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Home , Rhododendron, Rhododendron canadense, Rhododendron indicum, Rhododendron luteum, Section (biology)

植物研究雑誌 J. J. Jpn. Bo t. 73: 73: 143-154(1998)

Investigation Investigation of Sectional Relationships in the () sased on matK Sequences

Y 吋iKURASHIGE a，Masaki MINE bペNobuo KOBA Y ASHl bぺ Takashi HANDA b，Kenji TAKAYANAGl b and Tomohisa YUKAWA c

aLaboratory aLaboratory of ，Ak agi Nature Park ， Yuhikami Yuhikami 892 ，Minami- Ak agisan ，Ak agi-mura ，Seta-gun ，Gunma ， 379-1113 ; blnstitute blnstitute of Agriculture and Forestry ，University of Tsukuba ， Tennodai Tennodai 1-1-1 ，Tsukuba ，Ibaraki ， 305-0005 JAPAN; CTsukuba CTsukuba Botanical Garden ，National Science Museum ，Amakubo 4-1-1 ，Tsukuba ，Ibaraki ， 305-0005 JAPAN; dl wate Biotechnology Research Center ，Narita 22-174-4 ，Ki takami ，Iwate ， 024-0003 JAPAN;

巴Tatebayashi Research Station ，Hanayama 3258 ，Tatebayashi ，Gunma ， 374-0005 JAPAN (Received (Received on October 20 ， 1997)

Owing to the enormous number of and conflicting ideas of classification proposed proposed by many taxonomists ， the genus Rhododendron poses systematic problems at various various infrageneric levels. Sequence data derived from matK and trnK introns were used to to examine relationships among all 8 subgenera and 12 sections of Rhododendron ，and additional additional genera Ledum ，Menziesia ， and all Elliottia. The major results from this study are are as follows: (1) Menziesia and Ledum are nested within the genus Rhododendron. (2) Thus Rhododendron is paraphyletic. (3) R. camtschaticum forms a basallineage of Rhodoreae. Rhodoreae. (4) Tsutsusi is monophyletic; however ， R. tashiroi makes both sections sections Tsutsusi and Brachycalyx para-/polyphyletic. (5) R. tsusiophyllum is a member ofthe ofthe subgenus Tsutsusi Tsutsusi. (6) Subgenus Rhododendron is monophyletic. (7) (7) Subgenus Pentanthera is polyphyletic. (8) Subgenus Pentanthera section Sciadorhodion is is polyphyletic. (9) Subgenus Azaleastrum is polyphyletic.

In In the mountain of East to Southeast nus in various ways. On the basis of these ， the genus Rhododendron L. (Ericaceae) earlier works ，Sleumer (1949 ， 1980) proposed holds holds the essential position. The diversity of a framework system of Rhododendron with a this this group is represented by the immense main emphasis upon relative positions of number of species (e. g. over 1 ，000: Chamber- and leafbuds ，characteristics oflepidote scales lain lain et al. 1996) as well as variation in their on the abaxial surface of ，and persist- habita t. Consequently ， this group presents in- ence or deciduousness of leaves as primary tricate tricate taxonomic problems ， in particularthose diagnostic characters. He recognized the fol- on infrageneric circumscr ヤtion. lowing eight subgenera: Azaleastrum ， Since Linnaeus (1 753) established Rhodo- Hymenanthes ，Pentanthera ，Pseudazalea ， dendron ，taxonomists such as Don (1 834) ， Pseudorhodorastrum ， Rhododendron ， Planchon (1854) ，Maximowicz (1870) ，Wilson Rhodorastrum ，and Tsutsusi (Table 1). and Rehder (1 921) ，and Copeland (1 943) have W orkers of the Royal Botanic Garden ，Ed- attempted over the to subdivide the ge- inburgh ，subsequently started to revise

一一 143 一一 144 144 植物研究雑誌第73 巻第3号 平成10 年6月

Table 1. Comparison oftwo subdivisions ofthe genus Rhododendron

Sleumer (1980) Chamberlain et al. (1 996)

Genus Rhododendron Genus Rhododendron Subgen.Rhododendron Sec t. Pogonanthum Sec t. Pogonanthum Subgen. Rhododendron Sec t. Rhododendron Sec t. Rhododendron Sec t. Vireya Sec t. Vireya

Subgen. Subgen. Pseudazalea Sec t. Pseudazalea

Subgen. Subgen. Pseudorhodorastrum Sec t. Rhabdorhodion Sec t. Rhodobotrys Sec t. Trachyrhodion

Subgen. Subgen. Rhodorastrum Sec t. Rhodorastrum

Subgen. Subgen. Hymenanthes Sec t. Hymenanthes Sec t. Ponticum Subgen. Hymenanthes

Subgen. Subgen. Tsutsusi Sec t. Brachycalyx Sec t. Brachycalyx Subgen. Tsutsusi Sec t. Tsusiopsis Sec t. Tsutsusi Sec t. Tsutsusi

Subgen. Subgen. Pentanthera Sec t. Pentanthera Sec t. Pentanthera Subgen. Pentanthera Sec t. Rhodora Sec t. Rhodora Sec t. Sciadorhodion Sec t. Viscidula Sec t. Viscidula

Subgen. Subgen. Azaleastrum Sec t. Azaleastrum Sec t. Azaleastrum Subgen. Azaleastrum Sec t. Choniastrum Sec t. Choniastrum

Sec t. Mumeazalea Subgen. Mumeazalea

Sec t. Candidastrum Subgen. Candidastrum

Genus Therorhodio l1 Subgen. Therorhodion

Genus TSltsiophylllll l1

Sleumer' Sleumer' s system. Cullen (1980) stressed the (1990) united the monotypic section Tsusiopsis taxonomic taxonomic importance of lepidote scales; he sensu Sleumer with section Tsutsusi ，and they thus thus united all the lepidote (scaly) rhododen- also reduced the monotypic genus drons drons into a single subgenus Rhododendron ， Tsusiophyllum to section Tsutsusi. Judd and while while Sleumer (1 980) scattered them among Kron (1995) moved parts of sections four four subgenera: Rhododendron ，Pseudazalea ， Brachycalyx (of subgenus Tsutsusi) and Pseudorhodorastrum ，and Rhodorastrum. Rhodora (of subgenus Pentanthera) to section Philipson Philipson and Philipson (1986) assigned sec- Sciadorhodion (of subgenus Pentanthera). tions tions Mumeazalea and Candidastrum (of Chamberlain et al. (1996) compiled these re- subgenus subgenus Azaleastrum sensu Sleumer) to a sults and recognized the following eight subgeneric subgeneric rank ，and they reduced genus subgenera: Azaleastrum ，Candidastrum ， Therorhodion Therorhodion to a subgeneric rank of the ge- Hymenanthes ，Mumeazalea ，Pentanthera ，Rho- nus nus Rhododendron. Chamberlain and Rae dodendron ，Therorhodion ，and Tsutsusi (Ta- June June 1998 Journal of Japanese Botany Vo l. 73 No. 3 145 ble ble 1). We will use this system as the reference ， recognized by Chamberlain et al. (1 996) were because because (1) it covers the whole genus ，and (2) sampled; since tribe Rhodoreae (of it it took account of the results from recent stud- Rhododendroideae) comprises three genera ， les. les. Rhododendron ，Ledum ，and Menziesia ，we There are many studies that intended to chose one species each from the latter two clarify clarify the phylogenetic relationships within genera. A single species of Elliottia (sub- Rhododendron ，e.g. ， in terms of secondary Rhododendroideae tribe Clado- substances substances (Harborne 1980 ，Harborne and thamneae) was also included. A single species Williams Williams 1971 ，King 1980) ，interspecific cross of Cassiope (subfamily Vaccioideae tribe compatibility compatibility (Williams et al. 1985 ，Yamaguchi Cassiopeae) was selected as the outgroup based et et al. 1985) ， ontogeny (Palser et al. 1985 ， on the results of an analysis of rbcL (Kr on and Philipson Philipson 1980) ，and micromorphology (Seith Chase 1993) and 18s rDNA (Kr on 1996) se- 1980). 1980). However ， these results have been of quences. Table 2 shows the materials exam- limited limited value in determining the phylogeny of ined and the sources where the were Rhododendron. Rhododendron. Therefore ，it is clear that we grown. All the voucher specimens are depos- require require another approach to resolve ited in TNS. m 仕ageneric systematic problems of Rhodo- Total DNA was extracted from fresh tissue dendron. dendron. following the methods of Kobayashi et al. Comparison of DNA sequences of matK ， (1995). Sequences were determined with PCR- the the maturase-encoding gene located in an intron amplified the matK gene and its flanking trnK of of the chloroplast gene trnK ， the former of introns from a total DNA extract by use ofthe which has evolved approximately three times primers shown in Fig. 1 and Table 3. Single- faster faster than the chloroplast gene rbcL (Johnson stranded DNA for dideoxy sequencing was and So It is 1994) ， has proven to be a powerful produced in a second round of amplification tool tool for phy logenetic reconstruction within using the double-stranded product as a tem- angiosperm families and genera (Johnson and plate. Both the forward and reverse strands Soltis Soltis 1994 ，Steele and Vilgalys 1994 ，Soltis et were sequenced for all taxa. l. al. 1996). In this study ，we compared matK and All parsimony analyses were conducted trnK introns sequences to investigate with PAUP ，Phylogenetic Analysis U Par- sing subgeneric subgeneric and sectional relationships in Rho- simony ， Version 3.1 (Swofford 1993). The dodendron. dodendron. heuristic search option with 100 random repli- cates cates (Maddison 1991) was used to perform Materials Materials and Methods Fitch parsimony analyses (Fitch 1971). Branch Twenty-two species representing all eight lengths for were calculated by ACCTRAN subgenera subgenera and 12 sections of Rhododendron optimization (Swofford and Maddison 1987).

trnK trnK 3914F trnK 1MF matK 462F matK 3MF ーー... -.. ーー ... ーー ...

m o. tK

matK 174R m o. tK1MR matK 1848R trnK 2R 司咽ー- ，.._ -時一 4 ‘一ー

Fig. Fig. 1. Relative position of the PCR amplification and sequencing primers used for matK and trnK introns. introns. Arrows indicate the direction of strand synthesis. Boxed areas represent coding regions. 146 146 植物研究雑誌第73 巻第3号 平成 10 年6月

Table Table 2. Species of Rhododendron and its related genera used for matK sequencing. Subdivision of Rhododendron is is based on Chamberlain et al. (1 996). Classification of all other taxa is based on Stevens (1 971)

GenBank Species Species Subgenus Section Voucher access lO n

Subfamily Subfamily Rhododendroideae Tribe Tribe Rhodoreae Rhododendron ovatum (Lind l.) Maxim Aza1eastrum Aza1eastrum Wi1son 1391 AB012729 Rhododendron stamineum Franch. Aza1eastrum Choniastrum Akagi N ature Park 94/0084 AB012730 Rhododendron albiflorum Hook. Candidastrum Akagi Nature Park 95/0359 AB012731 L. Hymenanthes Ponticum Apo1d ，Cox & Hutchinson 205 AB012732 Rhododendron semibarbatum Maxim. Mumeaza1ea Kurashige 264 AB012733 Sweet Pentanthera Pentanthera Akagi Nature Park 89/0177 AB 0l 2734

Rhododendron canadense (L.) To 汀. Pentanthera Rh odora Akagi N ature Park 93/0150 AB012735 Rhododendron schlippenbachii Maxim. Pentanth 巴ra Sciadorhodion Akagi N ature Park 92/0280 AB012736 Rhododendron αlbrechtii Maxim. Pentanthera Sciadorhodion Kurashige 349 AB012737 Rhododendron pentaphyllum Maxim. Pentanthera Sciadorhodion Kurashige 166 AB012738 Rhododendron nipponicum Matsum. Pentanthera Viscidu1a Kurashige 241 AB012739

Rhododendron primuliflorum BUfi 巴au & Franch. Rhododendron Pogonanthum SICH 143 AB012740 Rhododendronferrugineum L. Rhododendron Rhododendron Akagi Nature Park 90/0001 AB012741 Rhododendronjavanicum (B1ume) Benn Rhododendron Vireya Akagi Nature Park 86/0004 AB012742 Rhododendron santapaui Sastry & al. Rhododendron Vireya Cox & Hutchinson 459 AB012743 Rhododendron camtschaticum Pa1 1. Therorhodion Kurashige 458 AB012744 Rhododendronfarrerae Rhododendronfarrerae Tate apud Sweet Tsutsusi Brachyca1yx Akagi Nature Park 89/0021 AB012745 Rhododendron wadanum Makino Tsutsusi Brachyca1yx Kurashige 168 AB012746 (L.) Sweet Tsutsusi Tsutsusi Kurashige 195 AB012747 Rhododendron kaempj セri P1anch. Tsutsusi Tsutsusi Ki 520-1 AB012748 Rhododendron tashiroi Maxim. Tsutsusi Tsutsusi Kurashige 100 AB012749 Rhododendron tsusiophyllum Sugim. Tsutsusi Tsutsusi Kurashige 771 ABOl2750 Ledum palustre L. divers subsp. 伊ilosum (Nakai) H.Hara Kurashige 476 AB012751 Menziesia Menziesia multiflora Maxim. Kurashige 356 AB012752 Tribe Tribe C1adothamneae Elliottia Elliottia paniculata (Siebo1d & Benth. Zucc.) & Hook. Kurashige 256C AB012753 Subfamily Subfamily Vaccinioideae Tribe Tribe Cassiopeae Cassiope Cassiope lycopodioides (Pa1 1.) D.Don Kurashige 1124B AB012754

For For assessment of the re1ative robustness for su1ted in the same topo1ogy. The phy10genetic clades found in each Fitch parsimony ana1ysis ， ana1ysis resu1ted in 84 most parsimonious the the bootstrap method (Fe1senstein 1985) was trees each of 426 steps. These trees had a used used on 600 rep1icates. consistency index (CI) excluding uninforma- tive tive characters of 0.695 and a retention index Results Results (RI) ofO.754. The strictconsensus andone Our matK and its f1 anking trnK sequences of the most parsimonious trees are shown in provided provided a matrix of 2113 bp. A tota1 of 106 Figs. 2 and 3，respective1y. nucleotide nucleotide positions were phy10genetically All of the most parsimonious trees sup- informative informative in the matrix. Of the 20 inde1s ported the basa1 position of Elliottia (sub- identified identified from the a1igned sequences ， seven fami1y Rhododendroideae tribe C1ado- were informative and unambiguous. These thamneae). The remaining genera in tribe inde1s inde1s were not used to construct the Rhodoreae ，Ledum andMenziesia were nested phy10genetic phy10genetic trees shown here ， but the ana1y- within genus Rhododendron. Consequently ， sis sis combining the inde1 information a1so re- the genus Rhododendron was shown to be June June 1998 Joumal of Japanese Botany Vo l. 73 No. 3 147

Table Table 3. Location and base composition of amplification and sequencing primers used for matK and trnK introns

Primer Primer 5' sequence 3' Designed by

trnK3914F GGG GTT GCT AAC TCA ACG G Learn trnK trnK lMF GAT AAG TTT ACC GAG GTA GC Yukawa matK 462F AAT ACC CTA [C 庁]CC C[A/ G]T [C 庁]CA TC Chase matK3MF GTG GTC TCA ACC AAG AAG G Yukawa matK 174R CGA [G 庁]TA ATI AA[C 泊] CGT TTC AC Chase matK lMR GTA GAA AAA ATC GTA ATA GC Yukawa matK 1848R TAT CGA ACT TCT TAA TAG C Johnso n/ Soltis trnK2R AAC TAG TCG GAT GGA GTA G Steele

p訂 aphyletic. (99 % bootstrap value). Within this ， the Rhododendron subgenus Therorhodion relationship of section Rhododen- formed the lineage of tribe Rhodoreae dron to section Pogonanthum (bootstrap value with with a bootstrap value of 99 %. In the core of of 93 %) as well as the of section tribeRhodoreae ，twomajorclades wereappar- Vireya (bootstrap value of 98 %) were sug- ent ent in all of the most parsimonious trees ， but gested. both both clades are only weakly supported by Subgenera Pentanthera and Azaleastrum bootstrap bootstrap values. The first clade comprised were shown to be polyphyletic in this study. A Rhododendron subgenera Candidastrum ， part of subgenus Pentanthera section Tsutsusi ， Azaleastrum (in part) ， Pentanthera Sciadorhodion fell into Clade 1， but the rest of (in (in part) ，and the genus Menziesia (Clade 1). the same section formed a clade with the genus Among the members of this clade ， subgenus Ledum in Clade 2. Rhododendron subgenus Tsutsusi Tsutsusi formed a clade with a 99% bootstrap Pentanthera sections Pentanthera ，Rhodora ， value. value. Within this clade ，R. tashiroi (subgenus and Viscidula are also placed in Clade 2. The Tsutsusi Tsutsusi section Tsutsusi) was nested within a former two sections show a sister group rela- clade clade made by the members of subgenus tionship (59 % bootstrap value) ，and the latter Tsutsusi Tsutsusi section Brachycalyx (100 % bootstrap formed a clade with the common ancestor of value). value). Although some taxonomists (e.g. ， subgenus Mumeazalea and subgenus Stevens Stevens 1971 ，Sleumer 1980 ，Yamazaki 1996) Azaleastrum section Choniastrum (58 % treated treated R. tsusiophyllum as a separate genus ， bootstrap value). Besides ， subgenus Tsusiophyllum ，this species was grouped with Azaleastrum section Choniastrum was s住ongly the the members of subgenus Tsutsusi section grouped with subgenus Mumeazalea (bootstrap Tsutsusi Tsutsusi with a 99 % bootstrap value. value 99 %) rather than with subgenus The second m 司or clade consisted of the Azaleastrum section Azaleastrum ，which was following following taxa: Rhododendron subgenera united with subgenus Tsutsusi in this study. Pentanthera (i n part) ，Rhododendron ， Hymenanthes ，Mumeazalea ， Azaleastrum (in Discussion part) ，and the genus Ledum (Clade 2). Among Generic relationships in tribe Rhodoreae the the members of this clade ， a particularly well The results of this study strongly suggest supported supported group was subgenus Rhododendron the of Rhododendron ， because gen- 148 148 植物研究雑誌第73 巻第3号 平成10 年6月

Subgenus in Section in Rhododendron Rhododendron Elliottia Elliottia

Menziesia Menziesia

R. R. albiflorum Candidastrum

R. R. farrerae Tsutsusi 8rachycalyx

R. R. tashiroi Tsutsusi Tsutsusi 10

R. R. wadanum Tsutsusi 8rachycalyx

R. R. tsusiophyllum Tsutsusi Tsutsusi

R. R. indicum Tsutsusi Tsutsusi

R. R. kaempferi Tsutsusi Tsutsusi

R.ovatum Azaleastrum Azaleastrum

R. R. pentaphyllum Pentanthera Sciadorhodion

R. R. schlippenbachii Pentanthera Sciadorhodion 4 R. R. albrechtii Pentanthera Sciadorhodion

Ledum

R. R. canadense Pentanthera Rhodora

R. R. luteum Pentanthera Pentanthera n R. R. ferrugineum Rhododendron Rhododendron 一 釦 1 991 L_ R. primuliflorum Rhododendron Pogonanthum a.

(1) (1) R. R. javanicum Rhododendron Vireya

ト3 R. R. santapaui Rhododendron Vireya

R. R. ponticum Hymenanthes Ponticum

R. R. nipponicum Pentanthera Viscidula

R. R. semibarbatum Mumeazalea

R. R. stamineum Azaleastrum Choniastrum

R. R. camtschaticum Therorhodion

Cassiope

Fig. Fig. 2. Strict consensus of 84 most parsimonious Fitch trees based on matK and trnK intron sequences ， length = 426; consistency index (excluding uninformative characters) = 0.695; retention index = 0.754. 0.754. Bootstrap values from 600 replicates are provided above clades. June June 1998 Journal of Japanese Botany Vo l. 73 No. 3 149

58 58 Elliottia Elliottia 9 Menziesia Menziesia 7 R. R. albiflorum 3 R. R. farrerae

R. R. tashiroi 2 「ーイ 」イ R. R. wadanum

R. R. tsusiophyllum

R. R. indicum

R. R. kaempferi

R.ovatum 5 R. R. pentaphyllum 5 13 13 R. schlippenbachii 4 3 「一一- R. albrechtii 4 Ledum 9 2 「一一一 R. canadense

R. R. luteum 5 76 76 3 r-一一- R. ferrugineum

R. R. primuliflorum 6 「寸 13 R. R. javanicum

R. R. santapaui

R. R. ponticum 17 17 R. R. nipponicum 2 6 R. R. semibarbatum

R. R. stamineum 28 28 R. R. camtschaticum

Cassiope Cassiope

Fig.3. Fig.3. One of 84 most parsimonious Fitch trees based on matK and trnK intron sequences. Number of nucleotide substitutions supporting each monophyletic group is indicated (ACCTRAN optimi- zation). zation). 150 150 植物研究雑誌第73 巻第3号 平成10 年6月 eraLedum andMenziesia fall within the mem- generic rank (e.g. ，Stevens 1971 ， Sleumer 1980 ， bers bers of Rhododendron. However ， if Ledum Yamazaki 1989). Stevens (1 971) ，however ， and M enziesia are included into Rhododen- suggested the close relationship of M enziesia dron ，this genus becomes monophyletic. The to R. tsusiophyllum (genus Tsusiophyllum sensu paraphyly paraphyly of Rhododendron is also suggested Stevens) as both have anthers dehiscing through by a cladistic study based on morphological vertical short slits. By contrast ， the remaining characters characters (Kron and Judd 1990) ， while place- members of tribe Rhodoreae have anthers ment of Ledum and M enziesia by Kr on and dehiscing by ovate pores. Kr on and Judd

Judd (1 990) is different from ours. (1990) ， in their cladistic analysis of morpho 四 Our results suggest that Rhododendron logical characters ， also agreed with Stevens' subgenus subgenus Therorhodion forms the basalline- (1971) placement of M enziesia with R. age age of tribe Rhodoreae (99 % bootstrap value). tsusiophyllum. Our results suggest that The following characters may wa 口ant a sep 仕 Menziesia falls among the members of Rhodo- rate rate generic status of subgenus Therorhodion: dendron ， whereas R. tsusiophyllum does not (1) (1) results of this study; (2) chromosome num- form a clade with Menziesia. It is thus likely bers: bers: n=12 in subgenus Therorhodion ，n=13 in that a similar type of anther dehiscence in all all other members of tribe Rhodoreae; (3) an Menziesia andR. tsusiophyllum evolved inde- elongated elongated axis with -like pendently. Further studies are required to as- bracts bracts and bracteoles ，a unique combination of sess the accurate phylogenetic position of characters characters in tribe Rhodoreae. A cladistic M enziesia and the evolution of anther dehis- analysis analysis using mo 叩hological characters of cence. tribe tribe Rh odoreae (Kr on and Judd 1990) also supports supports the basal position of Rhododendron Infrageneric relationships in Rhododen- subgenus subgenus Therorhodiem. dron The genus Ledum fell within Rhododen- (1) Placement of R. tashiroi and R. dron dron and formed a clade with R. albrechtii tsusiophyllum (subgenus (subgenus Pentanthera section Sciadorhodion) The monophyly of subgenus Tsutsusi was with with a 92 % bootstrap value. However ，no strongly supported by a 99 % bootstrap value. anatomical anatomical or morphological features uniting The position of R. tashiroi ，however ，makes these these two taxa have been found ye t. On the both sections Tsutsusi and Brachycalyx other other hand ，Kr on and Judd (1990) reduced the p紅 a-/polyphyletic. Based on morpho1ogical genus genus Ledum to a subsectional rank of section information ， the taxonomic position of R. Rhododendron based on the following tashiroi has not been stabilized. Having mono- synapomorphies: synapomorphies: complex ，multicellular ，glan- mo 中hic leaves is a diagnostic character of dular ，peltate scales; non-lacerate scales and section Brachyca1yx ，and having persistent simple simple fringing scales; and revo- leaves is that of section Tsutsusi. Sleumer lute lute leaves in leaf . However ， our l1 wtK (1 980) thus recognized R. tashiroi as an inter- analysis analysis does not reveal the close relationship mediate taxon between sections Tsutsusi and of of Ledum to section Rhododendron. Although Brachycalyx ，and established a monotypic sec- the the exact placement of Ledum in Rhododen- tion Tsusiopsis (of subgenus Tsutsusi). On the dron dron is uncertain ，we are convinced that Ledum other hand ， Chamberlain and Rae (1990) moved should should be treated as an infrageneric taxon of this species to section Tsutsusi ， because they the the genus Rhododendron. gave emphasis to the taxonomic importance of Menziesia Menziesia has been widely recognized at a its persistent leaves and flattened hairs on the June June 1998 Journal of Japanese Botany Vo l. 73 No. 3 151 young stems. On the contrary ，Yamazaki (1996) mostly in . Our results showed a sister united united this species with the members of sec- group relationship between sections Rhodo- tion tion Brachycalyx based on its three dendron and Pogonanthum. This close rela- pseudowhorled pseudowhorled leaves at the shoot apex and tionship is a1so supported by the following filiform filiform fringed hairs on both the leaves and synapomorphies: entire sca1es on the abaxia1 stems. stems. He designated this group section surface of leaves ， crenulate or undulate Sciadorhodion Sciadorhodion (the definition differs from that corollae ， and simple trichomes fringing the ofChamberlaineta l. 1996). Ourresults strongly flower bud scales. support support Yamazaki's (1 996) view ， and allow (3) of sub 二genus， Pent α nthera the the conclusion that both and per- Subgenus Pentanthera is characterized by sistent sistent leaved species exist in section its deciduous leaves with indumentums of Brachycalyx. Brachycalyx. mu1ticellular gland-headed and/or glandular Rhododendron Rhododendron tsusiophyllum was strongly hairs and unicellular hairs; terminal racemes grouped grouped with all other species of subgenus and 1eafy shoots usually from axillary buds; Tsutsusi Tsutsusi section Tsutsusi (99 % bootstrap and lack of gossypetin (Judd and Kron 1995). value). value). Although some taxonomists (e.g. ， However ，ourresults showed the polyphyly of Stevens Stevens 1971 ， Sleumer 1980 ，Yamazaki 1996) subgenus Pentanthera: sections Rhodora and treated treated this species as a separate genus ， Pentanthera were shown to be sister groups in Tsusiophyllum ， with emphasis on its vertical Clade 2; section Sciadorhodion was divided dehiscing dehiscing anther and a three-loculated ， into C1ades 1 and 2; the monotypic section our our results support the placement of this spe- Viscidula formed a clade with the common cies into into cies section Tsutsusi proposed by Cham- ancestor of subgenus Mumeazalea and berlain berlain and Rae (1 990). Rhododendron subgenus Azaleastrum section Choniastrum tsusiophyllum tsusiophyllum and Rhododendron section in Clade 2. Several morphological characters Tsutsusi Tsutsusi share the following characters: mul- a1so suggest the para-/polyphy1y of this ticellular ，flattened ，and ferruginous hairs; subgenus. Especially ，the composition of 1eaf flower flower and leaf buds enclosed with terminal and flower buds of section Sciadorhodion is bud sca1es. Therefore it seems best to infer that not uniform: in R. schlippenbachii both leaf the the three-1ocu1ar ovary and the vertical and flower buds are enclosed with terminal dehiscing dehiscing anther of R. tsusiophyllum are bud scales ，namely ， the condition of a mixed autapomorphies autapomorphies of this species. bud; on the other hand ， R. albrechtii and R. (2) (2) Monophyly of subgenus Rhododendron pentaphyllum develop an inflorescence from All All of the sections of subgenus Rhododen- the terminal bud and a vegetative shoot from dron dron formed a monophyletic group (99 % the lateral buds. Since the mixed bud is a bootstrap bootstrap va1ue). Section Vireya showed a diagnostic character of subgenus Tsutsusi ， sister sister group relationship to the common Sleumer(1980) submergedR. schlippenb αchii ancestor ancestor of sections Rhododendron and into subgenus Tsutsusi section Brachycalyx. Pogonanthum. The following morphological On the other hand ， he included R. αlbrechtii characters characters are known to be unique to section and R. pentaphyllum into subgenus Pentanthera Vireya: Vireya: with appendages (l onger than section Rhodora because the number of sta- seed bodies) at both ends; valves mens and the corolla shape of these taxa are twisted twisted at the time of dehiscence; placentas identica l. Judd and Kr on (1 995) moved these separating separating as thread-like structures from the three species to subgenus Pentanthera section central central axis ， as well as the isolated distribution Sciadorhodion on the basis of the following 152 152 植物研究雑誌第73 巻第3号 平成10 年6月 synapomorphies: synapomorphies: lack of gossypetin ，decidu- Choniastrum is mainly distributed in southern ous ous leaves ，and lack of stiff ，ferrugineous ， ， andhas persistentleaves ，funnel-shaped flattened ，scale-like hairs on the abaxial sur- corollae ， ten ，and elongate capsules. face face of leaves. However ， the strict consensus Although the relationships among lateral- flow- tree tree of this study is inconsistent with the recog- ered taxa was not definitely elucidated in this nition nition of Sleumer (1 980) and Judd and Kr on study ，it is probable that the character of lateral (1 995). For elucidation of the relationships in inflorescence evolved more than once in Rho- section section Sciadorhodion ， accumulation of more dodendron. phylogenetic phylogenetic signals from both molecular and conventional conventional data as well as inclusion of R. We thank Mark W. Chase for matK se- quinquefolium ， another member of this sec- quence primers ，Hiroichi Katsuyama for tech- tion ， are required. At this moment ， our best nical advice ， and Shigeru Okamoto and Takumi resolution resolution suggests that the mixed bud evolved Hashimoto for skillful cultivation of independently independently in R. schlippenbachii and materials for this study. Special thanks are due subgenus subgenus Tsutsusi. Jun-ichiro Etoh for technical assistance. ( 4) Re lations hips in the late ral タowered group ， subgenera Azaleastrum ，Mumeazalea ， References and Candidastrum Chamberlain D. F. and Rae S. J. 1990. A revision of Rhododendron IV. subgenus Tsutsusi. Edinb. J. Bo t. The monophyly of subgenus Azaleastrum 47: 89-200. was not supported in this study ， because sec- 一一一一， Hyam R.， Argent G. ，Fairweather G. and Walter tion tion Azaleastrum was grouped with subgenus K. S. 1996. The genus Rhododendron ，its classifica- Tsutsusi Tsutsusi in Clade 1， and section Choniastrum tion and synonymy. Roy. Bo t. Gard. Edinburgh ， Edinburgh. Edinburgh. showed a sister group relationship to subgenus Copeland Copeland H. F. 1943. A study ，anatomical and taxo- Mumeazalea in Clade 2. Since Sleumer (1949 ， nomic of the genera of Rhododendroideae. Amer. 1980) 1980) emphasized the taxonomic importance Mid l. Naturalist 30: 533-625. of of the inflorescence from axillary buds ，he Cullen J. 1980. A revision of Rhododendron. 1. Subgenus thus thus recognized these species within a single Rhododendron sections Rhododendron and Pogonanthum. Pogonanthum. Notes Roy. Bo t. Gard. Edinb. 39: 1- subgenus ，namely ，Azaleastrum ， and subdi- 207. vided this subgenus into four sections Don G. 1834. A general history ofDichlamydeous plants. Azaleastrum ，Choniastrum ，Mumeazalea ，and Vo l. 3，Calyciflorae. London. Candidastrum Candidastrum on the basis of the number of Felsenstein J. 1985. Confidence limits on phylogenies: an an approach using the bootstrap. Evolution 39: 783- stamens stamens and deciduousness or persistence of 79 1. leaves. leaves. However ，ourresults do not supportthe Fitch W. M. 1971. Toward defining the course of evolu- monophyly of subgenus Azaleastrum sensu tion: Minimum change for a specific tree topology. Sleume r. The close relationship of subgenus Sys t. Zoo l. 20: 406 -4 16. Harbome Harbome J. B. 1980. pigments as both taxo- Azaleastrum section Choniastrum with nomic nomic and phyletic markers in the genus Rhododen- subgenus subgenus Mumeazalea rather than with the dron. In: Luteyn J. L. and Q'Brien M. E. (eds.) ， remaining remaining lateral-flowered taxa is inconsist- Contributions toward a classification of Rhododen- ent ent with results of previous studies based on dron ，pp. 145-161. New Y ork Bo t. Gard. ，New Y ork. morphology and geographic distribution. For 一一一- and Williams C. A. 1971. Leaf survey of flavonoids and simple phenols in the genus Rhodo- example ， subgenus Mumeazalea ，a monotypic dendron. Phytochemistry 10: 2727-2744. group group endemic to Japan ， has deciduous leaves ， Johnson L. A. and Soltis D. E. 1994. matK DNA se- rotate rotate corollae ， five dimorphic stamens ， and quences and phylogenetic reconstruction in globose capsules; in contrast ， section Saxifragaceae s. str. Sys t. Bo t. 19: 143-156. June June 1998 Journal of Japanese Botany Vo l. 73 No. 3 153

Judd Judd W. S. and Kr on K. A. 1995. A revision of Rhodo- Planchon J. E. 1854. Sur 1'histoire botanique et horticule dendron V I. subgenus Pentanthera (sections des plantes dites Azalees des l'Inde. rev. Hor t. se r. 4 Sciadorhodion ，Rhodora and Viscidula). Edinb. J. 3: 42 -4 9. Bo t. 52: 1-54. Seith nee von Hoff ， A. 1980. Rhododendron hairs and King B. L. 1980. The systematic implications of . In: Luteyn J. L. and O'Brien 乱tI. E. (eds よ flavonoids flavonoids in Rhododendron subgenus Pentanthera. Contributions toward a classification of Rhododen- In: In: Luteyn J. L. and O'Brien M. E. (eds よContribu- dron ， pp. 89-115. Bo t. Gard. ，New Yor k. tions tions toward a classification of Rhododendron ， pp. Sleumer H. 1949. Ein system der gattung Rhododendron 163-185. 163-185. New York Bo t. Gard. ，New Yor k. L. Bo t. Jahrb. Sys t. 74: 511-553. Kobayashi Kobayashi N. ， Takeuchi R.， Handa T. and Takayanagi K. 一一一一 1980. Past and present taxonomic systems of 1995. 1995. identification of Azalea Rhododendron. Based on macromo 中hological char- with with RAPD method. J. Jap. Soc. Hor t. Sci. 64: 611- acters. In: 一Luteyn J. L. and O'Brien M. E. (eds.) ， 616. 616. Contributions toward a classification of Rhododen- Kron K. A. 1996. Phylogenetic relationships of dron ， pp. 19-26 ，New York Bo t. Gard. ，New York. Empetraceae ， Epacridaceae ， Ericaceae ， Soltis D. E. ，Kuzoff R. F. ， Conti E. ， Gornall R. and Monotropaceae ，and Pyrolaceae: Evidence from nu- Ferguson K. 1996. matK and rbcL gene sequence clear clear ribosomal 18s sequence data. Ann. Bo t. 77: data indicate that Sax ザraga (Saxifragaceae) is 293-303. 293-303. polyphyletic. Ame r. J. Bo t. 83: 371-382. 一一一一 and Chase M. W. 1993. Systematics of the Steele K. P. and Vilgalys R. 1994. Phylogenetic analyses Ericaceae ，Empetraceae ， Epacridaceae and related of Polemoniaceae using nucleotide sequences of the taxa taxa based upon rbcL sequence data. Ann. Missouri plastid gene mat K. Sys t. Bo t. 19: 126-142. Bo t. Gard. 80: 735-74 l. Stevens P. F. 197 1. A classification of the Ericaceae: 一一一- andJudd W. S. 1990. Phylogeneticrelationships and tribes. Bo t. J. Linn. Soc. 64: 1-53. within within the Rhodoreae (Ericaceae) with specific com- Swofford D. L. 1993. PAUP: Phylogenetic analysis ments ments on the placement of Ledum. Sys t. Bo t. 15: 57- using parsimony ，version 3.1. Ill inois N atural History 68. 68. Survey ， Champaign.

Linnaeus Linnaeus C. 1753 ‘ . Stockholm. 一一一- and Maddison W. P. 1987. Reconstructing an- Maddison D. R. 1991. The discovery and importance of cestral character states under Wagner parsimony. multiple multiple islands of most-parsimonious trees. Sys t. Math. Biosci. 87: 199-299. Zoo l. 40: 315-328. Yamaguchi S. ， Kunishige M. and Tamura T. 1985. Maximovicz C. J. 1870. Rhododendreae Asiae Orientalis. Interspecific compatibility in Japanese Rhododen- Mem. Acad. Sci. s t. Petersbourg. se r. 7， 16: 1-53. drons. Bull. Veg. and Ornam. Crops Res. St r. Japan ， Palser Palser B. F. ，Philipson W. R. and Philipson M. N. 1985. se r. C ，8: 87-97. The ovary ， ovule and megagametophyte in Rhodo- Yamazaki T. 1989. Wild ofJ apan ，Woody plants dendron. dendron. Notes Roy. Bo t. Gard. Edinb. 43: 133-160. 11: 122-156. Heibonsha ，Tokyo (in Japanese). Philipson Philipson M. N. 1980. Cotyledons and Rhododendron 一一一一一 1996. A revision of the Genus Rhododendron in classification. classification. In: Luteyn J. L. and O'Brien M. E. Japan ， Tai wan ， and Sakhalin. 125 pp. Tsumura (eds.) ，Contributions toward a classification of Rho- Laboratory ， Tokyo. dodendron ， pp. 133-160. N ew Y ork Bo t. Gard. ，New Williams E. G. ，Rouse J. L. and Knox R. B. 1985. Yor k. Barriers to sexual compatibility in Rhododendron. Philipson Philipson W. R. and Philipson M. N. 1986. A revision of Notes Roy. Bo t. Gard. Edinb. 43: 81-98. Rhododendron. 111. Subgenera Azaleastrum ， Wilson E. H. and Rehder A. 1921. A Monograph of Mumeazale α，Cα ndidastrum and Therorhodion. Notes . Cambridge ，Massachusetts. Roy. Roy. Bo t. Gard. Edinb. 44: 1-23.

倉重祐二a，峰正樹bぺ小林伸雄bヘ半田高¥

高柳謙二b ，遊川知久 c .葉緑体遺伝子 matK による ツツジ属(ツツジ科)の分子系統学的検討 ことを目的とし，この体系によるツツジ属の全亜 約 1，000 種からなる大きな分類群，ツツジ属 属，全節の少なくとも各 1 種を含む 22 種，さらに (Rhododendron) は 属内外の系統関係についてさ ツツジ亜科のホツツジ属，イソツツジ属，ヨウラ まざまな見解がある.本研究では， Chamberlain et クツツジ属から各1種をサンプリングした.また外 l. al. (1996) の分類体系を分子系統学的に検討する 群としてスノキ亜科のイワヒゲ属を用いた.これ 154 154 植物研究雑誌第73 巻第3号 平成10 年6月

らについて，葉緑体 DNA 上の matK 遺伝子と，そ Tsutsusi) およびミツノ T ツツジ節 (section の上流，および下流域の trnK イントロンをあわせ Brachycalyx) は多/偽系統群である.サクラツツ た， 2， 113 bp の塩基配列マトリックスを比較した. ジ (R. tashiroi) をヤマツツジ節からミツバツツジ 解析の結果，以下の結果が示唆された. (1)イソツ 節に移すことで，両節は単系統になる. (6) ヒカゲ ツジ属 (Ledum) ， ヨウラクツツジ属 (M enziesia) は， ツツジ亜属 (subgenus Rhododendron) は単系統群 ツツジ属に含まれる. (2) したがって，ツツジ属は である. ( 7 )レンゲツツジ亜属 (subgenus 偽系統群である. (3) エゾツツジ属として扱われる Pentanthera) は多系統群である. (8) レンゲツツジ ことのあるエゾツツジ亜属 (Subgenus 属シロヤシオ節 (subgenus Pentanthera section Therorhodion) は，ツツジ連で最初に分岐する系統 Sciadorhodion) は多系統群である. (9) セイシカ亜 である. (4) 独立属 (Tsusiophyllum) とされること 属 (subgenus Azaleastrum) は多系統群である.

のあるハコネコメツツジは，ツツジ属ヤマツツジ (赤城自然園植物研究室， b 筑波大学農林学系，

節 (subgenus Tsutsusi section Tsutsusi) に含まれる. c 国立科学博物館筑波実験植物園， d 岩手県

(5) ツツジ亜属 (subgenus Tsutsusi) は単系統群であ 生物工学研究センター， e 館林市つつじ研 るものの，構成要素であるヤマツツジ節 (section 究事務所)