504 (TUS) V. humilis Kunth 1 DQ351830 (ITS 1, 2) — GenBank V. humilis Kunth 2 DQ351831 (ITS 1, 2) — GenBank V. hyaeniscyamus Mourterde AJ131073 AJ131074 — GenBank V. japonica A. Gray 1 AF333569 AF333570 — GenBank V. japonica A. Gray 2 HM026433 HM026453 HM026401 Choi 97205 (IUI) Korea, Gyeongsangbuk Prov., Isl. Ulleung, Buk- myeon V. johannis Tamamshian AJ131080 AJ131081 — GenBank V. kalakhensis Khattab & al. AJ131071 (ITS 1, 2) — GenBank V. leucophaea Greene DQ351833 (ITS 1, 2) — GenBank V. leucophaea Greene — — HM026409 Endo and Nemoto Mexico, Chihuahua, from Cajurichic to Huerachic 45502 (TUS) V. linearifolia Y. N. Lee AF333577 AF333578 — GenBank V. ludoviciana Nutt. — — AF522158 GenBank V. lutea L. — — AF522159 GenBank V. mexicana Hemsl. DQ351835 (ITS 1, 2) GenBank V. mexicana Hemsl. — — HM026410 Endo and Nemoto Mexico, San Rafael, Dos Arroyos 43502 (TUS) V. michauxii Spreng. AJ414585 (ITS 1, 2) — GenBank V. narbonensis L. AJ130834 AJ130833 — GenBank V. nigricans Hook. & Arn. DQ351829 (ITS 1, 2) AF522155 GenBank ssp. gigantea (Hook.) Lassetter & Gunn V. nigricans Hook. AF335205 AF335206 — GenBank ssp. nigricans V. nipponica Matsum. AF333571 AF333572 — GenBank V. nipponica Matsum. — — HM026404 Cho 81024 (IUI) Korea, Gangwon Prov., Inje-gun, Mt. Seolak V. nummularia Hand.-Mazz. HM026420 HM026440 HM026383 Endo and Iokawa China, Yunnan, between Degen (Shengping) and 550101 (TUS) Benzilan V. pannonica Crantz AJ851225 (ITS 1, 2) — GenBank V. pannonica Crantz ssp. HM026415 HM026435 HM026412 Kenicer 98 (TUS) Hungary, Nagycovacsi striata (M. Bieb.) Nyman V. paucijuga (Trautv.) AJ131078 (ITS 1, 2) — GenBank B. Fedtsch. V. pisiformis L. AF333581 AF333582 — GenBank V. pseudo-cracca Bertol. AJ010806 AJ010807 — GenBank V. pseudo-orobus Fisch. & C. A. AF333573 AF333574 — GenBank Mey. 1 V. pseudo-orobus Fisch. & C. A. HM026421 HM026441 HM026377 Kakinuma 6 Japan, Ibaraki Pref., Mito-shi Mey. 2 (TUS) V. pulchella Kunth 1 DQ351836 (ITS 1, 2) — GenBank V. pulchella Kunth 2 DQ351837 (ITS 1, 2) — GenBank V. sativa L. AJ010804 AJ010805 AF522160 GenBank V. sativa L. var. angustifolia (L. AF335213 AF335214 — GenBank ex Reichard) Wahlenb. V. sativa L. var. angustifolia (L. — — HM026378 no voucher Japan, Ibaraki Pref., Higashi-ibaraki-gun, Ibaraki- ex Reichard) Wahlenb. machi V. serratifolia Jacq. AJ131075 AJ131076 — GenBank V. sparsiflora Ten. HM026422 HM026442 HM026413 Kenicer 75c Hungary, Nagycovacsi (TUS) V. sylvatica L. AB185887 AB185888 — GenBank V. tetrasperma Schreb. DQ351828 (ITS 1, 2) — GenBank V. tetrasperma Schreb. — — HM026384 Kakinuma 5 Japan, Ibaraki Pref., Higashi-ibaraki-gun, Ibaraki- (TUS) machi V. tibetica Prain 1 HM026424 HM026444 HM026386 Endo and Iokawa China, Tibet, between Gamtog and Jomda 518101 (TUS) V. tibetica Prain 2 HM026425 HM026445 HM026387 Endo and Iokawa China, Tibet, between Jomda and Qamdo 521101 (TUS) V. tibetica Prain 3 HM026427 HM026447 HM026389 Endo and Iokawa China, Tibet, between Qamdo and Xiaolu 525101 (TUS) V. tibetica Prain 4 HM026426 HM026446 HM026388 Endo and Iokawa China, Tibet, between Jomda and Qamdo 522101 (TUS) V. tibetica Prain 5 HM026428 HM026448 HM026390 Endo and Iokawa China, Tibet, between Markam and Chubalung 535101 (TUS) V. tibetica Prain 6 HM026423 HM026443 HM026385 Endo and Iokawa China, Sichuan, between Xindu and Garze 509101 (TUS) 1) No data. 2) Data from GenBank. December 2010 Journal of Japanese Botany Vol. 85 No.6 341

Table 1. Continued Species ITS1 ITS2 matK Voucher specimen Collection site A. Braun 1 — — HM026402 Cho 80806 (IUI) Korea, Chungcheongbuk Prov., Danyang-gun, Mt. Sobaek V. unijuga A. Braun 2 HM026429 HM026449 HM026391 Endo and Iokawa China, Sichuan, between Xindu and Garze 509102 (TUS) V. unijuga A. Braun 3 HM026430 HM026450 HM026392 Endo and Iokawa China, Tibet, between Gamtog and Jomda 520101 (TUS) V. unijuga A. Braun 4 AF333575 AF333576 — GenBank V. unijuga A. Braun 5 AB107205 AB107206 — GenBank V. unijuga A. Braun var. AF335195 AF335196 — GenBank kaussanensis H. Lêv. V. venosa (Willd. ex Link) — — HM026403 Cho 80824 (IUI) Korea, Chungcheongbuk Prov., Danyang-gun, Maxim. ssp. cuspidata Mt. Sobaek (Maxim.) Y. Endo & H. Ohashi V. venosa (Willd. ex Link) AF335197 AF335198 — GenBank Maxim. ssp. cuspidata (Maxim.) Y. Endo & H. Ohashi 1 V. venosa (Willd. ex Link) AB107203 AB107204 — GenBank Maxim. ssp. cuspidata (Maxim.) Y. Endo & H. Ohashi 2 V. venosa (Willd. ex Link) AB107201 AB107202 — GenBank Maxim. ssp. cuspidata (Maxim.) Y. Endo & H.Ohashi var. cuspidata Maxim. f. minor (Nakai) Ohwi V. venosa (Willd. ex Link) AF335199 AF335200 — GenBank Maxim. ssp. cuspidata (Maxim.) Y. Endo & H. Ohashi var. glabristyla Y. Endo & H. Ohashi V. venosa (Willd. ex Link) AF335193 AF335194 — GenBank Maxim. ssp. yamanakae Y. Endo & H. Ohashi V. venosa (Willd. ex Link) Maxim. AF335201 AF335202 — GenBank ssp. stolonifera Y. Endo & H. Ohashi V. vicina Clos AF333579 AF333580 — GenBank V. villosa Roth — — AF522161 GenBank 1) No data. 2) Data from GenBank. from 0.478 in Pisum sativum to 0.517 in Vicia matK are shown as Figures 1 and 2, respectively. mexicana. Pairwise distances of the regions The states of stylar characters are optimized onto ranged from 0.0 to 0.129, with the highest in these trees as Figures 3 and 4. group difference between V. cracca and P. Both ITS and matK trees show the following sativum (0.129). The length of the matK gene phylogenetic relationships. varied from 1475 bp in V. americana to 1527 (1) The species with abaxially tufted stylar bp in V. nigricans subsp. gigantea. The aligned hair form a monophyletic group, i.e., Dabt-type length of sequence matrix of matK gene was stylar group (Figs. 3A, 4A). 1584 positions of which 1208 were invariant (2) The species with laterally compressed across the samples. The proportion of G and C stylar shape form a monophyletic group, i.e., Le- of matK gene ranged from 0.305 in V. hirsuta to type stylar group (Figs. 3B, 4B). 0.326 in V. amoena. The highest ingroup value (3) The East Asian species with a (0.049) of pairwise distances of the matK gene dorsiventrally compressed, or terete, and evenly was observed between V. hirsuta and Lathyrus hairy styles form a monophyletic group, i.e., aphaca. including a part of De-type stylar group and a part of the Te-type stylar group. We call this Phylogenetic relationships shown on both ITS group “EA group” (Figs. 3, 4). tree and matK tree (4) Lathyrus is monophyletic and its sister Molecular phylogenetic trees of ITS and group is Pisum. We call this group “LP group” 342 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

(Figs. 1, 2). Vavilovia is included in this group phylogenetic trees (Figs. 1, 2). The monophyly on ITS tree (Fig. 1). of sect. Amurense was not supported in the trees estimated from ITS sequences of nrDNA Phylogenetic relationships shown on ITS tree (Fig. 1) and from matK sequence of cpDNA but different from those shown on matK tree (Fig. 2). On the other hand, sect. Amurense are The following differences are recognized characterized by a terete style and a swollen between ITS tree and matK tree (Figs. 1, 2). mesocolpium of pollen grains (Endo and Ohashi, (1) EA group and Le group form a 1996b). The terete style is an apomorphy in monophyletic group on ITS tree, but EA group, Fabeae (Choi et al. 2006, Endo et al. 2008). Lens, and Dabt group form a monophyletic The swollen mesocolpium has been observed group on matK tree. only in the species of sect. Amurense in Vicia (2) and LP group form (Endo and Ohashi 1996a, 1996b). Therefore, a monophyletic group on ITS tree, but V. the monophyly of the section would be strongly tetrasperma, other Vicia species, and Lens form supported by these morphological characters. a monophyletic group on matK tree. Consequently, we treat that sect. Amurense (3) , Lens culinaris, and V. should be included in sect. Vicilla. However, crocea form a monophyletic group in the ITS the discrepancy between the morphological tree, but V. nigricans branched at basal node of distinctions from other sections of the subgenus the clade consisting of Vicia and Lens on matK Vicilla mentioned above and the results of the tree. molecular phylogenetic analysis needs to be These differences are mainly due to the solved by further comprehensive studies on the differences between the branching patterns species of sect. Amurense. at the basal nodes of ITS tree and matK tree. The reason of the incongruence at basal nodes Phylogenetic position of Vicia crocea between ITS tree and matK tree of the present Vicia crocea, V. venosa, V. unijuga, and study is unclear and may be down to homoplasy, V. kulingiana have etendrillous leaves with lack of discriminatory characters (implying a fast relatively large, broad, papery-coriaceous, radiation), or ancient hybridization events. Even reticulate-veined leaflets, paniculate though, we consider that the monophyly of each inflorescences with their lateral branches group, i.e., Dabt group, Le group, EA group, and subtended by bracts, and the pedicels subtended LP group, is supported in the present molecular by bracteoles (Kupicha 1976). Therefore phylogenetic study (Figs. 1, 2). The remaining Kupicha (1976) considered them to be closely species are V. articulata, V. crocea, V. ervilia, V. related to each other. The last three species are hirsuta, V. nigricans, V. sylvatica, V. tetrasperma, all distributed in East Asia (Endo and Ohashi and Lens spp. We need further investigation on 1996b). However, V. crocea is distributed in the the branching order at the basal nodes of the Caucasus and is characterized by its petal shapes, molecular phylogenetic tree of Fabeae. i.e., the keel and wing petals have a larger blade than those of the other Vicia species (Radzhi Phylogenetic position of the species of sect. 1971a, Endo 1985), and chromosome number x Amurense = 5 against those of other Vicia species, x = 6 or The members of sect. Amurensis, i.e., Vicia 7 (Fedorov 1969). V. crocea is phylogenetically amurensis, V. dichroantha, V. nummularia, distant from species of EA group based on the and V. tibetica (Endo and Ohashi 1996b), present molecular phylogenetic study (Figs. 3, are nested in EA group, most of which are 4). member of sect. Vicilla, in the present molecular Kupicha (1976) treated Vicia crocea as a December 2010 Journal of Japanese Botany Vol. 85 No.6 343

Fig. 1. Bayesian tree estimated from ITS sequences of nrDNA. Bayesian posterior probabilities are indicated above branches. Dabt. Dorsiventrally compressed and abaxially tufted stylar group of Vicia. EA. East Asian group of Vicia. Le. Laterally compressed stylar group of Vicia. LP. Lathyrus and Pisum group. Species marked by solid triangles are the species of the former sect. Amurense. 344 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

Fig. 2. Bayesian tree estimated from matK sequences of chloroplast DNA. Bayesian posterior probabilities are indicated above branches. Dabt. Dorsiventrally compressed and abaxially tufted stylar group of Vicia. EA. East Asian group of Vicia. Le. Laterally compressed stylar group of Vicia. LP. Lathyrus and Pisum group. Species marked by solid triangles are the species of of the former sect. Amurense. December 2010 Journal of Japanese Botany Vol. 85 No.6 345 member of sect. Vicilla with East Asian species 2006, Endo et al. 2008). Since no molecular of Vicia. We consider that V. crocea should be phylogenetic analysis has been performed on the removed from sect. Vicilla, which was mainly Mediterranean species of Vicia, differentiation of composed of EA group species. Radzhi (1971a) species in the center of distribution is unknown. treated V. crocea as a monotypic section, Crocea Origin of the stylar diversification ofVicia is also Radzhi. However, a close relationship of V. unknown. Based on these facts and the results of crocea, V. nigricans and Lens culinaris was the present study, the following evolution of the implied in the ITS tree (Fig. 1). Therefore we styles of Vicia can be inferred: need further investigation on the phylogenetic (1) Dabt-type and Le-type styles are derived position of V. crocea before we accept the from the De-type style. species as a member of a monotypic section. (2) After the evolution of these two style- types, the species of these two style-types EA group dispersed into the three regions mentioned The EA group includes East Asian Vicia above. species having a dorsiventrally compressed (3) After the two style-type species spread and evenly hairy style, or terete and evenly into the three regions, the Te-type style evolved hairy style (Figs. 1, 2). This EA group forms a independently in each of the three regions. monophyletic group with European Vicia species having a dorsiventrally compressed and evenly Taxonomic treatment hairy style, i.e., V. cassubica, V. pisiformis, and Genus Vicia L., Sp. Pl. 734 (1753). V. sparsiflora (Figs. 1–4). These species are Subgenus Cracca Peterm., Deutschl. Fl. 152 classified into sect. Vicilla or sect. Cassubicae (1847). Radzhi of subgenus Cracca by Kupicha (1976). Sect. Vicilla (Schur) Aschers. & Graebner, The other species of sect. Cassubicae, i.e., V. Syn. Mitteleur. Fl. 6(2): 916 (1909); Kupicha in orobus DC., and V. megalotropis Ledeb. are Notes Roy. Bot. Gard. Edinb. 34: 307 (1976), reported to make a monophyletic group with pro parte. V. cassubica, V. sparsiflora, and V. pisiformis Genus Vicilla Schur, Enum. Pl. Transsilv., by Jaaska (2005) based on her molecular 170 (1866), pro parte. phylogenetic study on the basis of isozyme Genus Vicia subgen. Vicilla sect. Cassubicae variation. Hence, we united these two sections, Radzhi in Novit. Syst. Pl. Vasc. 7: 230 (1971), sect. Vicilla and sect. Cassubicae, as sect. Vicilla pro parte.; Kupicha in Notes Roy. Bot. Gard. (Schur) Aschers. & Graebner. We remove V. Edinb. 34 : 309 (1976), pro parte. crocea, mentioned above, V. dumetorum, V. Genus Vicia subgen. Vicilla sect. Oroboidea sylvatica, and V. nigricans from this sect. Vicilla Stankevich in Tr. Prikl. Bot. Gen. Sel. 43: 123 by the results of the present molecular study, but (1970), pro parte. further studies are needed for determing their Genus Vicia subgen. Vicilla (Schur) Rouy new position. sect. Amurense Y. Endo & H. Ohashi in J. Jpn. Bot. 71: 259 (1996). Evolution of stylar features in Vicia Species included in the Vicilla group: Vicia Most Vicia species are indigenous to the amoena Fisch. ex Ser., V. angustepinnata Nakai, Mediterranean area, the Americas, or to East V. bifolia Nakai, V. cassubica L., V. chosenensis Asia (Kupicha 1976, Choi et al. 2006, Endo Ohwi, V. fauriae Franch., V. hirticalycina Nakai, et al. 2008) and each of the four style-types V. japonica A. Gray, V. kulingiana L. H. Bailey, can be observed in the Vicia species in each of V. megalotropis Ledeb., V. nipponica Matsum., the three regions (Kupicha 1976, Choi et al. V. orobus DC., V. pisiformis L., V. pseudo-orobus 346 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

Fig. 3. Stylar characters of Fabeae optimized onto the Bayesian tree obtained from the analysis of ITS sequences of nrDNA in the present study. A. Stylar hair patterns; CI = 0.75, RI = 0.97. B. Stylar shapes; CI = 0.33, RI = 0.67. Species marked by solid triangles are the species of the former sect. Amurense. December 2010 Journal of Japanese Botany Vol. 85 No.6 347

Fig. 4. Stylar characters of Fabeae optimized onto the Bayesian tree obtained from the analysis of matK sequences of chloroplast DNA in the present study. A. Stylar hair patterns; CI = 1.00, RI = 1.00. B. Stylar shapes; CI = 0.43, RI = 0.75. Species marked by solid triangles are the species of the former sect. Amurense. 348 植物研究雑誌 第 85 巻 第 6 号 2010 年 12 月

Fisch. & C. A. Mey., V. sparsiflora Ten., V. A. 2008. Phylogenetic relationships of New World unijuga A. Braun, V. venosa (Willd.) Maxim. Vicia (Leguminosae) inferred from nrDNA internal transcribed spacer sequences and floral characters. Species included in the Amurense group: Syst. Bot. 33: 356–363. Vicia amurensis Oetting., V. dichroantha Diels, Fedorov A. A. 1969. Choromosome numbers of flowering V. nummularia Hand.-Mazz., V. tibetica Fisch. . pp. 322–325. Academy of Sciences of the USSR. The authors thank Jung-Hyun Lee and Gunn C. R. and Kluve J. 1976. Androecium and pistil characters for tribe Vicieae (). Taxon 25: Won-Bum Cho (Inha University, Korea) for 563–575. laboratory assistance; Ji-Yeon Lee (National Jaaska V. 2005. Isozyme variation and phylogenetic Institute of Biological Resources, Korea) for relationships in Vicia subgenus Cracca (Fabaceae). permission to use unpublished sequences data Ann. Bot. 96: 1085–1096. Kupicha F. K. 1976. The infrageneric structure of Vicia. and materials of Korean Lathyrus; Tomoyuki Not. Roy. Bot. Gard. Edinb. 34: 287–326. Nemoto (Ishinomaki Senshu University, Japan), Kupicha F. K. 1981. Tribe 21. Vicieae (Adans.) DC. (1825), Yu Iokawa (Joetsu University of Education, nom. conserv. prop. In: Polhill R. M. and Raven P. H. Japan), and Tadashi Kajita (Chiba University, (eds.), Advances in Legume Systematics. pp. 377–381. Japan) for their help in collecting materials. This Royal Botanic Gardens, Kew. Lock J. M. and Maxted N. 2005. Tribe Fabeae. In: Lewis work was partially supported by a Grant-in Aid G., Schrire B., Mackinder B. and Lock M. (eds.), for Scientific Research (B) (15405013) to Y. Legumes of the World. pp. 505–509. Royal Botanic Endo from the Japan Society for the Promotion Gardens, Kew. of Science. Maddison D. R. and Maddison W. P. 2003. MacClade version 4.06: Analysis of phylogeny and character evolution. Sinauer Associates, Sunderland. References Maxted N. 1992. New combinations and names in the Choi B.-H., Seok D.-I., Endo Y. and Ohashi H. 2006. genus Vicia (Leguminosae: Vicieae). Kew Bull. 47: Phylogenetic significance of stylar features in genus 129–130. Vicia (Leguminosae): an analysis with molecular Radzhi A. D. 1971a. Conspectus systematis specierum phylogeny. J. Pl. Res. 119: 513–523. Caucasicarum generis Vicia L. Nov. Sys. Pl. Vasc. 7: Endo Y. 1985. A study on the infrageneric system of Vicia 228–240 (in Russian). (Leguminosae). Doctoral thesis, Tohoku University (in Radzhi A. D. 1971b. Evolution of the genera of the tribe Japanese). Vicieae Adans. Bot. Zhurn. 56: 978–981 (in Russian). Endo Y. 1994. Pistil morphology of Vicia amurensis Steele K. P. and Wojciechowski M. F. 2003. Phylogenetic (Leguminosae) and its allied species. J. Jpn. Bot. 69: analyses of tribes Trifoieae and Vicieae, based on 379–382. sequences of the plastid gene matK (Papilionoideae: Endo Y. and Ohashi H. 1995. The morphology of styles Leguminosae). In: Klitgaard B. B. and Bruneau A. and stigmas in Vicia (Leguminosae), and its systematic (eds.), Advances in Legume Systematics Part 10, implications. J. Pl. Res. 108: 17–24. Higher Level Systematics. pp. 355–370. Royal Botanic Endo Y. and Ohashi H. 1996a. The pollen morphology of Gardens, Kew. Vicia (Leguminosae). Am. J. Bot. 83: 955–960. Trautvetter E. R. a. 1875. Catalogus Viciearum Rossicarum. Endo Y. and Ohashi H. 1996b. The infrageneric positions of Act. Hort. Petrop. 3: 33–83. East Asian species of Vicia (Leguminosae). J. Jpn. Bot. Venora G., Blangiforti S., Frediani M., Maggini F., Gelati 71: 254–262. M. T., Ruffini C. M. and Cremonini R. 2000. Nuclear Endo Y. and Ohashi H. 1997. The morphology and DNA contents, rDNAs, chromatin organization, and anatomy of styles in Vicieae (Leguminosae). J. Jpn. karyotype evolution in Vicia sect. Faba. Protoplasma Bot. 72: 9–18. 213: 115–125. Endo Y. and Ohashi H. 2001. Phylogenetic relationships Wojciechowski M. F., Sanderson M. J., Steele K. P. and among East Asian species of Vicia (Leguminosae). In: Liston A. 2000. Molecular phylogeny of the “temperate Cho D. S. (ed.), Proceedings of the 19th symposium on herbaceous tribes” of Papilionoid legumes: a supertree biology, biodiversity − status, conservation and approach. In: Herendeen P. S. and Bruneau A. (eds.), restoration. pp. 153–158.The Catholic University of Advances in Legume Systematics, Part 9. pp. 277–298. Korea, Puchon, Korea. Royal Botanic Gardens, Kew. Endo Y., Choi B.-H., Ohashi H. and Delgado-Salinas December 2010 Journal of Japanese Botany Vol. 85 No.6 349

遠藤泰彦a,*，崔 秉煕b，柿沼大介a，G. ケニサーc，朱 d e 相雲 ，大橋広好 ：マメ科ソラマメ属ノハラクサフジ節の分 子系統 ジ節としては独立しないことが明らかとなった．ノハラクサフ マメ科ソラマメ属ノハラクサフジ節は主に中国西部に分 ジ節の花部形態と花粉形態の特徴の単元性は今回の分子系 布し 4 種からなる．花柱の横断面が円形となり，柱頭がド 統解析の結果からは支持されなかった． ーム型で，花粉の溝間域が膨らむという特徴を共有する． 本研究では，ノハラクサフジ節の種の系統関係を明らかに （a,* 茨城大学理学部 , するため，他のソラマメ属の種および近縁属の種と共に， b 韓国・仁荷大学校理科大学生物学科 , nrDNA の ITS 領域および葉緑体の matK 遺伝子の塩基配 c 王立エジンバラ植物園 , 列を比較し分子系統解析を行った．その結果，ノハラクサ d 中国科学院植物研究所 , フジ節は，東アジアの固有種が大部分を占める Vicilla 節の e 東北大学植物園津田記念館） 種とともに単系統群を成し，この中に含まれ，ノハラクサフ