J. Jpn. Bot. 85: 213–229 (2010)

Phylogeny of (Leguminosae) Based on Chloroplast trnL-trnF Sequences

a, b c d Tomoyuki Nemoto *, Jun Yokoyama , Tatsuya Fukuda , Yu Iokawa e and Hiroyoshi Ohashi

aDepartment of Basic Sciences, Faculty of Science and Engineering, IshinomakiSenshu University, 1, Shinmito, Minamisakai, Ishinomaki, 986-8580 JAPAN; bDepartment of Biology, Faculty of Science, Yamagata University, Yamagata, 990-8560 JAPAN; cFaculty of Agriculture and Agricultural Science Program, Graduate School of Integrated Arts andScience, Kochi University, Nankoku, Kochi, 783-8502 JAPAN; dDepartment of School Education, Joetsu University of Education, Joetsu, Niigata, 943-8512 JAPAN; eBotanical Garden, Tohoku University, Sendai, 980-0862 JAPAN *Corresponding author: [email protected]

(Received on March 2, 2010)

Phylogenetic relationships of 35 species and one subspecies of Lespedeza and four species representing related genera (2 species of , 1 species of and 1 species of ) in tribe (Leguminosae) were explored with chloroplast DNA sequences of trnL intron and trnL-trnF intergenic spacer regions. Maximum parsimony, neighbor joining and maximum likelihood analyses constructed trees of almost the same topologies resolving 1) the monophyly of Lespedeza and Kummerowia, 2) the two main clades of Lespedeza consisted of Asian species and North American species, 3) two main clades in North American species, and 4) the monophyly of subgenus Macrolespedeza. The monophyly of the Lespedeza and that of the subgenus Lespedeza were not supported by the present results. The two main clades dividing the genus Lespedeza are supported by seedling morphology. Moreover, the two main clades of North American species are congruent with the traditional separation into two groups based on the color and morphology of flowers. The intercontinental relationships between North American and Asian species proposed by Maximowicz (1873) were not supported by the present study, while the separation of Asian species from North American ones by Schindler (1913) was almost supported. The present result suggests that earlier divergence had occurred in Asia resulting into two lineages in Lespedeza, and then one of them remained in Asia and the other migrated to North America. After the isolation of both lineages speciation had occurred independently in Asia and North America.

Key words: Asia, cpDNA, disjunct distribution, Kummerowia, Leguminosae, Lespedeza, molecular phylogeny, North America, trnL-trnF.

The genus Lespedeza comprises ca. 35 Malesia and eastern North America (Ohashi species of shrubs and perennial herbs distributed 2005). Although Lespedeza had been attributed in two disjunct areas: Asia to Australia through to tribe Desmodieae subtribe Lespedezinae

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Table 1. Four series and species classified in section Lespedeza (= subgenus Lespedeza in the present study) by Maximowicz (1873) Series Violaceae Series Junceae Series Lespedezariae* Series Pilosae L. elegans (A) L. caraganae (A) L. gerardiana (A) L. pilosa (A) L. floribunda (A) L. chinensis (A) L. medicaginoides (A) L. virgata (A) L. juncea (A) (= L. daurica) L. repens (NA) L. sericea (A) L. tomentosa (A) L. reticulata (NA) (= L. cuneata) L. trichocarpa (A) (= L. virginica) (= L. daurica) L. stuevei (NA) L. variegata (A) L. violacea (NA) L. capitata (NA) L. hirta (NA) (A): Asian species. (NA): North American species. *Lespedeza lanata Benth. was originally included in series Lespedezaria. But this species was regarded as a synonym of Paratephrosia lanata (Benth.) Domin (1912) and recently as a synonym of Tephrosia lasiochlaena Cowie (2004). Therefore, L. lanata is excluded from this table. together with Campylotropis, Kummerowia, Akiyama 1988) or as subgenera (Ohashi Neocollettia and Phylacium (Ohashi et al. 1981), 1982, Ohashi et al. 2009a, 2009b). The main RFLPs analysis of cpDNA (Nemoto et al. 1995) difference of these two groups is the presence or showed that Phylacium is very different from absence of cleistogamous flowers. The subgenus Lespedeza, Campylotropis and Kummerowia, Lespedeza produces cleistogamous flowers and and that Phylacium and Neocollettia should be is distributed in Asia and eastern North America, removed from the tribe Desmodieae together while the subgenus Macrolespedeza only in with Brya and Cranocarpus of subtribe Bryinae Asia. With respect to species of the subgenus of Desmodieae on the basis of the presence Lespedeza Maximowicz (1873) recognized of rpl2 intron (Bailey et al. 1997) and rbcL four groups, that is, series Violaceae, Junceae, sequence data (Doyle et al. 1997). Phylacium Lespedezariae and Pilosa (Table 1), which was and Neocollettia were shown to be moved to followed by Taubert (1894). Series Junceae tribe on the basis of morphological, and Pilosa consist only of Asian species, while palynological and molecular evidence (Doyle et series Violaceae and Lespedezariae consist of al. 2000, Kajita et al. 2001, Schrire 2005), and Asian and North American species. Schindler Brya and Cranocarpus to tribe from (1913), on the other hand, presented a diagram molecular evidence (Lavin et al. 2001) as well as showing three lineages divergent from a putative morphological one such as trichomes and pollen ancestor of the subgenus Lespedeza, one of (Klitgaard and Lavin 2005). In consequence, the which is composed of Asian species, another of remaining genera of the tribe Desmodieae was some North American species and one Asian subdivided into three groups, Lespedeza group, species L. tomentosa, and the other of only group and group North American species (Fig. 1). Although (Ohashi 2005). The Lespedeza group comprises Maximowicz (1873) considered some Asian Lespedeza, Kummerowia and Campylotropis. species of the subgenus Lespedeza to be related In Lespedeza two groups have been with some North American species in his series recognized: Lespedeza (sometimes as Eu- Violaceae and Lespedezariae, Schindler (1913) lespedeza) and Macrolespedeza, which have separated almost all Asian species from North been treated as sections (Maximowicz 1873, American ones except L. tomentosa. As to the Taubert 1894, Schindler 1913, Nakai 1927, relationships among species of the subgenus August 2010 The Journal of Japanese Botany Vol. 85 No. 4 215

A B

Fig. 1. Phylogenetic diagrams of Lespedeza presented by Schindler (1913) . A. Section Macrolespedeza, showing three lineages (①,② and ③ ) which partly correspond to three taxa recognized by Akiyama (1988), series Formosa, Macrolespedeza and Heterolespedeza, respectively. B. Section Lespedeza (as Eulespedeza), showing three lineages originated from an ancestor in Asia (S. China): the lineage ① consisting of only Asian species, the lineage ② of three North American species and one Asian L. tomentosa, and the lineage ③ of only North American species. Lespedeza friebeana in A has been treated as a synonym of L. maximowiczii (e.g., Ohashi et al. 2009b), L. dielsiana, L. sericea and L. trichocarpa in B as a synonym of L. floribunda, L. cuneata and L. daurica, respectively (e.g., Ohashi et al. 2009a), and also L. nuttallii and L. simulata in B as a synonym of L. hirta × intermedia and L. capitata × virginica, respectively (e.g., Clewell 1966c). The numbers ① , ② and ③ in A and B are all added here on the original diagrams.

Macrolespedeza. Schindler (1913) considered has been mentioned as one such group showing three lineages, which almost correspond to this distribution pattern (Gray 1846, Li 1952, three groups, Macrolespedeza, Formosae Boufford and Spongberg 1983, Wu 1983). and Heterolespedeza recognized by Akiyama Since 1990s molecular phylogenetic analyses (1988) at the rank of series under section have been carried out in many disjunct genera Macrolespedeza (Fig. 1). (reviewed by Wen 1998, 1999; Wen et al. Nemoto and Ohashi (1993b) examined 2002, Xiang et al. 1998, 2000, Nie et al. 2006). seedling morphology of Lespedeza and found These analyses did not support the presumed differences in leaf arrangement between intercontinental species pair relationship in many species of Asia and North America. The results genera, but support the placement of the eastern suggested closer relationships among species Asian species basal to the North American clade within the same continent than those within or two highly distinct clades corresponding to subgenera or other infrageneric groups. eastern Asia and eastern North America. Intercontinental disjunction of closely related The present study is intended to clarify taxa between eastern Asia and eastern the phylogenetic relationship among those North America has interested many botanists infrageneric taxa of Lespedeza using noncoding since the time of Linnaeus (Boufford and regions of cpDNA; the intron of trnL and the Spongberg 1983, Boufford 1998). Lespedeza intergenic spacer between trnL-trnF, these 216 植物研究雑誌 第 85 巻 第 4 号 2010 年 8 月 are among the most frequently used regions using the Taq DyeDeoxy Terminator Cycle (Shaw et al. 2005) and have often provided Sequencing Kit and Applied Biosystems useful phylogentic information at the lower Model 373A automated sequencer (Applied taxonomical levels (Raubeson and Jansen 2005). Bio Systems Division, PERKIN ELMER) following the manufacture's instructions. The Materials and methods primers for sequencing were the same ones used Plant material for amplification. Nucleotide sequences were A total of 35 species and one subspecies of aligned manually. Lespedeza were sampled. All of the 11 North American species (Clewell 1966b, Isely 1998) Phylogenetic analysis and most Asian species were included. Both Phylogenetic relationships were analyzed species of Kummerowia and one species of using three methods: the maximum parsimony Campylotropis of the subtribe Lespedezinae, or (MP) method, the neighbor-joining (NJ) method the Lespedeza group, were included together (Saitou and Nei 1987), and the maximum with Dendrolobium dispermum of the subtribe likelihood (ML) method (Felsenstein 1981). Desmodiinae, the Phyllodium group, as an For all the analyses, we used PAUP* 4.0b10 outgroup. Leaves for total genomic DNA (Swofford 2003). isolation were collected in the field, from For the MP analysis heuristic searches cultivated individuals in the Experimental were performed using 1,000 random addition Garden, Biological Institute, Faculty of replicates, tree-bisection-reconnection (TBR) Science, Tohoku University, or from herbarium branch swapping and MulTrees option in effect. specimens (Table 2). We used datasets without and with indels. In the former dataset, all indels were treated as missing DNA amplification and sequencing data. In the latter, indels were binary coded Total genomic DNA was isolated from 200 and treated as the fifth character, and gaps of to 300 µg of fresh, silica-gel-dried or herbarium different lengths were all treated as single events. specimens using the modified 2×CTAB All character states were equally weighted. procedure of Hasebe and Iwatsuki (1990). The Bootstrap analyses (Felsenstein 1985) in PAUP* isolated DNA was resuspended in 200 µl TE and were carried out using the heuristic search option stored at −20ºC until use. with 1000 replicates and 100 rounds of random PCR amplification was achieved using two sequence additions with MAXTREES set at non-coding chloroplast regions, trnL(UAA) 100,000 and REARRLIMIT set at 1,000,000. intron and trnL(UAA)–trnF(GAA) intergenic For the NJ analysis the Kimura’s 2-parameter spacer, using the universal primers (trnL-c, model of nucleotide substitutions (Kimura 1980) trnL-d, trnL-e and trnF-f) of Taberlet et al. was employed, and bootstrap analysis with 1000 (1991). The PCR were performed according replicates was performed. to the following thermocycle protocol: (94ºC For ML analysis, we applied TVM+G model for 2min) × 1 cycle; (94ºC for 1.5 min, 45ºC selected by AIC (Akaike Information Criterion) for 2 min, 60ºC for 3 min) × 30 cycles; (72ºC (Akaike 1974) using Modeltest 3.7 (Posada and for 15 min) × 1 cycle. The PCR products were Crandall 1998). Support values for nodes on separated by electrophoreses through 1% the ML tree were estimated with 100 bootstrap SeaKem GTG agarose gel (TaKaRa, Japan), and replicates. were purified using Gene Clean Kit II (Bio 101, La Jolla, CA). The purified PCR products were sequenced August 2010 The Journal of Japanese Botany Vol. 85 No. 4 217 AB538924 AB538925 AB538926 AB538927 AB538928 AB538929 AB538930 AB538961 AB538962 AB538931 AB538932 AB538933 AB538934 AB538935 AB538936 AB538937 AB538938 AB538939 AB538963 AB538940 AB538941 AB538942 AB538943 AB538944 AB538945 AB538951 AB538952 AB538953 AB538946 AB538947 AB538948 AB538949 AB538950 AB538954 AB538955 AB538956 AB538957 AB538958 AB538959 AB538960 Accession No. trnL - trnF 372 374 368 368 360 360 360 360 360 360 360 360 360 360 360 360 360 368 368 368 360 361 360 360 360 368 362 358 361 360 360 368 368 360 368 368 368 368 360 368 Length (bp) AB538911 AB538884 AB538885 AB538886 AB538887 AB538888 AB538889 AB538890 AB538922 AB538891 AB538892 AB538893 AB538894 AB538895 AB538896 AB538897 AB538898 AB538899 AB538923 AB538900 AB538901 AB538902 AB538903 AB538904 AB538905 AB538912 AB538913 AB538906 AB538907 AB538908 AB538909 AB538910 AB538914 AB538915 AB538916 AB538917 AB538918 AB538919 AB538920 AB538921 Accession No. trnL intron 532 547 528 530 530 535 530 530 530 530 530 530 530 530 530 535 530 532 530 530 530 530 530 530 530 530 530 530 530 530 506 530 535 530 530 530 530 530 530 530 Length (bp) Voucher Y. Iokawa 5393 (TUS) Y. Y. Iokawa 5388 (TUS) Y. Y. Iokawa 5240 (TUS) Y. Y. Iokawa 5239 (TUS) Y. T. Nemoto 8621 (TUS) T. T. Nemoto 8608 (TUS) T. T. Nemoto 8613 (TUS) T. T. Nemoto 8632 (TUS) T. T. Nemoto & al. 1006208 (TUS) T. H. Ohashi & Y. Tateishi 10739 (TUS) Tateishi Y. H. Ohashi & T. Nemoto 8634 (TUS) T. T. Nemoto 8615 (TUS) T. T. Nemoto 2884 (TUS) T. H. Ohashi & al. 96908407 (TUS) T. Nemoto & al. 97903106 (TUS) T. T. Nemoto 8605 (TUS) T. T. Nemoto 2885 (TUS) T. T. Nemoto & J. Yokoyama 95817018 (TUS) Yokoyama Nemoto & J. T. A. F. Clewell & R. K. Godfrey 2067 (TUS) A. F. T. Nemoto 5867 (TUS) T. T. Nemoto 2916 (TUS) T. T. Nemoto 2392 (TUS) T. T. Nemoto 8604 (TUS) T. T. Nemoto 8626 (TUS) T. T. Nemoto 5900 (TUS) T. T. Nemoto 8609 (TUS) T. T. Nemoto & al. 96729025 (TUS) T. T. Nemoto 2919 (TUS) T. T. Nemoto & al. 96803001 (TUS) T. H. Tabata & al. 3356 (TUS) Tabata H. T. Nemoto & J. Yokoyama 95817001 (TUS) Yokoyama Nemoto & J. T. T. Nemoto 5892 (TUS) T. P. Frana & M. J. Leoschke 37 (ISC) P. J. Yokoyama & al. s.n. (Sep. 1996) (TUS) Yokoyama J. T. Nemoto 8628 (TUS) T. T. Nemoto & J. Yokoyama 95816027 (TUS) Yokoyama Nemoto & J. T. H. Ohashi & al. 61092 (TUS) H. Ohashi & al. 60756 (TUS) T. Nemoto & K. 8625 (TUS) T. T. Nemoto & J. Yokoyama 95824015 (TUS) Yokoyama Nemoto & J. T. Locality A. F. Clewell in 28 Sep. 1959 (TUS) A. F. , Chinese Academy of Sciences as seed exchange. , Chinese Cult. (TAIWAN, Pingtung Co.) Cult. (TAIWAN, Cult. (CHINA)*1 JAPAN, Miyagi Pref., Sendai JAPAN, JAPAN, Miyagi Pref., Sendai JAPAN, JAPAN, Miyagi Pref., Tajiri-cho Miyagi Pref., JAPAN, JAPAN, Miyagi Pref., Sendai JAPAN, JAPAN, Miyagi Pref., Ishinomaki JAPAN, JAPAN, Miyagi Pref., Tajiri-cho Miyagi Pref., JAPAN, CHINA, Guangxi Prov., Ziyuan Co. CHINA, Guangxi Prov., Cult. (TAIWAN, Taichung Co.) Taichung Cult. (TAIWAN, Cult. (JAPAN, Shiga Pref. Takashima-gun, Kutsuki-mura) Takashima-gun, Shiga Pref. Cult. (JAPAN, JAPAN, Miyagi Pref., Ishinomaki JAPAN, Cult. (JAPAN, cult.) Cult. (JAPAN, KOREA, Kangwon Prov., Pyongchang-gun, Doam-myun, Mt. Odae KOREA, Kangwon Prov., KOREA, Chollanam Prov., Jurye-gun, Sandong-myun KOREA, Chollanam Prov., Cult. (JAPAN, Niigata Pref.) Cult. (JAPAN, Cult. (JAPAN, cult.) Cult. (JAPAN, U.S.A., Arkansas, Logan Co. U.S.A., U.S.A., Georgia, Seminde Co. U.S.A., Georgia, Cult. (U.S.A)*2 Cult. (CHINA) *1 Cult. (TAIWAN, Hualien Co.) Cult. (TAIWAN, JAPAN, Miyagi Pref., Ishinomaki JAPAN, Cult. (KOREA, Kyonggi Prov., Shihung-gun, Kunja ) Cult. (KOREA, Kyonggi Prov., Cult. (U.S.A., Indiana)*3 JAPAN, Miyagi Pref., Sendai JAPAN, CHINA, Yunnan Prov., Lijiang Pref. Co. Prov., Yunnan CHINA, Cult. (CHINA)*1 CHINA, Yunnan Prov., Lijiang Pref. Co. Prov., Yunnan CHINA, NEPAL, Dolpo distr., Chaurigoth Dolpo distr., NEPAL, U.S.A., Arkansas, Logan Co. U.S.A., Cult. (KOREA, Chungchongnam Prov.) U.S.A., Iowa, Winneshiek Co. Winneshiek U.S.A., Iowa, JAPAN, Miyagi Pref., Sendai JAPAN, Cult. (U.S.A., Arkansas)*4 Cult. (U.S.A., U.S.A., Missouri, Barry Co. U.S.A., Florida, Leon Co. U.S.A., Texas, Edwards Co. Texas, U.S.A., JAPAN, Iwate Pref., Takizawa-mura Iwate Pref., JAPAN, U.S.A., Iowa, Van Buren Co. Van U.S.A., Iowa, Tohoku University, Sendai, JAPAN. Origin is shown in parenthises. (JAPAN, cult.): Japanese cultivated species (Akiyama 1988). Origin is shown in parenthises. (JAPAN, Sendai, JAPAN. University, Tohoku (Nakai) S.Akiyama & H.Ohba Taxon (Maxim.) Makino C.K.Schneid. Franch. Subtribe Desmodiinae Engelm. (Bunge) Rehder macrocarpa (Pursh) Elliott Miq. (S.Watson) Britton (S.Watson) (Maxim.) Schindl. (L.) Britton L.H.Bailey (L.) Pers. Michx. Schindl. (Dum.Cours.) G.Don Franch. Nutt. (L.f.) Pers. Britton Dendrolobium dispermum (Hayata) Schindl. Dendrolobium Campylotropis K. striata (Thunb.) Schindl. Lespedezsa Subgenus Macrolespedeza (Maxim.) H.Ohashi L . bicolor Turcz. Miq. L . buergeri L . cyrtobotrya L . davidii (Thunb.) DC. L . virgata L . virginica (Vogel) Koehne subsp. formosa L . formosa (Vogel) L . formosa subsp. velutina L. homoloba Nakai L . japonica L . maximowiczii L . melanantha Nakai L . patens Nakai (DC.) Nakai L . thunbergii Subgenus Lespedeza L . angustifolia L . capitata L . caraganae Bunge L . chinensis G.Don L . cuneata L . daurica (Laxm.) Schindl. L . fasciculiflora L . intermedia L . juncea L . leptostachya L . floribunda Bunge L . forrestii Graham ex Maxim. L . gerardiana L . hirta (L.) Hornem. L . inschanica L . pilosa (Thunb.) Siebold & Zucc. Michx. L . procumbens (L.) Barton L . repens L . stuevei L . texana L . tomentosa (Thunb.) Siebold ex Maxim. L . violacea Tribe Desmodieae Tribe Subtribe Lespedezinae Cult.: Cultivated in Experimental Garden, Biological Institute, *1: Grown from seeds sent Beijing Botanical Garden, Institute of Botany *2: Grown from seeds sent Rivendell Botanical Garden, Beardstown, Illinois, U.S.A. as seed exchange. *3: Grown from seeds picked a specimen collected Indiana, U.S.A. by A. Hiratsuka. Arkansas, U.S.A. by *4: Grown from seeds gathered Johnson Co., Table 2. Sample information examined in the present study and sequence obtained Table 218 植物研究雑誌 第 85 巻 第 4 号 2010 年 8 月

Results dispermum (372 bp) have also longer sequences Sequencing comparison than Lespedeza in this region. Total of 17 indels The sequences of trnL intron and trnL-trnF were inferred from the alignment of all obtained spacer region were determined for 35 species sequences of the region. There are 69 variable and one subspecies of Lespedeza, two species nucleotide positions, of which 21 nucleotides of Kummerowia, one species of Campylotorpis (30.4% of all variable positions) are parsimony- and one species of Dendrolobium. All sequences informative. determined in the present study were submitted to DDBJ (accession numbers AB538884 to Phylogenetic analysis AB538963; see Table 2). The MP analysis without and with indels The obtained sequences of trnL intron varied found 4 (141 steps, CI = 0.915, RI = 0.940) and in length among the samples used in this study. 5 (169 steps, CI = 0.911, RI = 0.942) equally All but five samples ofLespedeza have the same most parsimonious trees, respectively. The strict length of the region (530 bp) and no insertions/ consensus trees of both cases are summarized deletions (indels) were observed among them. in Fig. 2, which showed the monophyly of Indels were observed in two Asian species, L. Lespedeza and Kummerowia (85/99% bootstrap buergeri and L. patens (535 bp), and in three support, BS, in analyses without/with indels). North American species, L. hirta (506 bp), L. Within this clade three monophyletic groups leptostachya (535 bp) and L. virginica (532 bp). were constructed: the clade of two species of Kummerowia stipulacea (528 bp) has a 2-bp Kummerowia (95/99% BS), the clade composed deletion compared with most of Lespedeza, of all eastern Asian species of Lespedeza though no indels were observed in the (Clade A, 84/92% BS), and the clade of all sequence of K. striata (530 bp). Campylotropis North American species of Lespedeza (Clade macrocarpa (547bp) has longer sequences than B, 99/100% BS). Within the Clade A seven Lespedeza and Kummerowia. The sequence of subclades were formed though their BS was low Dendrolobium dispermum (532 bp) is a little (<80% BS), except the clade of L. chinensie, L. longer than most Lespedeza. Total of 8 indels floribunda and L. virgata (Clade C, 86/87% BS). were inferred from the alignment of all obtained Within the Clade B six subclades were formed, sequences of the region, 5 of them inferred in among them two clades, Clade D and Clade E, Lespedeza. There are 53 variable nucleotide were grouped with good support (>95% BS). positions, of which 17 nucleotides (32.0% of all The NJ and ML trees showed almost the variable positions) are parsimony-informative. same topology as the MP trees (Figs. 3, 4). The sequences of trnL-trnF spacer region The Kummerowia clade and Clades A–E of were different in length between Asian and North Lespedeza were commonly grouped with good American species of Lespedeza. The North support (>76% BS). The NJ tree supported the American species have longer sequence (368 bp monophyly of all Lespedeza species (79% BS), in all samples) than the Asian species (360 bp in but other two trees did not. The MP and ML all samples except L. chinensis, L. floribunda, L. trees supported the monophyly of the subgenus virgata [361 bp] and L. fasciculiflora [358 bp]). Macrolespedeza species (61/62% and 69% BS, The length difference between these two groups respectively), but the NJ tree did not. Another resulted from 4 indels. Two Kummerowia subdivision of Lespedeza, subgenus Lespedeza, species have the same length in the region was not supported in the present analyses. as the North American Lespedeza and 3 of 4 Instead, species of subgenus Lespedeza were indels were common to them. Campylotropis grouped into two clades, A and B, one of which macrocarpa (374 bp) and Dendrolobium is composed only of Asian species and the other August 2010 The Journal of Japanese Botany Vol. 85 No. 4 219

Fig. 2. Strict consensus of five equally most parsimonious trees (169 steps, CI = 0.911, RI = 0.942) based on the chloroplast trnL intron and trnL-trnF intergenic spacer including indels for the species of Lespedeza and its closely related genera. Numbers above branches indicate bootstrap values (≥ 50%), excluding/including the indel characters, resulting from 1000 replicates. Clades A–G are notable clades in Lespedeza, and all of them correspond to those in Figures 3 and 4. 220 植物研究雑誌 第 85 巻 第 4 号 2010 年 8 月

Fig. 3. Neighbor-joining tree inferred from the chloroplast trnL intron and trnL-trnF intergenic spacer for the species of Lespedeza and its closely related genera based on Kimura’s two-parameter distance. Numbers above branches indicate bootstrap values (≥ 50%) resulting from 1000 replicates. Clades A–E and G are notable clades in Lespedeza, and all of them correspond to those in Figures 2 and 4. August 2010 The Journal of Japanese Botany Vol. 85 No. 4 221

Fig. 4. Maximum likelihood tree inferred from the chloroplast trnL intron and trnL-trnF intergenic spacer for the species of Lespedeza and its closely related genera (log-likelihood = ­−2164.4). Numbers above the nodes indicate bootstrap values (≥ 50%) resulting from 100 replicates. Clades A–G are notable clades in Lespedeza, and all of them correspond to those in Figures 2 and 3. 222 植物研究雑誌 第 85 巻 第 4 号 2010 年 8 月 only of North American species. The Asian almost whole Lespedeza also reveals the same species of subgenus Lesapedeza form a clade relationship of Kummerowia and Lespedeza. (Clade A) together with those of the subgenus The monophyly of Kummerowia was Macrolespedeza. Within the Asian Lespedeza strongly supported by the present study, while clade the monophyly of species attributed to the that of Lespedeza is still uncertain although both subgenus Lespedeza was not supported. Within genera are easily distinguished from each other the North American clade (Clade B), two robust in habit (annual vs. perennial), leaf venation clades were formed with good support (>90% pattern (primary lateral veins extending to leaf BS) in all trees. margin or not), inflorescence features (Nemoto and Ohashi 1993a, Nemoto et al. 1995), and Discussion so on. Relationships among the three major Phylogenetic relationships between Lespedeza clades, Kummerowia clade, Asian Lespedeza and allied genera clade (Clade A) and eastern North American Lespedeza, Kummerowia and Campylotropis Lespedeza clade (Clade B), remain incompletely were attributed to subtribe Lespedezinae of resolved. tribe Desmodieae together with Phylacium and Neocollettia mainly on the basis of 1-ovulate Infrageneric phylogeny in Lespedeza ovary and absence of hooked trichomes The present sequence data constructed (Ohashi et al. 1981). However, Phylacium two robust clades, Clade A and Clade B, and Neocollettia were recently moved to in Lespedeza, the former is composed of tribe Phaseoleae based on morphological, Asian species and the latter of eastern North palynological and molecular evidence (Doyle American species. Lespedeza has been classified et al. 2000, Kajita et al. 2001, Ye and Ohashi into two groups as sections or subgenera, 2005, Schrire 2005, Ohashi 2005). Lespedeza, Macrolespedeza and Lespedeza, on the basis Kummerowia and Campylotropis have remained of habit, morphology of calyx and presence or in the subtribe. The tribe Desmodieae was absence of cleistogamous flowers (Maximowicz recently distinguished into three groups, the 1873, Taubert 1894, Schindler 1913, Nakai Lespedeza, Phyllodium and Desmodium groups 1927, Ohashi 1982, Akiyama 1988, Ohashi et al. (Ohashi 2005). The subtribe Lespedezinae 2009a, 2009b). The subgenus Macrolespedeza, corresponds to the Lespedeza group (Ohashi which consists of shrubs without cleistogamous 2005). Dendrolobium dispermum used as an flowers and is naturally distributed only in outgroup in the present study is attributed to the Asia, was supported its monophyly in the Phyllodium group. present MP and ML analyses although the The MP, NJ and ML trees obtained in the branch was with lower-support. Subgenus present study all supported the monophyly Lespedeza, which consists of perennial herbs of Lespedeza with Kummerowia not with with cleistogamous flowers and is disjunctively Campylotropis. Kajita et al. (2001) represented distributed in Asia and eastern North America, sister relationship between Kummerowia and was shown to be paraphyletic. Asian species Campylotropis within a large tree of whole of subgenus Lespedeza make a common clade legumes based on rbcL sequences. Stefanović et with the subgenus Macrolespedeza, while al. (2009) recently indicated the monophyletic eastern North American species make a clade relationship of Lespedeza and Kummerowia by themselves. In contrast to North American based on trnL-F sequences of whole "phaseoloid species, the monophyly of Asian species of the legumes”. The present study including all two subgenus Lespedeza is still uncertain because Kummerowia species and species representing no informative variations were found in their August 2010 The Journal of Japanese Botany Vol. 85 No. 4 223 sequences examined in the present study. monophyletic group derived from a common Maximowicz (1873) proposed an infrageneric ancestor. system of Lespedeza of the world for the first The two clades (Clade A and Clade B) time, and was followed by Taubert (1894). found in Lespedeza in the present study are He recognized four groups as series in the well congruent with divergence in seedling subgenus Lespedeza, i.e., series Violaceae, morphology between Asian and North American Junceae, Lespedezaria and Pilosa (Table species shown by Nemoto and Ohashi (1993b), 1). Among them, series Junceae and Pilosa i.e., first two foliage leaves subsequent to consist only of Asian species, while Violaceae cotyledons are usually opposite in Asian species and Lespedezaria consist of both Asian and regardless of two subgenera, while they are North American species, i.e., series Violaceae alternate in North American species. Because comprising L. elegans, L. floribunda and L. only the opposite seedling type has been so virgata of Asia and L. repens, L. virginica (as L. far known in allied genera, Kummerowia and reticulata), L. stuevei and L. violacea of North Campylotropis (Lubbock 1892, Maekawa 1955, America, and series Lespedezaria comprising L. Ohashi 1968), and it is also common in other gerardiana, L. daurica (as L. medicaginoides or genera of the tribe Desmodieae (Ohashi 1973), L. trichocarpa), L. tomentosa and L. variegata the alternate seedling type can be regarded as of Asia and L. capitata and L. hirta of North being apomorphic in the tribe Desmodieae. America. The present result is incongruent Therefore the alternate seedling type is a with Maximowicz's (1873) treatment of series synapomorphy for North American species of Violaceae and Lespedezaria, but reveals the Lespedeza. separation of Asian species and North American ones in both series. Schindler (1913) proposed Relationship among Asian Lespedeza a phylogenetic diagram showing relationships Within the Asian clade (Clade A), three among species of the subgenus Lespedeza. In his clades (Clade C, Clade F, Clade G) were diagram three main lineages are derived from a resolved although their bootstrap values were common ancestor borne in Asia (Fig. 1B): the rather low. Clade F is composed of species first lineage consists of all Asian species except attributed to the subgenus Macrolespedeza. L. tomentosa; the second one consists only of Within this clade one or two clades (L. bicolor North American species with purple flowers; + L. formosa subsp. formosa + L. melanantha, the third one consists of North American species L. buergeri + L.patens) were found with low with white flowers and the Asian L. tomentosa. bootstrap value. These clades are incongruent Schindler (1913) separated all Asian species with interspecies relationships presented by except L. tomentosa from those two series of Schindler (1913) (Fig. 1A) or any infrageneric Maximowicz (1873) and combines almost all taxa (Nakai 1927, Akiyama 1988, Ohashi et al. Asian species into one lineage. His separation 2009b). Clade C and Clade G were resolved of Asian species from North American ones among Asian species of the subgenus Lespedeza. is almost supported by the present result. The Clade C composed of L. virgata, L. floribunda two lineages, that were recognized in North and L. chinensis is supported better than Clade G American species for the first time by Torrey composed of L. cuneata, L. daurica, L. forrestii, and Gray (1840) as sections Lespedeza and L. juncea, L. inschanica and L. tomentosa. Lespedezaria and were subsequently accepted Among these species in Clade G L. cuneata, L. by Maximowicz (1873) and Schindler (1913), juncea, L. inschanica and L. daurica have often are also supported by the present analysis. More been regarded as being closely related to each over, the two lineages were shown to form a other (Maximowicz 1873, Schindler 1913, Nakai 224 植物研究雑誌 第 85 巻 第 4 号 2010 年 8 月

1927, Ohashi 1982, Pramanik and Thothathri very difficult (Clewell 1966c). Isely (1955) 1983) (Fig. 1B), but no morphological characters considered the L. virginica – intermedia – sutevei critically characterizing these clades have been complex as a group of closely related species. found so far. Although the monophyly of L. virginica and L. stuevei was weakly supported, the relationship Relationships among North American Lespedeza of both species with L. intermedia was not in the The present results showed that North present study. American species have more informative variations in nucleotide substitutions than Asian Relation of sequence divergences to natural ones. Therefore, all analyses produced a better- hybridization in Lespedeza resolved phylogenetic hypothesis of cpDNA The sequence divergences among North relationships in North American species than in American species were found to be larger than Asian ones. those among Asian ones in the present study Two robust clades found in North American (Figs. 2–4). The American form a species, Clades D and E, correspond to two relatively close knit group of species whose taxa, sects. Lespedeza (as Eulespedeza) and precise intra-specific limits are sometimes Lespedezaria by Torrey and Gray (1840) difficult to define and it is frequently said that the or series Violaceae and Lespedezariae by American lespedezas hybridize with one another Maximowicz (1873), that have been often (Isely 1955). The species are mostly well- distinguished among North American species defined, but identifications are complicated by mainly on the basis of flower color (purple infraspecific polymorphism and by ubiquitous vs. white) and the relative length of corolla to interspecific hybridization (Isely 1998). Clewell calyx (corolla at least twice as long as calyx vs. (1966a) recognized 33 interspecific hybrid corolla slightly longer than calyx). Moreover, combinations of 55 possible ones. According Schindler (1913) also recognized two groups to him there are a few hybrids in nearly every corresponding to these two. Even within each population and 16 names have been applied to clade better resolved hypothesis is obtained, various hybrid combinations. Therefore, North although bootstrap values were relatively low. American species are taxonomically difficult In the clade of white-flowered species L. mainly due to such extensive hybridization. leptostachya, which is a state and federally Although the sequence divergences are threatened species and distributed only in the tall relatively large (eight mutations) between white- grass prairie region of four midwestern states (Clade D) and purple-flowered clades (Clade (Sather 1990), is distinguished from three other E), the natural hybridization has been known white-flowered species, L. angusutifolia, L. between the species of both clades. Moreover, capitata and L. hirta. Lespedeza leptostachya is the percentages of cells with univalent and assumed to be divided at the bottom of the clade. stainable pollen grains are variable in hybrids In the clade of purple-flowered species L. (Clewell 1966a) and there are no conspicuous virginica and L. stuevei, both of which share differences in such percentages between similar flower morphology, i.e., wings longer hybrid combinations within each clade and than keel-petals, and surface features of leaves between two clades. In contrast, although there (Clewell 1966b, 1966c), were weakly supported was only one mutation found in the present their monophyly by the present molecular data. sequences between Asian species of subgenera On the other hand, the monophyly of L. repens Macrolespedeza and Lespedeza, no hybrids and L. texana was not supported, although have been recorded so far between species of morphological distinction of them is sometime both subgenera, but only within each subgenus August 2010 The Journal of Japanese Botany Vol. 85 No. 4 225

(e.g., Makino 1913, Kitagawa 1934, Akiyama virgata (Pierce 1939) and L. gerardiana (Sandhu and Ohba 1982, 1983a, 1983b, Akiyama 2003, and Mann 1989). The former three species 2004). Thus, the ability of hybridization does not formed the same clade (Clade C) with rather correlate with the divergence or genetic distance good support in all analyses. The monophyly of cpDNA sequences in Lespedeza. of these three species was also supported by RFLPs analysis (Nemoto et al. 1995). Schindler Variation and evolution of chromosome number (1913) located L. chinensis, L. gerardiana and L. in Lespedeza virgata near the base of the Asian species lineage According to Goldblatt (1981), the chromo- and connected L. floribunda with L. chinensis in some base number of tribe Desmodieae is his phylogenetic diagram (Fig. 1B). However, fundamentally x = 11. The tribe Desmodieae the primitiveness of the Clade C composed of L. has been included into the clade of tribe chinensis, L. floribunda and L. virgata was not Phaseoleae by recent molecular data (Bruneau shown in the present study. et al. 1995, Doyle et al. 1997, Kajita et al. 2001, Schrire 2005, Ohashi 2005). The base number Biogeographical implications on disjunct of the tribe Phaseoleae is also regarded as x distribution of Lespedeza = 11 (Goldblatt 1981). In Campylotropis the Lespedeza has been known as a genus numbers of n = 11, 2n = 22, 23 (Index to Plant representing the well-known disjunct Chromosome Numbers Data Base accessed on distribution pattern between eastern Asia and 8 Jan. 2010) and in Kummerowia the numbers eastern North America (Gray 1846, Li 1952, of 2n = 20, 22 (Nishikawa 2008, Index to Plant Wu 1983, Boufford and Sponberg 1983, Wen Chromosome Numbers Data Base accessed on 1999). Like many other disjunct genera (Wen 8 Jan. 2010) have been reported. In Lespedeza 1998, 1999), the present data reveals no species the numbers of n = 9, 10, 11 and 2n = 18, 18+2, combinations suggesting direct (intercontinental) 20, 22, 36, 40, 42, ca. 44 have been reported sister relationships between the two continents (Kawakami 1930, Cooper 1936, Pierce 1939, in Lespedeza. Instead, the present results Young 1940, Hanson and Cope 1955, Clewell strongly suggest that early diversification was 1964, 1966a, 1971, Lee 1969, 1970, Mesicek and occurred in an ancestral lineage of Lespedeza. Sojak 1972, Sareen and Singh 1975, Nishikawa Because almost all genera of the allied 2008). Among these counts the number of n = subtribe Desmodiinae except Desmodium and 11 or 2n = 22 is assumed to be plesiomorphic, are distributed in the Old World and others to be apomorphic in Lespedeza. and most diverse in tropical S and SE Asia as Within Lespedeza the chromosome number of well as Campylotorpis and Kummerowia of the n = 11 or 2n = 22 was reported commonly in subtribe Lespedezinae (Ohashi 2005), we can subgenus Macrolespedeza (Akiyama 1988), infer that the ancestor of Lespedeza was borne in while apomorphic numbers of n = 10 or 2n Asia, early diversification occurred in Asia, and = 20 were dominantly reported in both Asian then one of the lineages diversified was followed and North American species of the subgenus by unidirectional migration/dispersion to North Lespedeza, especially the number 2n = 20 or n America, perhaps via the Bering land bridge like = 10 has been determined in all North American other temperate deciduous representing species (Clewell 1964, 1966a). However, the the same disjunct distribution pattern (Wen plesiomorphic chromosome number, 2n = 22 or 1999, Xiang et al. 2000, Donoghue and Smith n = 11, have been known in four Asian species 2004). The dispersed lineages of both continents of the subgenus Lespedeza, i.e., L. chinensis have been isolated from each other after the (Nemoto, unpublished data), L. floribunda, L. primary divergence, and then speciation in both 226 植物研究雑誌 第 85 巻 第 4 号 2010 年 8 月 continents has occurred independently with Miq. J. Jpn. Bot. 57: 232–240. neither reciprocal nor bidirectional migration/ Akiyama H. and Ohba H. 1983a. Studies on hybrids in the genus Lespedeza sect. Macrolespedeza (2) A hybrid dispersion between both regions. swarm between L. homoloba Nakai and L. kiusiana Nakai. J. Jpn. Bot. 58: 97–104. We are grateful to Profs. T.-C. Huang and C.- Akiyama H. and Ohba H. 1983b. Studies on hybrids in the F. Hsieh (Taiwan University) for their support genus Lespedeza sect. 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根本智行 a,*，横山 潤 b，福田達哉 c，五百川 裕 d，大 橋広好 e：葉緑体 DNA trnL-trnF 領域の塩基配列に基づく マメ科ハギ属の系統解析 や花序の形態と相関する 2 つのクレードが高いブートストラ マメ科ハギ属 (Lespedeza) は約 40 種からなり，アジアと ップ値で支持された．以上の結果は，ハギ属をヤマハギ亜 北アメリカに隔離分布する植物として知られている．種間系 属（あるいは節）およびハギ亜属（あるいは節）の 2 群に 統関係を明らかにするため，ハギ属 35 種 1 亜種および近 分類する従来の見解やヤマハギ亜属内の節（あるいは系） 縁属のヤハズソウ属 2 種，ハナハギ属 1 種，ナハキハギ の分類を支持しなかった．また，ハギ属の祖先群はアジア 属１種の計 40 分類群の系統解析を行った．解析には葉緑 に起源し，初期に 2 つの系統群に分化したこと，そのうちの 体 DNA の trnL イントロンおよび trnL-trnF 遺伝子間領域 一方が北アメリカに分布を拡大したこと，その後 2 つの系統 を用いた．解析の結果，以下のことが明らかになった．１） 群は両地域間で互いに交流することなく，独自の種分化の ハギ属とヤハズソウ属は単系統群を形成した．２）ヤハズ 過程を経たことを示唆した． ソウ属は単系統性が支持されたが，ハギ属の単系統性は支 持されなかった．３）ハギ属には 2 つの単系統群があり， (a石巻専修大学理工学部基礎理学科， 一方はアジアの種のみから，またもう一方は北アメリカの種 b 山形大学理学部生物学科， のみから構成される．この 2 つのクレードは，アジアと北ア c高知大学農学部大学院 メリカの両地域間で見られる芽生え形態の相違と一致した． 総合人間自然科学研究科農学専攻， ４）アジアのクレードには，ブートストラップ値は低いもの d上越教育大学大学院学校教育研究科学校教育学系， のさらに 3 つのクレードが認められ，このうちの 1 つはヤマ e東北大学植物園津田記念館 ) ハギ亜属と一致した．５）北アメリカのクレードには花の色