molecular phylogeny and evolution of the asianaconitum subgenusaconitum (ranunculaceae)

J. Plant Res. 108: 429-442, 1995 Journal of Plant Research �9 by The Botanical Society of Japan 1995

Molecular Phylogeny and Evolution of the Asian Aconitum Subgenus Aconitum (Ranunculaceae)

Yoko Ki ta TM, Kun ih i ko Ueda 1 and Yu ich i Kado ta ~

Department of Biology, Faculty of Science, Kanazawa University, Kanazawa, 920-11 Japan 2 Department of Botany, National Science Museum, Tsukuba, Ibaraki, 305 Japan

Classification of the genus Aconitum (Ranunculaceae) has long been considered quite difficult because its species show' high levels of morphological and ecological variability. The molecular phylogeny of Asian aconites, Aconitum subgenus Aconitum was, therefore, studied based on RFLP and sequences of the intergenic spacer between the trnL (UAA) 3'exon and trnF (GAA), and of the trnL intron, of the chloroplast DNA. Using Aconitum subgenus Lycoctonum as an outgroup, we obtained a statistically reliable molecular tree composed of six clades branched radiatively at the very base. There are three clades of Japanese aconites, a single clade of the species of u and Himalayas, and two clades of Siberian plants. All the tetraploid taxa of Japan we studied did not show any difference based on the molecular characters analyzed, though they have been classified into many taxa. Evolution and phytogeography of the Asian aconites as well as the phylogeny are discussed.

Key words: Aconitum Subgenus A c o n i t u m - Chloro- plast DNA - - Evolution E Molecular phylogeny E Plant geography - - Taxonomy

Taxonomy of the genus Aconitum L. subgenus Aconitum (Ranunculaceae) has been considered to be very difficult because aconites are highly variable in their morphologies. This has led to the elevation of many morphologically similar forms (races) to the species level by many authors. Few monographs or revisions of the genus have been published except for several regional works. Therefore it is difficult to compare the taxa between Japan and the area(s) concerned, even if the plant names are the same there.

Three hundred or more species have been reported in Aconitum, which are widely distributed throughout tem- perate and sub-frigid zones in the Northern Hemisphere (Kadota 1987). Three subgenera, Aconitum, Lycoctonum Tournefort and Gymnaconitum (Stapf.) Rapaics are commonly accepted in the genus, and the former two are distributed in Japan (Wang 1979, Kadota 1987, Tamura

* Present Address: Department of Biology, Faculty of Science, Chiba University, Chiba, 263 Japan

1990). Wang (1979) reported 162 species, belonging to 3 subgenera, 4 sections and 11 series, in China, which are about half of all the species in the world. In the Korean Peninsula, 20 species were reported (Lee 1989).

Nakai (1953) treated Asian members of subgenus Aconitum in usual sense as the independent genus Aconitum. In Aconitum, he recognized 2 subgenera, 7 sections, 4 series, 60 species and 7 varieties in Japan. Tamura and Namba (1959a, b, 1960) considered Aconitum sensu Nakai as the subgeneric rank, and organized Japanese taxa into 34 species and 22 varieties. Kadota (1987, 1991) revised Japanese aconites and classified them into 2 sections, 4 series, 1 subseries, 17 species, 11 subspecies and 4 varieties.

The opinions of authors concerning the taxonomy of this group have varied greatly depending on the features that were considered significant. Nakai (1953) attached much importance to the flowering sequence of the inflorescences as a diagnostic character and distinguished the plants with "indeterminate" floral axes from those with "determinate" ones determining subgeneric rank in his Aconitum. On the other hand, Tamura and Namba (1959a, b, 1960) emphasized variability in the conditions of pedicel pubescence in developing their taxonomy of the genus. In turn, Kadota (1987) regarded the morphology of petals (nectaries) and sepals, and the ploidy level as significant taxonomic diagnostic characters. Because of these differences in character evaluation among intrageneric taxa, the classification systems are dramati- cally different from each other. For example, Tamura and Namba (1960) revised Nakai's taxa and reduced five species belonging to 2 subgenera and 4 sections FA. nipponicum Nakai (Subgen. Napellus (DC.) Nakai Sect. Nipponica Nakai), A. micranthum Nakai and A. sakuraii Nakai (Subgen. Cammarum (DC.) Nakai Sect. Maxima (Steinb.) Nakai), A. kobusiense Nakai (Subgen. Cammarum Sect. Japonica Nakai), and A. Iongistylum Nakai (Subgen. Cammarum Sect. Napiformia Nakai)], into a single species, A. sakuraii. In his treatment, Kadota (1987), for example, placed the several species that had been classified into "the group of A. sanyoense" by Tamura and Namba, into two sections, Sect. Flagellaria (Steinb.) Nakai Ser. Latifolia Nakai and Sect. Euchylodea Reichb. Ser. Japonica (Nakai) Kadota Subsers. Japonica and

430 Y. Kita, K. Uede and Y. Kado ta

Table 1. Materials, and their systematic position, acronyms, ploidy level, sources and voucher specimens

Taxon Acronym Ploidy Collector *, Voucher and Locality

Japanese materials

Sect. Flagellaria (Steib.) Nakai

Ser. Yuparensia Kadota

A. yamazakii Tamura & Namba YAM 2X

A. yuparense Takeda

var. yuparense YUP 2X

var. apoiense (N~akai) Kadota APO 2X

Ser. Latifolia Nakai

A. sanyoense Nakai

'A. iide-montanum'

'A. nagisoense'

Sect. Euchylodea Reichb.

Ser. Euchylodea

A. kiyomiense Kadota TET 4X

A. ciliare DC. TET 4X

A. sachalinense Fr. Schm.

ssp. sachalinense TET 4X

ssp. yezoense (Nakai) Kadota TET 4X

A. maximum Pallas ex DC.

ssp. kurilense (Takeda) Kadota TET 4X

Ser. Japonica (Nakai) Kadota

Subser. Japonica

A. okuyamae Nakai TET 4X

A. japonicum Thunb. ex Murray

ssp. subcuneatum (Nakai) Kadota TET 4X

ssp. napiforme (Lev. et Van't.) Kadota TET 4X

Subser. Nipponica (Nakai) Kadota

A. zigzag Lev. et Van't.

ssp. ryohakuense Kadota TET 4X

A. senanense Nakai

ssp. senanense TET 4X

ssp. paludicola (Nakai) Kadota TET 4X

A. nipponicum Nakai

ssp. nipponicum TET 4X

ssp. micranthum (Nakai) Kadota TET 4X

A. kitadakense Nakai TET 4X

Siberian materials

Sect. Nepellus DC.

A. krasnoboroffii Kadota

A. baicalense Turcz. ex Rapaics

Sect. Catenata Steinb.

A. decipiens Worsch. et Anfalov

Sect. Euchylodea Reichb.

Ser. Euchylodea

A. villosum Reichb.

Ser. Glandulosa Worosch.

A. pascoi Worosch.

YK KANA180040-44: Mt. Nipesotsu, Hokkaido, Japan

KANA180022-29: Mt. Yupari, Hokkaido, Japan

YK KANA180075-84: Horoman Riv., Hokkaido, Japan

SAN 2X YK KANA180095-96: Asiu, Kyoto, Japan

II D 2X YK KANA191948-49: Mts. lide, u Japan

TET 4X ~" Kadota s.n. (TNS): Nagiso, Nagano, Japan

KRA 4X

BAI 4X

DEC '4X

4X

4X

V I L

PAS

YK KANA180110-12: Kiyomi-mura, Gifu, Japan

Kadota10632 (TNS): Aso-gun, Kumamoto, Japan

YK KANA180063-70: Nemuro, Hokkaido, Japan

YK KANA180045-49: Mt. Yupari, Hokkaido, Japan

YK KANA180055-60: Iwaobetsu Spa, Hokkaido, Japan

KD TNS9027426: Iwate, Japan

YK KANA180015-17: Hokkaido Univ. Bot. Gard., Japan

KD TNS9027438: Saijyo, Hiroshima, Japan

YK KANA180097: Mt. Hakusan, Ishikawa, Japan

YK KANA191943-47: Mt. Kitadake, Yamanashi, Japan

KD TNS9027427: Mt. Kashimayari-ga-take, Nagano, Japan

YK KANA180103, 06: Mt. Shirouma-dake, Nagano, Japan

YK KANA180091-94: Mt. Kisokoma-ga-take, Nagano, Japan

YK KANA191938-41: Mt. Kitadake, Yamanashi, Japan

KD TNS9027189: Mts. West Tannu-Ola, W. Sayan, Tuwa, Siberia

KD TNS9027196: Mts. Tumat Taiga, Sayan, Tuwa, Siberia

Wakabayashi et al. 9327102 (TN$) : Mts. West Tannu-Ola, W. Sayan, Tuwa, Siberia

KD TNS9237144: Mts. Tumat Taiga, W. Sayan, Tuwa, Siberia

Wakabayashi et al. 9327216 (TNS): Mts. West Sayan, Tuwa, Siberia

Molecular Phylogeny and Evolution of Asian Aconi tum 431

Table 1. Continued

Taxon Acronym Ploidy Collector*, Voucher and Locality

Kamchatka's materials A. delphinifolium DC

Yunnan materials

Sect. Aconitum

DEL

Ser. Rotundifolia Steib.

A. hookeri Stapf HOO -- Ser. Bullatifolia W.T. Wang

A. nagarum Stapf NAG -- Ser. Stylosa W.T. Wang

A. taronense (H.-Mazz.) Fletch. & Lauen. TAR -- A. rockii Fletch. et Lauener ROC -- A. Iongtouense T.L. Ming LON --

Ser. Brachypoda W.T. Wang A. pendulum Busch PEN

Series unknown

'A. sino-profiferum' SIN

Nepal materials aff. Ser. Bullatifolia

A. ferox Wall. ex Seringe

Outgroup Subgenus L ycoctonum

A. gigas Lev. et Van't. A. barbatum Pers.

A. sajanense Kumin.

A. septentrionale Koelle

FER 4X

OUT1 2X OUT2 2X

OUT2 2X

OUT2 2X

IM KANA191950-51: ca. 400 km NW of Magadan, NEmost. Siberia

Kadota21366 (TNS); Mt. Baima xueshan, Yunnan (alt. 4550 m)

Kadota21473 (TNS): Dali, Yunnan (alt. 2840 m)

Kadota21196 (TNS): Tianchi, Zhongdian, Yunnan (alt. 3880 m) Kadota21381 (TNS): Weixi, Yunnan (air. 3150 m) Kadota21178 (TNS): Mt. Tianbaoshan, Zhongdian, Yunnan (alt. 3470 m)

Kadota21224 (TNS): Wufengshan, Zhongdian, Yunnan (alt, 3300 m)

Kadota21215 (TNS): Bitahai, Zhongdian, Yunnan (alt. 3550 m)

Minaki et al. 9100909 (TI): Mt. Shiwapuri (alt. 2700 m), C. Nepal

IM KANA180011 : Hokkaido, Japan Wakabayashi et al. 9327097 (TNS): Mts. Tannu-Ola, W. Sayan, Tuwa, Siberia Wakabayashi et al. 9327200 (TNS): N. Buida River, W. Sayan,Tuwa, Siberia Wakabayashi et al. 9327177 (TNS): Mrs. Sangiten, W. Sayan, Tuwa, Siberia

* YK, Y. Kita; KD, Y. Kadota; IM, M. Imazu. ** Present original data.

Nipponica (Nakai) Kadota. At present, therefore, we do not yet have a commonly

accepted basic plan for the systematics of the subgenus Aconitum. To elucidate these divergent hypotheses concerning the phylogeny of subgenus Aconi tum in Japan, we developed a new molecular data base from restriction fragment length polymorphisms (RFLP) and sequences of non-coding region of chloroplast DNA (cpDNA). Further, to clarify the origin and evolut ion of the Japanese aconites, we investigated Asian cont inental aconites, a l though we were able to treat only a part of them among over 200 species.

Materials and Methods

In identi f icat ion of materials, we fo l lowed the taxonomy of Japanese aconites of Kadota (1987, 1991), except for the cases of 'Aconi tum i idemontanum' and 'A. nagisoen-

se' as mentioned in the discussion. 'Aconitum s ino- prol i ferum' from China wil l be described by Kadota.

Numbers of taxa are counted as follows: treat as 1 species and 1 subspecies in the cases such as A. nipponicum ssp. nipponicum and ssp. micranthum (Nakai) Kadota, but treat as 1 species if only a single taxon, for example, A. max imum Pallas ex DC. ssp. kuri lense (Ta- keda) Kadota occurs in an area concerned.

Japanese taxa as well as the plants from Yunnan (China), Nepal (Himalayas) and West Sayan (Siberia) were analyzed (Table1). To insure accurate identif ications, samples were obtained only during anthesis. Ploidy level for each taxon shown in Table 1 was cited from Kurita (1955, 1956, 1957) and Kadota (1987) except for a new count.

In Japan, Kadota (1987, 1991) recognized 5 diploid taxa, and 13 tetraploid species, 11 subspecies and 3 varieties. Of these, we were able to study all diploid taxa (except for

432 Y. Kita, K. Uede and Y. Kado ta

Table 2. Restriction fragment length polymorphisms of chloroplast DNA

No. Enzyme Region* Flagment Taxa**

1 Apal B2, 28 12.4---10.8+1.6 2 Apal B2, 28 6.9---5.6+1.3 3 Apal B2 10.8+6.9---17.7 4 BamHI B2 8.7+4.6---13.3 5 BamHI B28,19 2.4+0.4---2.8 6 BamHI B19 11.4+1.2---11.1 +6.0 7 BamHI B25 8.6+3.0---11.6 8 Bglll B19 10.0+1.5---11.5 9 Bglll B19 4.9+1.1 ---6.0

10 Bglll B25 9.8---6.2+3.6 11 Dral B25 3.4+4.6---9.0 12 Dral B25 4.6---3.4+(1.2) 13 Dral B25 14.4---3.8+10.6 14 Dral B25 11.7---6.4+5.3 15 Dral B25 2.9---2.6+(0.3) 16 Dral B2 5.0--2.3+1.7+1.0 17 Dral B28 13.7--11.2+2.5 18 Dral B28 12.1 ---9.0+3.1 19 Dral B28 1.9+0.9---2.8 20 Dral B19 5.6---3.9+1.7 21 Dral B19 3.5+2.0---5.5 22 EcoRI B25 2.5---2.0+0.5 23 EcoRI B25 3.7---3.1 + 0.5 24 EcoRI B19,:25 1.6+0.9---2.5 25 EcoRI B19 3.4+(0.5)---3.9 26 EcoRI B19 2.1 +1.6--2.0+1.7 27 EcoRI B28 1.9+1.1---3.0 28 EcoRI B28 1.6+(1.7)---3.3 29 EcoRI B2 3.4+3.0--5.1 +1.3 30 EcoRI B2 3.0---2.0+1.4 31 EcoRl B2 3.4---2.0+1.4 32 EcoRI B2 3.4---3.0+0.4 33 EcoRV B19 12.0---10.2+1.8 34 EcoRV B19 16.5---2.9+13.6 35 EcoRV B28, B2 18.3---15.5+2.8 36 EcoRV B28 3.2+0.9---3.1 +1.0 37 EcoRV B2 10.2---8.6+1.6 38 Hindlll B25 6.2+2.2---5.9+2.5 39 Mlul B19 26.0---18.7 +7.3 40 Mlul B19 26.0---11.0 +15.0 41 Mlul B2 15.0+5.0---20.0 42 Pstl B2, 28 18.0---14.0+6.0 (--2.0) 43 Pstl B25 15.8---9.6+6.2 44 Pvul B2 1.3+1.0---1.2+1.3 (-- 2.0) 45 Sacll B25 6.6+4.2---10.8 46 Smal B25 15.7+4.6---14.0+6.3 47 Smal B25 29.0---20.0 + 9.0 48 Xbal B2 19.3---7.2+? 49 Xbal B2 3.2---2.2+1.0 50 Xbal B19 5.0--3.2+1.8 51 Xbal B19 5.0+1.7---6.8 52 Xhol B25 8.9+3.1---12.0 51 Xhol B25 8.5+3.5---8.9+3.1 54 Haelll B2 2.8+0.8---3.6 55 Haelll B2 1.'8+0.5---2.3 56 Haelll B2 1.5+(0.3)---1.8

TET YUP, APO OUT1, OUT2 OUT1, OUT2 OUT1, OUT2 OUT1, OUT2 OUT1, OUT2 OUT1, OUT2 OUT1, OUT2 OUT2 TET liD FER NAG HOO OUT1, OUT2 HOO HOO PEN LON, TAR, SIN, ROC OUT1, OUT2 TET OUT1, OUT2 YAM, YUP, APO, liD YAM, YUP, APO,IID FER, HOO, NAG, TAR, ROC, PEN, LON, SIN FER, HOO, NAG, TAR, ROC, PEN, LON, SIN DEC YAM, APO, YUP OUT1, OUT2 liD LON, TAR, SIN, ROC YAM OUT1, OUT2 OUT1, OUT2 OUT1, OUT2 PAS OUT1, OUT2 FER, HOO, NAG, TAR, ROC, PEN, LON, SIN OUT1, OUT2 OUT1, OUT2 YUP APO YUP OUT1, OUT2 OUT1, OUT2 LON, TAR, SIN, ROC YUP OUT1, OUT2 YAM, YUP, APO, liD OUT1, OUT2 FER, PEN, NAG, HOO OUT1, OUT2 YAM, YUP, APO, liD OUT1, OUT2 SAN

* See text. ** See Tablet.

Molecular Phylogeny and Evolution of Asian Aconitum 433

A. metajaponicum s. str.)including 'A. iidemontanum', and the tetraploids of 11 species and 4 varieties including 'A. nagisoense'. The tetraploids investigated are most of the Japanese tetraploid species covering every series and subseries in Japan. Adding to these Japanese taxa, we studied the following Asian plants: 5 species from West Sayan, Siberia; 7 species from Yunnan, China; 1 species from Nepal, and 1 species from Okhotsk. Chinese plants were identified based on Wang (1979) and Ming (1985), and Russian species were based on Woroshilov (1945, 1966, 1982) and Kadota (1987, 1994).

RFLP analyses were performed for all the materials mentioned above except for A. delphinifolium DC. from Okhotsk. The intergenic spacer region between the trnL (UAA) 3'exon and trnF (GAA) of cpDNA was sequenced for all the materials except for two Japanese tetraploid taxa, A. senanense Nakai ssp. paludicola (Nakai) Kad0ta and A. nipponicum ssp. micranthum. Sequences of the trnL intron were also analyzed for all the materials except for A. delphinifolium and 6 Japanese tetraploids: A. maximum ssp. kurilense, A. japonicum Thunb. ex Murray ssp. subcuneatum (Nakai) Kadota and ssp. napiforme (Lev et Van't.) Kadota, A. zigzag Lev. et Van't. ssp. ryohakuense Kadota, A. senanense ssp. paludicola and A. nipponicum ssp. micranthum.

As an outgroup, we adopted A. gigas Lev. et Van't. because the subgenus Lycoctonum has consistently been considered as a separate taxon from the subgenus Aconitum. For RFLP analyses, three other Lycoctonum species from West Sayan, A. barbatum Pers., A. sajanense Kumin. and A. septentrionale Koelle were also used as the outgroup.

DNA preparation Total DNA was isolated from the living or dried leaf

tissues collected from the living materials (Table 1), using

modification of standard protocols of CTAB method for each species (Doyle and Dickerson 1987, Doyle and Doyle 1987).

Restriction site variation DNA samples of 34 taxa were digested with the follow-

ing 17 restriction endonucleases using the specifications of the suppliers: Apal, BamHI, Bglll, Dral, EcoRI, EcoRV, Hindltl, Mtul, Pstl, Pvull, Sacl, Sacll, Safll, Scal, Xbal , Xhol and Haelll. DNA fragments were separated on 0.7% agarose gels, denatured, and transferred to a nylon membrane (Amersham). Twelve cpDNA subclones from tobacco were used as hybridization probes; these were kindly provided by Prof. T. Sugiura of Nagoya University. They were grouped into 4 sets: two of them from the LSC region (here named B25 (B25, B7 and B20) and B19 (B19, B29, B22 and B1)), and the IR as B28 (B28, B15, B 10 and B8) and the SSC as B2. Labeling assay of probes was carried out by the ECL gene labeling/detection system (Amersham) following the manufacturer's specifications.

For inference of the phylogenetic relationships among the materials, three methods were employed: maximum parsimony (MP), neigbor joining (N J) and maximum likeli- hood (ML) methods. The basic data for these analyses are shown in Table 2. MP trees were obtained by PAUP 3.1.1 (Swofford 1993) using the branch and bound search strategy. Bootstrap confidence values for each internode in the phylogenetic tree were calculated using 100 times bootstrap replicates. Genetic distance matrix shown in Table 3 was calculated using the site data and following the formulas of Nei and Li (1979). The resultant d-value matrix was used for NEIGHBOR (PHYLIP 3.5c; Felsenstein 1993) to construct the NJ tree. For the ML tree, RESTML (PHYLIP 3.5c) was first used with the Global option and Jumble option (100 times) based on the data in Table 2. Then we compared the tree mentioned

Table 3. Genetic distance matrix

TET YUP APO YAM SAN liD FER LON HOO NAG PEN PAS DEC VIL OUT1 TET YUP .0085 APO .0067 .0030 YAM .0061 .0036 .001B SAN .0024 .0073 .0055 .0049 I I D .0054 .0054 .0036 .0030 .0042 FER .0054 .0104 .0085 .0080 .0042 .0073 LON .0060 .0109 .0091 .0085 .0048 .0078 .0030 HOO .0054 .0104 .0085 .0080 .0042 .0073 .0012 .0030 NAG .0066 .0116 .0097 .0091 .0054 .0085 .0024 .0041 .0024 PEN .0055 .0105 .0086 .0080 .0043 .0074 .0012 .0030 .0012 .0024 PAS .0024 .0072 .0054 .0048 .0012 .0042 .0042 .0048 .0042 .0054 .0042 DEC .0024 .0073 .0055 .0049 .0012 .0042 .0042 .0048 .0042 .0054 .0061 .0012 V I L .0054 .0067 .0048 .0042 .0006 .0036 .0036 .0042 .0036 .0048 .0054 .0006 .0006 OUT1 .0245 .0259 .0239 .0233 .0192 .0225 .0225 .0229 .0225 .0237 .0245 .0190 .0192 .0184 OUT2 .0210 .0265 .0245 .0239 .0198 .0231 .0231 .0235 .0231 .0243 .0251 .0196 .0198 .0190 .0006

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above, the NJ tree and 17, 450 trees obtained from the MP tree by complete dissolving its polychotomous branching into strict dichotomies to get the ML tree using RESTML with user-tree option.

Sequencing of two non-coding regions of cpDNA DNA samples were amplified by polymerase chain

reaction (PCR) using primers homologous to the trnL (UAA) intron, and the intergenic spacer between tmL (UAA) 3'exon and tmF (GAA). Sequenses of the two pairs of primers follows; the intron: 5'-CGAAARCGGTAGACG- CTACG-3' and 5'-GGGATAGAGGGACTTGAAC-3', the intergenic spacer: 5 ' -GGTTCAAGTCCCTCTATCCC- 3' and 5'-ATTTGAACTGGTGACACGAG-3' (Taberlet et al. 1991). Less than 50 ng of total DNA were introduced for a 35 cycle of ampf!ification (lmin 94C, lmin 60C, 2min 72C) in a total volume of 100/zl, containing 80mM KCI, 10mM Tris-HCI pH 8.9, 1.5raM MgCI2, 200~M of each dNTP, 1 M of each primer, and 1unit of Tth polymerase (TOYOBO). The PCR products were purified by the GeneClean II Kit (BIO 101). DNAs obtained were then prepared for A.L.F. II autosequencing machine (Phar- macia) using Autocycle Sequencing Kit (Pharmacia) or AutoSequencer Core Kit (TOYOBO). The both directions of each region were read.

Sequence data were aligned using ClustalV (Higgins 1991) and a part of the resulted sequence are shown in Tables 4 and 5. To construct a phylogenetic tree using both the RFLP data and sequence data, any algorithm except parsimony cannot be utilized. Therefore we used PAUP (Swofford 1993) to construct a tree based on the RFLP data mentioned above together with substitution and gap data as 2 state characters (Tables 2, 4 and 5).

Results

RFLP data Three hundred and sixty-eight fragments were detected

on average for each taxon investigated here, and 56 polymorphisms were revealed (Table2). These RFLP data indicated no differences among 1) all the Japanese tetraploids investigated, 2) 4 species from Yunnan (A. taronense (Hand.-Mazz.) Fletcher et Lauener, A. rockii Fletcher et Lauener, A. Iongtouense T.L. Ming and 'A. sino-proliferum'), and 3) 3 species from West Sayan (A. villosum Reichb., A. krasnoboroffii Kadota and A. baicalense Turcz. ex Rapaics). Because there was no RFLP variation within each group, each group was treated as a single operational taxonomical unit (OTU) during computer analyses. We have investigated Japanese tetraploids of 11 species and 4 varieties and discovered no differences in their RFLP patterns. These represent most of the Japanese tetraploid species. Because of this lack of variability, all Japanese tetraploid taxa were treated as a single complex.

The molecular phylogenetic analyses using MP and ML methods were based on the data shown in Table 2. Infer- red phylogenetic trees are shown in Figs. 1A and lB.

MP analysis produced only one most parsimonious tree using the branch and bound algorithm and the Collapse zero-length branches option, but the tree contains many polychotomies (Fig. 1A). The consistency index (CI) of the tree is 1.0 (i.e., there is no homoplasy). The MP tree shows: 1) Aconitum subgenus Aconitum is a monophyletic clade relative to the outgroup (subgenus Lycoctonum) at a 100% bootstrap probability; 2) A. ferox Wall. ex Seringe of Nepal and all the taxa from Yunnan were considered monophyletic with a bootstrap value of 94%, and within this group, A. ferox and A. pendulum Busch, A. nagarum Stapf and A. hooked Stapf (----A. pulchellum Wang (1979), non Hand.-Mazz.) comprise a weakly supported monophyletic group (69%); 3) Japanese diploids, except for A. sanyoense Nakai comprise a strongly supported monophyletic group (100%) and all taxa from Hokkaido are monophyletic (86%); and 4) A. pascoi Worosch., A. decipiens Worosch. et Anfalov, and A. villosum/A. krasnoboroffii/A, baicalense (all of which are taxa from West Sayan) represent a separate clade.

The ML tree is shown in Fig. lB. If we treated branches that are not supported statistically as zero length branches (shown as fine lines with an arrow), it shows the same topology as that of MP tree.

NJ analyses were performed using the data shown in Table 2. We produced a genetic distance matrix (Table 3) using the method of Nei and Li (1979). The range of genetic distances among all the taxa including the outgroup was 0.0-0.0265. Genetic distances among taxa of subgenus Aconitum ranged 0.0-0.0116. The NJ tree based on these data is depicted in Fig. lC. It is practi- cally impossible to show any statistical confidence value for NJ trees based on RFLP data. However, if we desig- nate short length inner branches (indicated by arrows) as zero length branches, the NJ tree also shows the same topology as in the MP tree.

Sequences of two non-coding regions of cpDNA The length of intergenic spacer between trnL and trnF

varies from 396-460 bp and that of the intron of trnL varies from 475-490 bp. Alignments are shown in Tables 4 and 5 and the calculated lengths of each are 485 bp and 498 bp, respectively.

Analysis of the intergenic spacer region between tmL and trnF of cpDNA shows following results: there are no differences in sequences among 1) Japanese tetraploid complex (A. senaense ssp. paludicola and A. nipponicum ssp. micranthum were not sequenced); 2) two diploid taxa, A. yuparense Takeda var. yuparense and var. apoiense (Nakai) Kadota; 3) Yunnan taxa, 'A. sino-proliferum', A. Iongtouense and A. taronense; 4) Sil~erian taxa, A. vil- Iosum, A. krasnoboroffii and A. pascoi and 5) Siberian taxa, A. baicalense and A. decipiens. We also sequenced the region in the outgroup taxa and discovered no difference between A. gigas and A. sajanense. Five base substitutions and three indels (two 4 bp and 6 bp) were observed between subgenera Aconitum and Lycoctenum. Within subgenus Aconitum, we found

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eleven substitutions, and eight indels (1 bp to 6 bp) and a 63 bp gap. The regions being compatible with the 63 bp gaps (55th to 117th in Table 4) were omitted from further analyses, though there are two substitutions in this region between the two subgenera. Of these, nine characters are useful for phylogenetic analyses (Table. 4).

Sequencing of the intron of tmL revealed no difference among 1) Japanese tetraploid taxa (6 taxa: A. maximum ssp. kurilense, A. japonicum ssp. subcuneatum and ssp. napiforme, A. zigzag ssp. ryohakuense, A. senanense ssp. paludicola and A. nipponicum ssp. micranthum were not sequenced); 2) two diploid taxa, A. yuparense vars. yuparense and apoiense; 3) Yunnan taxa, 'A. sino-profiferum', A. Iongtouense and A. taronense; and 4) Siberian taxa, A. baicalense and A. krasnoboroffii (Table5). We also determined the sequence in A. gigas. Five substitutions and two 3 bp indels were found between the two subgenera. Among the species of subgenus Aconitum, 10 substitutions and 6 indels (4 bp to 6 bp) were identified. Only 2 characters are synapomorphic (Table 5).

Gaps seen in non-coding regions have often been recorded in various taxa (e.g, Taberlet et al. 1991). Each of these gaps was considered to be originated by a single event. We treated gaps found in aconites in the present study, which are AT rich in their sequences, as a single characters for phylogenetic analyses, together with characters of base substitutions and RFLP data for MP analysys (Tables 4 and 5). The single MP tree resulted following the branch and bound search and the Collapse zero-length branches option (Fig. 2). The CI is 0.991 and there is a single homoplasy. Bootstrap confidence values for each internode in the phylogenetic tree were calculated using 142 times bootstrap replicates. Basi- cally the tree resembles the MP tree based on only RFLP data, although it provides additional phylogenetic information. The following four groups emerged as monophyletic: 1) Yunnan aconites (A. pendulum, A. nagarum and A. hookeri); 2) Yunnan aconites ('A. sino-profiferum', A. taronense and A. Iongtouense); 3) Siberian taxa (A. pascoi, A. villosum and A. krasnoboroffii), the former two are also monophyletic; and 4) Siberian A. baicalense and A. decipiens.

Discussion

Comparison among three kinds of data Three kinds of molecular data, RFLP, base substitutions

and gaps were used for constructing the phylogenetic trees. Zurawski and Clegg (1984) and Clegg and Zuraws- ki (1991) stated that evolutionary rates of introns are rather slower than that of intergenic spacers and 'are similar to that of protein coding regions. However, Gilley and Taberlet (1994) pointed out that both rates are similar. In the present study, we found 15 substitutions and 11 gaps in the spacer region, and 15 substitutions and 8 gaps in the intron. The total number of variations in subgenus Aconitum is 20 in the intergenic spacer and 16 for the intron. There are only 2 synapomorphies in the trnL

intron compared to 5 in the intergenic spacer. Therefore, the level of variation for the intron is somewhat lower. RFLP analysis appeared to be much more phylogenetically informative than analysis of the non-coding regions (Tables 2, 4 and 5, Fig. 2).

Several interesting features were discovered in the present phylogenetic trees. As seen in Fig. 2, no sequence data support the branches that are strongly supported by RFLP data (arrows 1 and 4). On the other hand, sequence data clarify two clades for Siberian aconites (arrows 2 and 3), but there are no informative RFLP along these branches. Apomorphic sequence data tend to concentrate on several particular branches whereas data from analyses of RFLP data provides information on the whole chloroplast DNA. Evolutionary rates appear to vary among clades, and the RFLP data rather correctly indicate the evolutionary history of cpDNA, but these non- coding regions of about 900 bp appear to provide biased view of molecular evolutionary processes. Despite the potential for discrepancy, there was no phylogenetic contradiction between RFLP and sequence data, and when both kinds of information are considered, greater detail concerning evolutionary processes is revealed.

The ML and NJ methods cannot simultaneously incor- porate all of the data from the RFLP, base substitutions and gaps. As noted in the results, the MP, ML and NJ trees based on RFLP data show fundamentally the same topology, and the MP tree based on only RFLP data and the MP tree using all the data did not show any inconsist- ency (Figs. IA, 1B, 1C and 2).

We will discuss the molecular tree for the subgenus Aconitum being based on the MP tree in Fig. 2.

Aconitum metajaponicum, and 'A. iidemontanum' and 'A. nagisoense'

A. metajaponicum Nakai was first described by Nakai (1914) based on a specimen collected from Mt. On-take, Nagano Prefecture, Japan. Kurita (1957) reported the chromosome number as 2n=16 based on the specimens from Mt. On-take. Kadota (1987) circumscribed this species based on the specimens from two localities: the type locality and Mrs. lide-san, Yamagata Prefecture. Kadota reported 2n=16 for lide's materials. Later Kadota (1990) added a new locality, Nagiso (Nagano Pref.) for this species.

Molecular data of cpDNA of the materials from Nagiso, however, are strictly the same as those in the Japanese tetraploid complex, and the chromosome number was revealed to be 2n=32. Therefore Nagiso plants are considered not to be identical to A. metajaponicum s. str. that is a diploid. Molecular data of lide plants are quite different from the tetraploid species including Nagiso plants. Their morphologies are also fairly different from one another (Kadota 1990). Hence it seems that the plants from three different localities represent a different taxon, that is, diploid plants of Mt. Ontake, tetraploid Nagiso plants, and diploid lide plants. We have called the aconite from Mts. lide as 'A. iidemontanum', and the

438 Y. Kita, K. Uede and Y. Kadota

tetraploid Nagiso plants as 'A. nagisoense" here. We could not yet obtain a diploid mateiral from Mt. Ontake, referrable to A. metajaponicum.

Molecular phylogeny and evolution in the Japanese diploids

We compared our molecular tree with traditional classi- fications of Kadota (1987), Wang (1975), Ming (1985) and Woroshilov (t945, 1966, 1982) (Fig. 2).

The tree sho~ved three clades in subgenus Aconitum from Japan: 1) diploids in Hokkaido (two species and one variety) and 'A. iidemontanum" from Honshu, which are supported by a 99% bootstrap value; 2) A. sanyoense; and 3) Japanese tetraploid complex (11 species and 4 varieties). Any combinations of these do not have closer relationships. The distribution areas of the three clades

are shown in Fig. 3. Kadota (1987) classified all the Japanese diploids into

Sect. Flagellaria, and placed those from Hokkaido into Ser. Yuparensia Kadota, while A. sanyoense and A. metajaponicum were classified into Ser. Latifolia. Kadota distinguished Ser. Latifolia from Set. Yuparensia by features that might be environmentally influenced such as stem length and quantity of the branchings. Hokkaido taxa are small-sized, whereas Honshu ones, including A. metajaponicum and 'A. iidemontanum', are large-sized. If 'A. iidemontanum' is closely related to A. metajaponicum (cf. Kadota 1987, 1990), the systematic position of these two species should be changed to Ser. Yuparen- sia from Ser. Latifolia (Fig. 2).

Genetic distances between 'A. iidemontanum' and Hokkaido diploids are great and range from 0.0030-

yamazakii

, 99 ~ 1 ~ yuparense

~ J a p sany~

anese Tetraploid Complex

I 69 ~ villosum

l ~ - ~ - ~ a " l F pascol

} ~- krasnoboroffii

3 . ,~ ~ baicalense

1 " decipiens

I Ser. Yuparensia

I Set. Latifolia

Ser. Japanica Ser. Euchylodea

I Ser. Glandulosa

rox I aft. Set. I Bullatifolia

rum { Ser. Bullatifolia

pendulum I Ser. Brachypoda

" I 1 [ ~ ~ hooked I Ser. Rotundifolia

~ n rock# touense eRse

' s~o-proliferum"

I Ser. Stylosa

affinity not yet published gigas

Sect. Flagellaria

Sect. Euchylodea

Sect. Napellus

Sect. Catenata

Sect. Aconitum

Fig. 2. The single most parsimonious tree obtained following a branch and bound search based on both RFLP and seqeunce data for the Asian aconites and compared with the traditional system. C1=0.991. Branch length represents the number of characters. Numbers along inner branches are percent indication of bootstrap probability based on 142 times replications. Rectangular marks on branches show RFLP data (Fig. IA), open circles represents sequence po~ymorphism (Tables 4 and 5), and black circles show homoplasy. Characters between subgenera Aconitum and Lycoctonum are omitted for convenience sake. Arrows with a number indicate characteristic clades explained in text.

Molecular Phylogeny and Evolution of Asian Aconitum 43P

�9 phini fol ium .... ::~i~iiiiiii!ii-~-'~ *'~ ~ ""

tetraploid complex

e

A. sanyoense 'A. i idemontanum' A. maximum ssp. maximum

Fig. 3. Distribution map of Japanese aconites with those of A. dolphinifolium and N, maximum ssp. maximum. A. maximum ssp. kurilonso belongs to Japanese tetraploid complex.

0.0054 (Table 3)~ Among Hokkaido diploids, the genetic distances range from 0.0018-0.0030. Hawaiian silversword and the alliances (3 genera and 33 species) were considered to be derived from a single species about half to one and half million years ago (Witter and Carr 1988). Baldwin et al. (1991) reported the genetic distances and the highest is 0.0046. The genetic distances between Hokkaido and lide aconites are certainly larger than values among those in the silversword alliances. Judg- ing from this comparison, every diploid species examined is a distinct species, and our data suggest that A. yuparense var. yuparense and var�9 apoiense should be treated as distinct sister species.

A. delphinifofium occurs from Alaska to the Aleutians, Kamchatka, and Siberia (Fig. 3). It includes several intraspecific taxa and is said to be composed of diploid and tetraploid populations. In this connection, it is interesting to evaluate the relationships between diploid A. delphinifolium, and diploids in Hokkaido and 'A. iidemontanum' (Figs. 2 and 3). The intergenic spacer between trnL and trnF of A. delphinifolium was sequenced (Table4), unfortunately, however no phylogenetically informative variation was found against Japanese diploids.

Taxonomy and evolution of Japanese tetraploid complex Morphological features such as pedicel pubescence,

degree of branchings in inflorescences, degree of dissec- tion of leaves, etc. in the Japanese tetraploid complex are highly variable and have led to the proposal of numerous separate taxa. Thus it was quite surprising to discover that there are no molecular polymorphisms among members of the Japanese tetraploids complex. Perhaps the most remarkable case involves A. ciliare DC., a single vine species among Japanese taxa. A. ciliare shows, however,

the same molecular profile as the other members of the Japanese tetraploid complex.

Because we have investigated only chloroplast DNA, the molecular tree represents only the maternal lineage. This observation suggests, however, that the Japanese tetraploid complex can be considered to have originated from a single taxon. Probably there has not been suffi- cient time for this single maternal progenitor to diversify and generate molecular variations. Unfortunately, our tree shows that the Japanese tetraploid complex does not exhibit any closer relationship with any diploid species examined, which means that the diploid maternal parent was not included in our sample here or it has become extinct.

In addition, there are few potential candidates for the paternal lineage of tetraploids, because there is a relative- ly small number of diploids (whether maternal or paternal) in Japan and the surrounding regions. There is also no evidence that the Japanese tetraploid complex originated following several hybridization events and subsequent introgression.

Consequently, the best hypothesis for the evolution of the Japanese tetraploid complex is as follows: a single original tetraploid species has become adapted to various environments, including growing in forest floors or edges, or sunny herbaceous lands, in montane or alpine areas. These adaptations to different habitats have led to variation in the external morphology and each population has been more or less isolated�9 This adaptive diversification has resulted in the production of many "forms" scatterd throughout Japan, but they have not yet become genetically diversified in their cpDNA.

The Japanese tetraploid complex occurs widely in Japan from Hokkaido to Kyushu, and most of them are endemic species. In Hokkaido, they grow in herbaceous


zones of montane regions, and, in Honshu, Shikoku and Kyushu, they occur in subalpine herbaceous zones and in montane forest edges. In general, the tetraploids are rather rare in southwestern Japan but often seen in northern Japan. A. japonicum ssp. napiforme, A. ciliare and A. jaluense occur not only in Japan but also in Korean Peninsula, and A. maximum (all subspecies) occurs in the area from Hokkaido to Alaska through the Chishima (the Kuriles), Kamchatka and the Aleutians (Fig. 3). A. maximum ssp. kurilense of Hokkaido belongs to Japanese tetraploid complex according to the present study. The center of diversity of this tetraploid complex is apparently Japan.

Chinese and Himalayan species In his revision of Chinese aconites, Wang (1975) recog-

nized Sect. Sinaconitum W. T. Wang (with a single species) and Sect. Aconitum (with 11 series) in subgenus Aconitum in China. Four series from Yunnan, a center of aconites' diversification in China, were included Jn our study. A. ferox of Nepal is closely related to A. nagarum Stapf. (Sect. Bullatifolia W. T. Wang) of Yunnan.

The 8 species from Yunnan and Nepal constitute a monophyletic clade (Fig. 2). A. hooked (Ser. Rotundifolia Steib.), A. pendulum (Ser. Brachypoda W.T. Wang) and A. nagarum are monophyletic, and further, together with A. ferox from Nepal, form a monophyletic group. Species of Ser. Stylosa W.T. Wang (A. rockii, A. Iongtouense and A. taronense) and 'A. sino-proliferum' constitute a monophyletic clade. Other than A. rockii, the latter three are molecularly identical.

Among these Chinese and Himalayan species examined, A. pendulum, A. nagarum, A. rockii, A. Ion- gtouense, A. taronense and A. ferox have "indeterminate" inflorescences. This characteristic of inflorescence does not circumscribe a natural taxon judging from our results, although this inflorescence character was once treated as a key subgeneric character by Nakai (1953).

"A. sino-proliferum' appears closely related to Korean A. proliferum Nakai, because these species are vines which produce roots when their apices touch the ground. 'A. sino-proliferum' is, however, not related to Siberian A. villosum and above mentioned Japanese A. ciliare, which are also vining species, but is related to Chinese and Himalayan species with erect stems. The latter two vine species also closely related to the species with erect stems belonging to the different clades, respectively (Fig. 2). Kadota (1987) supposed that vine taxa had been evolved independently in various aconites groups. The present data support this interpretation.

Although these Yunnan and Nepal species examined show a high degree of morphological diversity (Wang 1975), but they are monophyletic.

Siberian (West Sayan) species Five Siberian species, all of which are tetraploids,

showed some variation in RFLP but could be resolved with sequence data as ((A. pascoi, A. villosum), A. krasnoborof-

fii) and (A. baicalense, A. decipiens), although we found one homoplasy (Figs. 1A and 2). Kadota (1994) classified both A. krasnoboroffii and A. baicatense into Sect. Napellus which is inconsistent with the present tree (Fig. 2).

Plants of subgenus Aconitum produce a new tuber every year and tubers in the previous year decay. A. decipiens (Sect. Catenata Steinb. Ser. Grandituberosa Steinb.; Woroshilov (1945)) has peculiar tubers like a string of beads, because the old ones do not decay. It is, however, monophyletic with A. baicalense, which has a normal tuber morphology, in our tree. There are several species having such peculiar type of tuber such as A. decipiens in the Russian Federation and China.

A. pascei, occurring in West Sayan, Siberia, was classified in Sect. Euchylodea by Woroshilov (1945). Kadota (1987) includes all the Japanese tetraploids in Sect. Euchylodea along with Siberian A. kusnezoffii Reichb. (not analyzed here) and A. villosum. A. pascoi and A. villosum belong to the same clade atong with A. krasnoboroffii of Sect. Napellus in the present tree (Fig. 2), although the former two species have been classified in Sect. Eu- chylodea. They constitute the separate clade, from Japanese tetraploid species (Fig. 2). These conflicting data suggest that the taxonomy of Sect. Euchylodea should be re-investigated.

Origin of alpine species A. kitadakense, a member of Japanese tetraploid com-

plex, occurs only in an alpine region, around 3000 m in altitude, on Mt. Kitadake, central Japan, and is represent- ed by plants shorter than 15 cm tall: Some taxonomists considered this dwarf species to be a northern-origined, relict species of Quaternary glacial age (e.g., Hotta 1974), but it shows no molecular differences from the other tetraploid species. Therefore A. kitadakense may be derived from montane or subalpine populations of the Japanese tetraploid complex.

It is phytogeographically noteworthy that aconites from Yunnan and Himalayas included in our study form a monophyletic group. A. pendulum, A. nagarum and A. hooked occurring in Yunnan, Sichuan and Himalayas constitute a monophyletic group, along with Himalayan A. ferox. A. rockii, A. Iongtouense and A. taronense, all endemic to Yunnan, and 'A. sino-profiferum' are also monophyletic (the last three showed no molecular differences). Aconitum hookeri is an alpine species occurring around 4500 m in altitude in the Himalayas and Yunnan areas. Judging from this and our molecular tree, it is not a northern-origined relict in Quaternary glacial age but is derived from Yunnan and/or Himalayan non-alpine aconites and then adapted to alpine area. Different from A. kitadakense, it was probably diversified from an ancestral stock in fairly old time, because it has many molecular autoapomorphies.

We express our cordial thanks to Prof. T. Shimizu for his guidance throughout this study, and to Dr. C.H. Haufler for


his valuable suggestions. Anonymous reviewers for this article are acknowledged for their valuable criticism. A part of figures were kindly depicted by Mr. M. Umebaya- shi. Our hearty thanks are extended to Drs. M. Imazu and M. Suzuki for providing us important materials and to Prof. M. Tamura for his suggestions. This study was finan- cially supported, in part, by a Grant- in-Aid from the Ministry of Education, Science and Culture, Japan (05304009 to Ueda; leader, H. Tobe). Fieldwork in the Himalayan and Siberian mountains were also f inancial ly supported by the Grants from Monbusho International Scientif ic Programs (Field Research) (04041087 to Kadota; leader, M. Wakabayashi).

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(Received April 24, 1995: Accepted July 26, 1995)

molecular phylogeny and evolution of the asianaconitum subgenusaconitum (ranunculaceae)

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