NUCLEAR DNA, CHLOROPLAST DNA, AND PLOIDY

Transcription

1 American Journal of Botany 92(9): NUCLEAR DNA, CHLOROPLAST DNA, AND PLOIDY ANALYSIS CLARIFIED BIOLOGICAL COMPLEXITY OF THE VANDENBOSCHIA RADICANS COMPLEX (HYMENOPHYLLACEAE) IN JAPAN AND ADJACENT AREAS 1 ATSUSHI EBIHARA, 2,9 HIROSHI ISHIKAWA, 2,3 SADAMU MATSUMOTO, 4 SU-JUAN LIN, 5 KUNIO IWATSUKI, 6 MASAYUKI TAKAMIYA, 7 YASUYUKI WATANO, 8 AND MOTOMI ITO 2 2 Department of System Sciences, Graduate School of Arts and Sciences, the University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan; 3 Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba , Japan; 4 Tsukuba Botanical Garden, National Science Museum, Amakubo, Tsukuba , Japan; 5 Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue-shi, Shimane , Japan; 6 The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba , Japan; 7 Graduate School of Science and Technology, Kurokami, Kumamoto , Japan; and 8 Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba , Japan Species complexes consisting of ill-defined species are widely known among ferns, and their involvement with reticulate evolution is expected. Nevertheless approaches to reticulation history with DNA markers are not yet commonly adopted. We have successfully elucidated the biological status of the Vandenboschia radicans complex in East Asian islands by combining analyses of ploidy level, a cpdna marker (rbcl), and a nuclear DNA marker (GapCp). The results based on 266 individuals collected from 174 localities throughout Japan and Taiwan suggest that complicated hybridizations have occurred involving at least three parental diploid species from within the V. radicans complex and Vandenboschia liukiuensis, which was formerly considered to be distinct from this complex. Triploids are the most common cytotype, but they show no evidence of apogamous reproduction, while all nonhybrid diploids are rare and have very limited distribution. Possible accounts of this phenomenon will be briefly discussed including the possibility of relict distribution and occasional apogamous reproduction. Key words: GapCp; hybrid; Hymenophyllaceae; Japan; rbcl; reticulate evolution; single-strand conformation polymorphism (SSCP); Vandenboschia. Species complexes that have a wide variety of morphological characters with ambiguous boundaries between species often result from reticulate evolution accompanied by hybridizations and polyploidizations (Stebbins, 1974; Grant, 1981). In ferns, polyploids occur at a significantly high frequency (Grant, 1981; Soltis and Soltis, 1999). As a consequence, there are a large number of recognized species complexes (Lovis, 1977). Historical approaches to clarify reticulate evolution, as exemplified by the study of Appalachian Asplenium triangle (Wagner, 1954), adopted chromosome counting, observation of chromosome pairing during meiosis, and morphological studies (Lovis, 1977). Recent studies utilizing modern markers (isozymes, RAPD, nrdna sequences, etc.) have confirmed the 1 Manuscript received 13 January 2005; revision accepted 13 May The authors thank the following persons who helped collect materials: K. Akai, S. Aoki, S. Fujimoto, N. Hamada, M. Hasebe, S. Hennequin, Y. Horii, M. Hyodo, R. Ikehata, Y. Inoue, S. Ishizawa, T. Iwata, T. Jinbo, F. Kasetani, H. Kariya, T. Kikuchi, T. Kuramata, T. Maruno, K. Mizote, A. Narita, S. Nishiyama, K. Ohora, M. Ohta, T. Oka, T. Okamoto, C.-I Peng, H. Sada, Y. Saito, M. Sasagawa, S. Sato, K. Seto, Y. Sonehara, T. Sudo, Y. Sugimura, T. Suzuki, T. Takara, S. Tamura, M. Tanaka, Y. Tosaka, H. Tsuji, K. Tsujii, S. Tsutsui, S. Watanabe, K. Yamaoka, and T. Yasui. The authors also thank Dr. Fred Rumsey (The Natural History Museum, London, UK) for checking and correcting the English manuscript and two anonymous reviewers for providing useful comments. A grant to A. E. from the Japan Society for the Promotion of Science partly supported this study. Field work in Yaku Island was partly funded by Research Institute for Humanity Nature Project (P2-2). 9 Author for correspondence ( ebiharak@dolphin.c.u-tokyo.ac.jp) 1535 classical diagram of reticulation, and some studies have implied unexpected evolutionary histories such as the existence of extinct progenitors (Haufler et al., 1995; Van den Heede et al., 2003; Sara et al., 2004). Unlike other ferns, filmy ferns (Hymenophyllaceae) have rarely been the subject of a study of reticulate evolution. Even the family s very few described probable hybrids (Jermy and Walker, 1985; Manton et al., 1986) have been recognized only recently through cytological studies. Nevertheless this does not mean that species delimitation of Hymenophyllaceae is an easy task. There are many cosmopolitan species already recognized that have a wide range of morphological variation. Recent molecular phylogenetic studies utilizing chloroplast DNA (Dubuisson, 1997; Pryer et al., 2001; Ebihara et al., 2004) have gradually been clarifying evolutionary lineages within this family that used to be controversial, and thus favorable conditions for studies at the level of microevolution are being created. Vandenboschia radicans (Sw.) Copel. and its close relatives are widely distributed from the tropics to the cool-temperate zone. The V. radicans complex as recognized here encompasses a range of taxa (some of which have yet to be formally combined within the segregate genus Vandenboschia) including the European V. speciosa (Willd.) G. Kunkel, African Trichomanes giganteum Willd., North American T. boschianum Sturm, and southeast Asian V. birmanica (Bedd.) Copel. Formerly most of these taxa were not distinguished from V. rad-

2 1536 AMERICAN JOURNAL OF BOTANY [Vol. 92 TABLE 1. A comparison of taxonomic treatments of Japanese Vandenboschia radicans complex. Form (Japanese name) Large form (O-haihoragoke) Medium form (Haihoragoke) Small form (Hime-haihoragoke) Yaeyama form (Koke-haihoragoke) (V. subclathrata) Ito (1949) Genus Vandenboschia V. radicans (Sw.) Copel. var. naseana (H. Christ) H. Ito V. radicans var. orientalis (C. Chr.) H. Ito V. radicans var. nipponica (Nakai) H. Ito Nakaike (1975) Genus Lacosteopsis L. orientalis (C. Chr.) Nakaike var. naseana (H. Christ) Nakaike Iwatsuki (1995) Genus Crepidomanes C. radicans (Sw.) K. Iwats. var. naseanum (H. Christ) K. Iwats. L. orientalis var. orientalis C. birmanicum (Bedd.) K. Iwats. L. orientalis var. abbreviata (H. Christ) Nakaike/var. angustata (H. Christ) Nakaike L. subclathrata (K. Iwats.) Nakaike C. amabile (Nakai) K. Iwats. C. subclathratum (K. Iwats.) K. Iwats. icans, which was originally described based on Jamaican material. In the Japanese Archipelago, plants that belong to this complex are common in wet, shaded places (e.g., on rocks along streamlets; cliffs around waterfalls), and distributed nearly throughout the country including Ryukyu and Bonin islands (Kurata and Nakaike [1987] mapped their distribution.). Morphologically, this wide-ranging complex varies greatly (e.g., the size of a mature frond ranging from 2 cm to more than 30 cm in length), which has caused taxonomic confusion. Nakaike (1975) recognized four varieties (var. orientalis, var. angustata (H. Christ) Nakaike, var. abbreviata (H. Christ) Nakaike, and var. naseana (H. Christ) Nakaike) under a species Lacosteopsis orientalis (C. Chr.) Nakaike and a distinct species L. subclathrata (K. Iwats.) Nakaike, while Iwatsuki (1995) recognized four taxa Crepidomanes amabile (Nakai) K. Iwats., C. birmanicum (Bedd.) K. Iwats., C. radicans var. naseanum (H. Christ) K. Iwats., and C. subclathratum (K. Iwats.) K. Iwats. (Table 1; we hereafter call them by the forms in the table). In both cases, many collections have features intermediate between the defined taxa. Aside from the taxonomy, there is a distinct cline in morphology: small-sized plants are more often found in areas of heavy snowfall (northern Honshu and the Sea of Japan side of the island), and largesized plants are more often found in the areas under the influence of warm currents (Pacific side of Honshu southward). Cytological observation of this complex by Mitui (1966, 1967, 1968, 1973, 1975, 1976a, b, 1980, 1986) and Kurita (1976) provided interesting results; half of their samples were triploids with irregular meioses, while sexual diploids and tetraploids were less common (Table 2). Although their sampling was limited and not sufficient to draw firm conclusions, it is still quite exceptional that sterile triploids are dominant in a certain species or species-complex of pteridophytes. In Japan, many pteridophyte species consist of triploids, but most of them reproduce by apogamy (Takamiya, 1996). There are also many sterile hybrids, but they are usually much less common than their parental fertile species. Mitui (1967) hypothesized that a meiosis-irregular triploid found in Yaku Island could be a hybrid between var. naseana and var. orientalis. From the cytological viewpoint, it is quite likely that hybridization occurred among several biological species included in this complex, but no other researchers have recognized any hybrid taxa ever since. In this study, we intend to clarify the biological status of entities within this complex with ample sampling covering nearly their entire distribution range in Japan. Analysis with a biparentally inherited marker is necessary to trace the true history of a group that contains any hybrid or organism of hybrid origin. A maternally inherited marker, which is common at higher-level phylogeny, is also useful for determining sexual directionality in cases of hybridization. We therefore combine the following three approaches, ploidy-level determination, cpdna analysis, and nuclear DNA analysis. MATERIALS AND METHODS Materials In total, 266 individuals of the V. radicans complex were collected from 34 of the 47 prefectures of Japan, Cheju Island (South Korea), and Taiwan (Appendix 1). Materials from different patches within the same locality are shown by the same locality code but with different branch codes (e.g., AC-2a and AC-2b). It is very difficult to define individuals in this complex due to the plant s long-creeping habit, therefore we usually chose clearly discontinuous patches when collecting more than one sample from a TABLE 2. Previous cytological observations of Japanese and Taiwanese Vandenboschia radicans complex: Sex. normal chromosome pairings were observed in meiosis, Irr. irregular chromosome pairings are observed in meiosis. Identifications are standardized in varietal ranks of V. radicans. Ploidy Sex./Irr. Identification Locality Reference Voucher 2x Sex. var. amabilis Oirase, Aomori Pref. Mitui, 1986 Not cited 2x Sex. var. naseana Yaku Isl., Kagoshima Pref. Mitui, 1967 TNS x Sex. var. naseana Mt. Yonaha, Okinawa Pref. Mitui, 1975 TNS x Sex. radicans var. orientalis? Taiwan Mitui, 1968 Not cited 3x Irr. var. amabilis Sado Isl, Niigata Pref. Mitui, 1968 Not cited 3x Irr. var. amabilis Hanyuda, Niigata Pref. Mitui, 1980 Not cited 3x Irr. var. orientalis Yaku Isl., Kagoshima Pref. Mitui, 1967 TNS x Irr. var. orientalis Mt. Chibusa, Hahajima Isl., Bonin Isls. Mitui, 1973 TNS x Irr. radicans var. orientalis Mt. Kiyosumi, Chiba Pref. Kurita, 1976 Not cited 4x Irr. var. amabilis Jikohji, Niigata Pref. Mitui, 1976a TNS x Sex. radicans var. orientalis Izuhara, Tokushima Pref. Mitui, 1966 TNS

3 September 2005] EBIHARA ET AL. VANDENBOSCHIA RADICANS COMPLEX IN JAPAN 1537 locality. Vandenboschia auriculata (Blume) Copel. (one sample) and V. liukiuensis (Y. Yabe) Copel. (three samples that sympatrically grow with plants of the complex), attributed to the genus Vandenboschia sensu Ebihara (A. Ebihara, J.-Y. Dubuisson, K. Iwastuki, S. Hennequin, and M. Ito, unpublished manuscript), were also included in this study. All the voucher specimens are deposited in TNS, unless otherwise specified. Ploidy analyses According to Bennett and Leitch (2003), the only known genome size within the Hymenophyllaceae is that of V. speciosa (which is attributed to the V. radicans complex) in the study of pteridophyte C-values by Obermayer et al. (2002). They failed to measure the genome size of V. speciosa by flow cytometer and resorted to Feulgen microdensitometry instead. In contrast, our preliminary analysis of Hymenophyllaceae by flow cytometry was successful, and hence we carried out the ploidy analyses by this method. For our analyses, fresh Vandenboschia leaf tissue (c. 400 mm 2 ) was chopped by a razor in 1.0 ml of propidium iodide (PI) buffer (1.0% Triton X-100, 140 mm 2-mercaptoethanol, 50 mm Na 2 SO 3, 50 mm Tris-HCl [ph 7.5], 25 g/ml PI, 40 mg/ml polyvinyl-pyrrolidone [PVP-40], and 0.1 mg/ ml ribonuclease) together with leaf tissue of Nicotiana tabacum L. cv. Xanthi (c. 25 mm 2 ) for internal standard. The crushed tissue was placed on ice for 5 min and filtered through 30- m nylon mesh (Partec, Münster, Nordrhein- Westfalen, Germany). The suspension was incubated at 37 C for 15 min, then 4 C for 30 min. Genome sizes were analyzed by Epics XL System (Beckman Coulter, Fullerton, California, USA). We also observed chromosome number during meioses in sporangia of fixed material of an example of the V. radicans complex [Ebihara , which was identified as V. subclathrata (K. Iwats.) Copel.] by the usual squashing method (Takamiya, 1993). This is a control for ploidy of V. radicans complex. Molecular analyses For maternal lineage marker, we selected the chloroplast rbcl gene, because a large number of sequences for Hymenophyllaceae have already been deposited in the GenBank database ( Total DNA was extracted from ca. 5 mg of leaf tissue with a DNeasy Plant Mini kit (Qiagen, Hilden, Germany). The PCR amplification of the rbcl gene was performed with primers rbcl-tkt-f1 (5 - ACCCAWGTCACCACAAACRGAG-3 ) and rbcl-tkt-r3n-2 (5 -CA- AGCGGCAGCCRAYTCAG-3 ), 35 cycles: 94 C (45 s), 52 C (45 s), 72 C (75 s) using diluted total DNA in 10 ng/ L as reaction templates. The PCR products were incubated at 37 C (50 min) and 80 C (15 min) with 5% ExoSAP-IT (usb, Cleveland, Ohio, USA) to remove single-strand DNA. Four primers (rbcl-af, -1300R, -HIF1 and -HIR1; see Ebihara et al., 2003) were used for cycle sequencing reactions with BigDye Terminator version 3.1 (Applied Biosystems, Foster City, California, USA). Each sample was sequenced using an ABI 310 genetic analyzer (Applied Biosystems). For the biparentally inherited marker, only a few studies utilizing nuclear DNA for pteridophytes have been published. The nuclear DNA region most frequently used for the analyses of other organisms; nuclear ITS is often said to be problematic (Alvarez and Wendel, 2003), especially for pteridophytes (H. Ishikawa, personal observation), although Hoot and Taylor (2001) and Van den Heede et al. (2003) reported successfully obtaining results in studies of Isöetes and Asplenium, respectively. In recent years, several studies effectively applied single- or low-copy nuclear sequences to analyses of reticulate evolution (Ferguson and Sang, 2001; Raymond et al., 2002). For ferns, Ishikawa et al. (2002) designed some primer sets for the nuclear single-copy PgiC gene, but they did not work on Hymenophyllaceae (H. Ishikawa, unpublished data). We therefore employed a region that includes another nuclear singlecopy gene, the subunit C gene of nuclear NAD -dependent glyceraldehydes- 3-phosphate dehydrogenase (GapC), for this study. For this GapC region, we designed four forward primers (GapC-6FA: 5 -GCTCCAATGTTTGTAATG GG-3, 6FB: 5 -GCTCCAATGTTTGTCATGGG-3, 6FC: 5 -GCTCCAATG TTTGTGATGGG-3 and 6FD: 5 -GCTCCAATGTTTGTTATGGG-3 ) and two reverse primers (GapC-9RA: 5 -CCCCATTCATTGTCGTACCA-3 and 9RB: 5 -CCCCATTCATTGTCATACCA-3 ) in positions of low-degeneracy amino acids based on the cdna sequence of Marsilea quadrifolia L. (GenBank accession AJ003783) compared with sequences from other organisms. Several combinations of these primers worked for the Hymenophyllaceae, but they also amplified fungal DNA. We therefore designed a primer set that specifically worked on Vandenboschia: GapC-7FA (5 -GAGGGTTT GATGACCACAGT-3 ) and GapC-CR-2 (5 -CTTTTCCACTCGACAAGTC AG-3 ). Fragments were amplified by PCR with these primers (35 cycles: 94 C [45 s], 57 C [45 s], 72 C [60 s]). Then, we performed single-strand conformation polymorphism (SSCP) analyses for detecting polymorphism of the PCR products generally following the method by Watano et al. (2004). The PCR products were denatured in formamide with loading dye at 96 C for 3 min and electrophoresed in 0.5 mutation detection enhancement (MDE) gel (Cambrex, East Rutherford, New Jersey, USA) containing 2% glycerol at 20 C for 14 h at 350 V, followed by silver staining. When polymorphism was detected in a sequence (i.e., more than three bands per sample were observed), each band was cut out and mashed between two slide glasses. Then the mashed gel was incubated at 65 C in250 L of the elution buffer of E.Z.N.A. Poly Gel DNA Extraction kit (Omega Bio-tek, Doraville, Georgia, USA) for 4 h. Each DNA was purified generally following the protocol of the kit, and finally it was eluted to 50 L of sterile water. Obtained DNA was directly used as templates for nested PCR with two primers, GapC-7FA and GapC- BR-1 (5 -GAATGCCATGCCAGTCAGT-3 ) with the same program as the former reaction. All subsequent procedures until sequencing followed that of the rbcl gene. Phylogenetic analyses Bayesian inference was used for phylogenetic analyses of both rbcl and GapC sequences. After alignments by Clustal X 1.83 (Thompson et al., 1997) and manual correction, the analyses were performed by MrBayes 3.0 (Ronquist and Huelsenbeck, 2003) based on generations (initial 4000 trees were discarded as of burn-in period) with four Markov chain Monte Carlo (MCMC) chains starting from random trees that were sampled every 100 generations, treating indels as missing characters. In the analysis of the rbcl gene, we added six Vandenboschia taxa [V. davallioides (Gaudich.) Copel., V. johnstonensis (F.M. Bailey) Copel., V. maxima (Blume) Copel., V. radicans, V. speciosa, and Trichomanes rupestre (Raddi) Bosch]; the last taxon has not yet been combined with Vandenboschia) used in our recent phylogenetic study of Trichomanes s.l. (Ebihara et al., in press) and also added six species of non-vandenboschia filmy ferns (Hymenophyllum polyanthos, belonging to well-defined sister genus of Trichomanes s.l. (Pryer et al., 2001), is assumed as an outgroup.). In the analysis of GapC, V. auriculata, which is clearly distinguishable from the V. radicans complex but is evidently included in Vandenboschia (Ebihara et al., in press), is applied as an outgroup taxon. RESULTS Ploidy analyses The only material available for chromosome counting showed n 36 bivalents in meiosis (Fig. 1). Because chromosome base numbers of Trichomanes s.l. are x 32, 33, 34 and 36 (Braithwaite, 1975), the material is considered a diploid. This is also the first cytological record for V. subclathrata. The genome size of this material was 3.13 times that of internal standard (Nicotiana tabacum). Considering the genome size of Nicotiana tabacum is ca pg (Narayan, 1987), genome size of this diploid material is estimated as ca pg. For other diploids, that of the medium form is ca pg on an average (N 16), that of the small form is ca pg (N 8), and that of the large form is ca pg (N 4) the last one is slightly but evidently larger than the other forms. We detected triploids and tetraploids along with diploids, and their genome sizes are approximately multiples of that estimated in the diploid (Fig. 2). Ploidy analyses by flow cytometer confirmed the dominance of triploids (Appendix 1); more than 50% of individuals of this complex used in this study are triploids. Furthermore the triploid individuals are found in nearly every portion of its

4 1538 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 2. Examples of peaks in fluorescence for three cytotypes (2x, 3x, and 4x) ofvandenboschia radicans complex from analyses with flow cytometer. The horizontal scale indicates fluorescent intensity (calibrated by the internal standard, Nicotiana tabacum) and the vertical scale indicates number of cells. Fig. 1. Chromosomes in meiosis of the Yaeyama form Vandenboschia subclathrata (sample code: OK-1a). (a) Light micrograph. (b) Diagram of chromosomes in (a). Scale bar 10 m. distribution range, while diploids and tetraploids, which probably include sexual reproductive types, are not common. Indeed, the restricted distribution of diploids is particularly striking. Chloroplast rbcl sequence In the V. radicans complex, five types of sequences (named types I, I, II, III, and IV) were found in 1206 bp of the rbcl gene (Fig. 3, Appendix 1; GenBank accessions are in Appendix 2). There is only one base pair substitution between the type I and the type I and seven substitutions between the type I and type II. The genetic distance between the type I and the type III is much greater: 20 bp of substitutions. The type IV sequence is also quite different from the other types. Apparently, these sequence types do not correspond exactly to the current morphologically based identification; the plants of the medium form in current definition contain various rbcl sequences including types I, I, II, and III. In the genus Vandenboschia, type I, I, and II sequences form a strongly supported clade with European V. speciosa and Hawaiian V. davallioides, while type III and IV sequences form another strongly supported clade together with V. liukiuensis, South American V. radicans, Pacific V. maxima, and Australian V. johnstonensis (this latter clade is placed sister to the former one). Nuclear GapC sequence The sequenced region is about 550 bp long, including intron 7, exon 8, and intron 8 (Fig. 4; introns and exons are numbered after GenBank accession AJ for a sequence from Pinus sylvestris L. by Meyer-Gauen et al., 1994, 1998). Obtained amino acid sequences clearly showed features of GapCp, which is a counterpart of GapC and codes GAPDH enzyme imported from the cytosol into the chloroplast (Meyer-Gauen et al., 1998; Petersen et al., 2003). Therefore, hereafter we should call our sequences GapCp. In most of the samples, sequence polymorphism was detected by SSCP analyses, and the GapCp sequence had greater variation than rbcl. In total, 45 different sequences were identified in samples of the complex. We recognized four groups of sequences (groups A, B, C, and D in Fig. 5 and Appendix 1), each of which is supported by high posterior probability of Bayesian inference in the phylogenetic tree obtained from the sequences (Fig. 5). The genotype of each sample is sum-

5 September 2005] EBIHARA ET AL. VANDENBOSCHIA RADICANS COMPLEX IN JAPAN 1539 Fig. 3. A consensus phylogram resulting from Bayesian analysis of chloroplast rbcl sequences of the genus Vandenboschia. Numbers at the nodes show posterior probability for supporting each clade. The scale bar indicates a branch length corresponding to 0.04 substitutions per site. marized in Appendix 1. We must note that our current method cannot identify gene dosage in polyploids, and therefore unknown genomes are shown by asterisks (e.g., A1*B1 means genotype A1A1B1 or A1B1B1; A1A*A2 means genotype A1A1A2 or A1A2A2). DISCUSSION To elucidate biological entities within species complexes that involve a polyploid series, the most essential approach is the identification and analysis of the diploid progenitors (Grant, 1981). When we focus on diploids, nuclear (GapCp) genotype A*A* corresponds to chloroplast (rbcl) types I and I, named genome. Similarly, nuclear genotype B*B* corresponds to chloroplast type II (genome ), and nuclear genotype C*C* corresponds to chloroplast type III (genome ). There are neither diploids having the group D sequence of GapCp nor those having the type IV sequence of rbcl, but D/IV sequences match those of V. liukiuensis (named genome ), which was reported to be diploid (Mitui, 1976b). It can be assumed that this complex (excluding V. liukiuensis) in Japan and the adjacent areas contain at least three biological diploid species, which correspond to the,, and genomes, respectively. The bases of this idea are (1) nonhybrid diploids are clearly distinguishable morphologically from one another (discussed later; characters are summarized in Table 3); (2) as already noted, the difference between the genome and the genome in the rbcl sequence is 20 bp, which is too large Fig. 4. Approximate primer positions on the GapCp gene of the Vandenboschia radicans complex.

6 1540 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 5. A consensus phylogram resulting from Bayesian analysis of nuclear GapCp sequences identified from the Vandenboschia radicans complex of Japan and adjacent areas and V. liukiuensis. Numbers at the nodes are the posterior probabilities for the support of each clade. The scale bar indicates a branch length corresponding to 0.1 substitutions per site. to be considered as intraspecific variation when compared with other examples of ferns (Yatabe et al., 2001); and (3) in our preliminary observation (A. Ebihara, unpublished data), the hybrid diploid of genomic formula is sterile (both irregular meiosis and spores observed), and therefore reproductive isolation has been established, even between the genome and the genome. Distribution and characteristics of each diploid species As stated, each diploid species can be recognized by its mor-

7 September 2005] EBIHARA ET AL. VANDENBOSCHIA RADICANS COMPLEX IN JAPAN 1541 TABLE 3. Morphological characters of each diploid form of Japanese Vandenboschia radicans complex. Character The large form The medium form The small form Yaeyama form (V. subclathrata) V. liukiuensis Genomic formula Frond length cm 3 13 cm 4 8 ( 11) cm 2 5 cm cm Frond width 4 10 cm cm cm cm 2 3 cm Lamina Flat Wing crisped Three-dimensional Almost flat Flat Leaf color Dark green Green Light green Pale green Light green Diameter of rhizome mm mm mm ca. 0.3 mm ca. 1.0 mm Sori Truncate a Truncate a Truncate a Truncate Lips distinctly flared a Lips are sometimes minutely flared. phological characters. The genome corresponds to both the medium form and the Yaeyama form (V. subclathrata). However, we currently cannot distinguish the two forms by molecular markers in spite of their distinct morphological differences. The genome diploids have so far been found only on the Pacific Coast of Kii Peninsula (Honshu), Shikoku, Yaku Island, Iriomote Island, and Bonin Islands, where the climatic conditions are mild throughout the year as a result of the warm ocean currents (Fig. 6). The diploids of Iriomote Island exclusively show the features of V. subclathrata: clathrate pagina, pale green fronds, and flat wings. On the other hand, those found in the other localities have nonclathrate pagina, darkercolored fronds, and waved wings. Considering that no probable specimens of these diploid forms are found in Okinawa Island, these two forms have speciated possibly by recent vicariance in Okinawa Island. The genome corresponds to the small form, and the genome has been found on northernmost part of the main island, Honshu (Aomori Prefecture, AM-1, -3a, -3c, -3d and -3e; Akita Prefecture, AK-3a and -3d) and on the Sea of Japan side of central Honshu (Niigata Prefecture, NG-8a and -10; Toyama Prefecture, TY-2a). Though this form is probably less common and the distribution is narrower than formerly considered, its localities could sporadically occur from northern Honshu Island toward the Sea of Japan side of central Honshu. This species has probably evolved through adaptation to habitats covered by heavy snow in the winter season. The most distinctive character of this type is its three-dimensionally developing segments and light green fronds. An unexpected fact is that plants with the genome (the small form) do not differ significantly from the genome (the medium form) in frond size. Finally, the genome corresponds to the large form. For the genome, we have found only two samples in Japan (Okinawa Island: OK-6b and OK-7), but the genome is evidently dominant in this complex in Taiwan. Future investigations will find the distribution range of this genome population further north several samples have only the genome but unknown ploidy (KG-1a; SZ-6a; TS-1) are possible diploids. This type is genetically far from the remaining types, even in the genus Vandenboschia (Figs. 3 and 5), and certainly more closely related to American V. radicans than to Japanese relatives. This is characterized by dark-green-colored fronds, broad and flat wings of rachis and stipe, and thicker rhizomes (more than 1 mm in diameter). Very short stipes, less than a quarter of the length of the frond are often observed in this type. Because taxonomic treatments of this complex are still required, especially of the type specimens of scientific names applied within this complex, we hereafter should call each possible species by their genomic constitution. And it must be noted that none of Japanese plants of V. radicans complex form a monophyletic group with South American V. radicans in the rbcl tree (Fig. 3). It implies that the Japanese species are not truely V. radicans but independent species. Hybrids and polyploids The most remarkable result is that hybrids which have multiple GapCp sequences belonging to two or more groups in a single material are much more widely and abundantly spread throughout their distribution range in Japan (Fig. 6). The most common combination of the hybrid is triploid between the genome and the genome (genomic formula: * ), which spreads from northern Honshu down to the southern island of Kyushu. Hybrids between the genome and the genome (genomic formula: * ) are also common in the Kanto region southward. In the extreme case, there are some triple hybrids (genomic formula: ) that consist of three different genomes derived from all three species. About 22% of our samples whose ploidies are available are allotetraploids of hybrid origin. Though we have observed their meiosis only in a small number of samples, some of the ** genome individuals have normal 72 bivalents (data not shown). It implies that they have genome and reproduce sexually as amphidiploids. Furthermore, our results revealed that quite a large form collected from Okinawa is a tetraploid of ** genome. This form probably originated from a chromosome doubling subsequent to hybridization between the large form ( genome) and V. liukiuensis ( genome), which was previously considered to be distinct from the V. radicans complex. This tetraploid apparently looks like V. radicans complex plants, but reflects characters of V. liukiuensis, such as distinctly flared lips of sori and wiry rhizomes. The inferred reticulation process within this complex is illustrated in Fig. 7. There are also autotriploids; a few of them have only one GapCp sequence, but most of them have two different GapCp sequences that belong to the same group. Because autotetraploids have not been discovered, the origins of these triploids are unclear at this moment. But it is possible that they arose from hybridizations between haploid gametes and unreduced (diploid) gametes. Geographic distribution of sequences The,, and genomes as present in hybrids or amphidiploids have distinctly wider geographical distributions than their parental diploids. The genome (A/I, I sequences) is the most widespread in the studied area, but nearly confined to Japan. However, our data showed that it evidently reached Taiwan (TW-4) as an

8 1542 AMERICAN JOURNAL OF BOTANY [Vol. 92 Fig. 6. A distribution map of cytotype and genomic formula of the Vandenboschia radicans complex of Japan and adjacent areas. Shapes of symbols indicate cytotypes: circle diploid, triangle triploid, square tetraploid, pentagon unknown. Colors of symbols indicate genomes circumference color for maternal lineages (rbcl) and internal color for nuclear (GapCp) genotypes: yellow the genome (A/I, I sequences), blue the genome (B/II sequences), red the genome (C/III sequences), green the genome (D/IV sequences, V. liukiuensis). autotriploid. The genome (B/II sequences) is dominant in the Sea of Japan side of the archipelago, but its southernmost limit reached southern Kyushu (KG-1c). The genome (C/III sequences) is apparently an element of the Pacific Sea side, but it is unexpectedly found in the San-in area of the Sea of Japan side (SM-4, TT-2, TT-3a, and TT-3b). It is likely that the last type is closely related to Chinese and southeast Asian counterparts of this complex.

9 September 2005] EBIHARA ET AL. VANDENBOSCHIA RADICANS COMPLEX IN JAPAN 1543 Fig. 7. A hypothesized process of reticulate evolution in the Japanese Vandenboschia radicans complex. All the fronds are fertile, taken from voucher specimens of the current study. The bold line shows chromosome doubling, the broken line shows polyploidization that concerns unreduced spores, and the dotted line shows alternative hybridization process of the genotype. Scale bar 3 cm.

10 1544 AMERICAN JOURNAL OF BOTANY [Vol. 92 Reproductive system The reproductive system of this complex is most problematic. There has so far been no evidence of apogamous reproduction; all the preceding reports and our own observations of the meioses showed irregular chromosome pairings in triploids in spite of their dominance. More careful observation is necessary because occasional apogamy may contribute largely to their reproduction. However, considering some of the results of recent studies, it is unlikely that apogamy is the main method of reproduction within this complex because hybrids with different genomic formula occur sympatrically in many cases (e.g., both,, *, and * are at KN-1). Even if sympatrically occurring individuals have the same genomic formula, the maternal lineages, which is suggested by the rbcl sequence, are often different (e.g., all the six samples collected from the locality FI-3 have a genomic formula **. In the rbcl sequence, FI-3a, -3d, -3e, and -3f have type II, while FI-3b and -3c have type I). Results of genetic variations also suggest their recurrent origins rather than clonal (apogamous) reproduction; each genomic formula includes various combinations of alleles found in GapCp sequences (e.g., a genomic formula consists of genotypes, A1A2B3, A1A2B1, A1A6B2, A5A7B1, etc.), and a certain allele appears in several different genomic formula (e.g., the allele A4 appears in,, and ** ). This fact suggests that most of the alleles were already present before the formation of hybrids and that certain hybrids that have the same genomic formula but different alleles originated from independent events. Therefore, enormous independent hybridization events have to be considered to explain the current genetic diversity of the complex in spite of low frequency of occurrence of sexual parents. One of the possible explanations for this phenomenon is that the hybrids are vegetatively reproducing relicts of the time when the fertile races were distributed widely and abundantly in climates warmer than today, as suggested in tropical ferns at rockhouses in eastern North America (Farrar, 1998). But it must be noted that the geological conditions of the habitats of Japan and those of North America, especially the physical stability of the rock face, are not identical. Hybrids that occur without being accompanied by their fertile parents in habitats heavily disturbed by recent human activities (e.g., SZ-8, ca. 140-yr-old stone yard) suggest that they are not relicts but of recent origin. Conclusion Our study combining ploidy analysis (utilizing flow cytometry), nuclear DNA analysis (GapCp), and chloroplast DNA analysis (rbcl) effectively clarified the biological status of the species complex originated through reticulate evolution. Furthermore, the suggested evolutionary history within the complex well explained its morphological variation. These methods should be applicable to other species complexes, especially those of ferns that contain developed polyploid series and many hybrids. However, a big problem still remains unsolved about the reproductive methods of members of the complex. Our next step is to focus on this issue through careful observation of their fertility and more detailed genetic variation in local populations. It is noteworthy that independent gametophytes are more widely distributed than sporophytes, both in the North American counterpart of the complex, T. boschianum (Farrar, 1998), and in the European counterpart, V. speciosa (Rumsey et al., 1998). Though there is no record of independent gametophytes of V. radicans complex in the studied areas so far, we should also consider the possibility of their involvement in hybrid formations. LITERATURE CITED ALVAREZ, I., AND J. F. 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Any GapCp genotypes that were identified by sequencing are in boldface; otherwise, they were deduced from comparisons of band positions in SSCP gels. When ploidy is unknown, the genotype is in brackets. For samples with unknown genome dosage, unidentified genomes are marked by asterisks. Taxon Sample code; ploidy; genotype: GapCp, rbcl; genomic formula; voucher; locality. Vandenboschia radicans complex AC-1: 3x; A1*B1, I; * ; Ebihara ; Kuragari Valley, Nukata-cho, Aichi Pref. AC-2a: 4x; A1**B3, II; ** ; Ebihara ; Natsuyama, Nukata-cho, Aichi Pref. AC-2b: 3x; A1A2B3, I; ; Ebihara ;. AC-2c: 3x; A1*B3, I; * ; Ebihara ;.AK-1: 3x; A1*B1, I; * ; T. Kikuchi ; Mt. Nanakura, Futatsui-machi, Akita Pref. AK-2: 3x; A1A2B1, II; ; T. Kikuchi ; Ohkuzo, Hinai-cho, Akita Pref. AK-3a: 2x; B1B1, II; ; Y. Horii ; Takinogashira, Oga-shi, Akita Pref. AK-3b: 3x; B1B1B1, II; BBB; Y. Horii ;. AK-3c: 3x; B1B*B2, II; ; Y. Horii ;.AK-3d: 2x; B2B3, II; BB; Y. Horii ;. AK-4: 3x; A1B1B2, I; ; Y. Horii ; Ishizawa-ohtaki Fall, Honjo-shi, Akita Pref. AM-1: 2x; B1B2, II; ; Ebihara ; Sugenuma, Towadako-machi, Aomori Pref. AM-2: 3x; B1B*B2, II; ; Ebihara ; Oirase, Towadako-machi, Aomori Pref. AM-3a: 2x; B1B4, II; ; T. Oka ; Komagome, Aomori-shi, Aomori Pref. AM-3b: 3x; B1B*B2, II; ; T. Oka ;.AM-3c: 2x; B1B4, II; ; T. Oka ;.AM-3d: 2x; B1B1, II; ; T. Oka ;.AM-3e: 2x; B1B1, II; ; T. Oka ;. CB-1a: NA; [B1/B2], II; [ / ]; Ebihara ; Hachiro-zuka, Kimitsu-shi, Chiba Pref. CB-1b: 3x; B1B1B1, II; ; Ebihara ;. CB-2: 3x; B1B2C1, II; ; Ebihara ; Mt. Mitsuishi, Kimitsu-shi, Chiba Pref. CB-3a: 4x; A13B1C4*, III; *; Ebihara ; Uchiura, Amatsu-kominatomachi, Chiba Pref. CB-3b: 4x; A1A5B1C2, I ; ; Ebihara ;. EH-1: 3x; A3A*A12, I ; ; M. Hyodo 11415; Tomidani, Ohzu-shi, Ehime Pref. EH-2: 3x; A1*B1, I; * ; M. Hyodo ; Kusukubo, Tanbara-cho, Ehime Pref. FI-1a: 4x; A1A2B1B3, I; ; K. Akai ; Mt. Monju, Fukui-shi, Fukui Pref. FI-1b: 4x; A1A2*B3, II; * ; K. Akai ;. FI-2a: 3x; A2B1B3, II; ; Y. Saito ; Iwaya, Katsuyama-shi, Fukui Pref. FI-2b: 3x; A2*B1, II; * ; Y. Saito ;.FI-2c: 3x; A2*B1, II; * ; Y. Saito ;.FI-2d: 3x; A2*B3, I; * ; Y. Saito ;.FI-3a: 4x; A2**B3, II; ** ; Y. Saito ; Komyoji, Eiheiji-cho, Fukui Pref. FI-3b: 4x; A2**B3, I; ** ; Y. Saito ;. FI-3c: 4x; A2**B3, I; ** ; Y. Saito ;.FI-3d: 4x; A2**B3, II; ** ; Y. Saito ;.FI- 3e: 4x; A2**B3, II; ** ; Y. Saito ;.FI-3f: 4x; A2**B3, II; ** ; Y. Saito ;. FO-1a: 3x; A2*B1, I; * ; S. Tsutsui ; Narutake, Nakagawa-machi, Fukuoka Pref. FO-1b: 3x; A3*C2, I; * ; S. Tsutsui ;.FO-2: 3x; A1A*A6, I ; ; S. Tsutsui ; Saruyamadani, Nakagawa-machi, Fukuoka Pref. FO-3: 3x; A1*B1, I; * ; Takamiya ; Iharayama, Maebaru-shi, Fukuoka Pref. FO-4: 3x; A1A2C1, I ; ; S. Tsutsui ; Kuwanokochi, Nakagawa-machi, Fukuoka Pref. GF-1: 3x; A1*B3, II; * ; Ebihara ; Momijigataki Fall, Mugegawa-cho, Gifu Pref. GF-2: 3x; A1B1B3, II; ; Ebihara ; Iwakado, Horado-mura, Gifu Pref. GF-3: 3x; A5A7B1, I ; ; Ebihara ; Kamagataki Falls, Gujoshi, Gifu Pref. HG-1: 3x; A3B1B3, II; ; T. Suzuki ; Kutani, Hamasaka-cho, Hyogo Pref. HG-2: 3x; A1*B1, I ; * ; T. Suzuki ; Yodo, Hamasaka-cho, Hyogo Pref. HG-3: 3x; A1*B3, I; * ; T. Suzuki ; Mt. Koganegadake, Sasayama-shi, Hyogo Pref. HG-4: 4x; A1**B1, II; ** ; Ebihara ; Yamagai, Aogaki-cho, Hyogo Pref. HG-5a: 3x; A1*B1, II; * ; Ebihara ; Torogawa-inari, Muraokacho, Hyogo Pref. HG-5b: 3x; A2B1B3, II; ; Ebihara ;. HR-1: 4x; A1A2*B2, II; * ; N. Hamada ; Mt. Ohyorogi, Takano-cho, Hiroshima Pref. HR-2: 3x; A1B1B2, II; ; N. Hamada ; Mendaki Falls, Takano-cho, Hiroshima Pref. KC-1: 3x; A1*B1, I ; * ; K. Yamaoka ; Kirimigawa, Ochi-cho, Kochi Pref. KC-2: NA; [A1], I ; [ ]; K. Yamaoka 0306; Yasui Valley, Ikegawa-cho, Kochi Pref. KC-3a: 3x; A2*C6, I; * ; Takamiya ; Kubotsu, Tosa-shimizushi, Kochi Pref. KC-3b: 2x; A1A1, I; ; Takamiya ;.KC- 4: 3x; A1*B1, II; * ; K. Yamaoka ; Mt. Imano, Tosa-shimizushi, Kochi Pref. KC-5: 3x; A2*C2, III; * ; A. Narita ; None River, Toyo-cho, Kochi Pref. KC-6: 3x; A1A2C2, I; ; H. Sada ; Kyoho, Sukumo-shi, Kochi Pref. KG-1a: NA; [C3/C6], III; [ / ]; Tak-

12 1546 AMERICAN JOURNAL OF BOTANY [Vol. 92 amiya s.n.; Takinoshita River, Kagoshima-shi, Kagoshima Pref. KG-1b:3x; A1C3C6, I; ; T. Maruno ;.KG-1c: 3x; A1*B1, I; * ; T. Maruno ;.KG-2a: 4x; A3**C2, I; ** ; Takamiya ; Jyusso, Ohkuchi-shi, Kagoshima Pref. KG-2b: 3x; A3*C2, I; * ; Takamiya ;. KG-3a: 2x; A1A1, I; ; Takamiya ; Hana-age River, Yaku Isl., Kagoshima Pref. KG-3b: 3x; A6A7C2, I; ; Takamiya ;.KG-4a: 2x; A1A1, I; ; Takamiya ; Suzu River, Yaku Isl., Kagoshima Pref. KG-5a: 2x; A2A2, I; ; Takamiya ; Koseda, Yaku Isl., Kagoshima Pref. KG-5b: 2x; A2A2, I; ; Takamiya ;.KG-6a: 3x; A2A*A6, I ; ; Takamiya ; Nagata, Yaku Isl., Kagoshima Pref. KG-6b: 3x; A2A*A6, I; ; Takamiya ;.KG-7: 3x; A2A2A2, I; ; Takamiya ; Hanayama Virgin Forest, Yaku Isl., Kagoshima Pref. KG-8a: 2x; A2A2, I; ; Takamiya ; Anbo Arakawa Dam, Yaku Isl., Kagoshima Pref. KG-8b: 2x; A2A2, I; ; Takamiya ;.KG-8c: 2x; A2A2, I; ; Takamiya ;. KG-9a: 2x; A1A1, I ; ; Takamiya ; Shiratani Unsuikyo Tsuji Pass, Yaku Isl., Kagoshima Pref. KG-9b: 3x; A1A*A6, I ; ; Takamiya ;.KG-9c: 2x; A2A2, I ; ; Takamiya ;.KG-10: 3x; A2*B1, I; * ; Takamiya ; Fuke, Ohkuchi-shi, Kagoshima Pref. KG-11: 2x; A1A1, I; ; Takamiya ; Mt. Motchomu, Yaku Isl., Kagoshima Pref. KM-1: 3x; A1A*A15, I ; ; Takamiya ; Nagaso, Kahoku-machi, Kumamoto Pref. KM-2: 3x; A1A1A1, I; ; Takamiya ; Oritachi, Itsuki-mura, Kumamoto Pref. KM-3: 3x; A1*C2, I; * ; Takamiya ; Fukuregi, Amakusa-machi, Kumamoto Pref. KM-4a: 3x; A1*B1, I; * ; Takamiya ; Mae, Yamae-mura, Kumamoto Pref. KM-4b: 3x; A3A*A9, I ; ; Takamiya ;.KM-4c: 3x; A1*B1, I ; * ; Takamiya ;. KM-5: 3x; A1A4B1, I; ; Takamiya ; Kanaji, Yabe-machi, Kumamoto Pref. KN-1a: 3x; A2B1B3, II; ; Ebihara ; Jinmuji, Zushi-shi, Kanagawa Pref. KN-1b: 4x; A2B1C4*, III; *; Ebihara ;.KN-1c: 3x; A1B1C1, III; ; Ebihara ;.KN-1d: 3x; A1B1C1, II; ; Ebihara ;. KN-1e: 3x; A3*C1, III; * ; Ebihara ;.KN-1f:3x; A1*B1, I ; * ; Ebihara ;.KN-1g: 4x; A1B1C2*, III; *; Ebihara ;.KN-2: 3x; B1*C1, III; * ; T. Oka ; Asahinacho, Yokohama-shi, Kanagawa Pref. KN-3: 4x; A2B1C1*, III; *; T. Oka ; Kawana, Fujisawa-shi, Kanagawa Pref. KR-1: 3x; A1A4B1, I ; ; Matsumoto SM ; Hyo-dong River, Cheju Isl., South Korea. KR-2: 3x; A1*B1, I ; * ; Matsumoto SM ; Donneko Valley, Cheju Isl., South Korea. KT-1: 4x; A1*B1B3, II; ; F. Kasetani ; Izuriha-cho, Kyoto-shi, Kyoto Pref. ME-1: 3x; A2A6C2, I ; ; Ebihara ; Sebara, Kiho-cho, Mie Pref. ME-2: 2x; A1A1, I; ; Ebihara [TI]; Sebara, Kiho-cho, Mie Pref. ME-3: 3x; A2A6C2, I ; ; K. Ohora ; Asari, Kiho-cho, Mie Pref. ME-4: 3x; A1A5B1, I ; ; F. Kasetani ; Kisohara, Miyagawa-mura, Mie Pref. ME- 5: 4x; A1A2*B1, I; * ; K. Seto 61750; Miyanotani, Iitaka-cho, Mie Pref. MZ-1: 4x; A1**C2, III; * ; Takamiya ; Daimaru River, Miyazaki-shi, Miyazaki Pref. MZ-2: 4x; A1*C1C2, III; * ; Takamiya ; Uchiumi River, Miyazaki-shi, Miyazaki Pref. MZ-3: 3x; A1A1A1, I ; ; Takamiya ; Kuruson Valley, Ebino-shi, Miyazaki Pref. MZ-4a: 3x; A1*C3, III; * ; S. Fujimoto ; Kobuse Fall, Nichinan-shi, Miyazaki Pref. MZ-4b: 3x;A1A1A1, I; ; S. Fujimoto ;.MZ-4c: 3x; A13A*A22, I ; ; S. Fujimoto ;. NG-1: 4x; A3*B1B3, II; * ; Ebihara ; Mt. Awagatake, Kamoshi, Niigata Pref. NG-2a: 3x; B1B*B3, II; ; S. Sato ; Minamisawa River, Yuzawa-machi, Niigata Pref. NG-2b: 3x; A1*B1, II; * ; S. Sato ;.NG-2c: 3x; A1*B1, I; * ; S. Sato ;. NG-3a: 4x; A1**B3, I; ** ; S. Ishizawa ; Shiratama-no-taki Falls, Niitsu-shi, Niigata Pref. NG-3b: 3x; A1*B3, I; * ; S. Ishizawa ;.NG-4: 3x; B1B*B2, II; ; H. Kariya ; Mt. Yahiko, Yahiko-mura, Niigata Pref. NG-5: 3x; A1*B3, II; * ; S. Ishizawa ; Tsukimizu-no-ike Pond, Itoigawa-shi, Niigata Pref. NG-6: 3x; A2B3B4, II; ; Y. Tosaka ; Kirinzan, Kanose-machi, Niigata Pref. NG-7a: 3x; A1B1B5, II; ; H. Kariya ; Jyujikyo Valley, Muika-machi, Niigata Pref. NG-7b: 3x; A1B1B2, II; ; S. Ishizawa ;.NG-8a: 2x; B2B4, II; ; S. Nishiyama ; Suyoshi, Nagaoka-shi, Niigata Pref. NG-8b: NA; [A1/A2/B1], II; [ / / ]; S. Nishiyama ;. NG-9: 2x; [A2/B2], II; [ / ]; M. Sasagawa ; Sugigawa, Muramatsu-machi, Niigata Pref. NG-10: 2x; B1*B2, II; ; H. Kariya ; Mt. Kakuda, Maki-machi, Niigata Pref. NN- 1: 3x; A2*B1, II; * ; S. Ishizawa ; O-irisawa, Sakae-mura, Nagano Pref. NR-1: 3x; A1*B1, I ; * ; F. Kasetani ; Mt. Kasuga, Nara-shi, Nara Pref. NR-2: 3x; A2*B1, I; * ; F. Kasetani ; Nishitani, Murou-mura, Nara Pref. NR-3: 3x; A4A7B3, II; ; F. Kasetani ; Kisadani, Yoshino-cho, Nara Pref. NR-4: 3x; A2*B1, I; * ; K. Ohora ; Kotochi, Kamikitayama-mura, Nara Pref. NR-5: 3x; A4A*A1, I ; ; F. Kasetani ; Nishigawa, Kawakami-mura, Nara Pref. NR-6: 3x; A1*B1, I ; * ; F. Kasetani ; Ohmata, Higashiyoshino-mura. NR-7: 4x; A1**B3, I; ** ; F. Kasetani ; Hikawase River, Nishiyoshino-mura, Nara Pref. NR-8: 3x; A1A6B2, I; ; F. Kasetani ; Kamitako River, Kawakami-mura, Nara Pref. NR- 9: 3x; A2*B1, I ; * ; K. Seto ; Sannokoh, Kawakami-mura, Nara Pref. NR-10: 4x; A2*B1B3, I; * ; F. Kasetani ; Takimoto-cho, Tenri-shi, Nara Pref. NR-11: 3x; A1A2B1, I ; ; K. Seto ; Tanigaito, Totsukawa-mura, Nara Pref. NR-12: 3x; A1*B1, II; * ; K. Seto ; Murouji Temple, Murou-mura, Nara Pref. NR- 13: 3x; A1*B1, I ; * ; F. Kasetani ; Ryuchin Valley, Haibaracho, Nara Pref. OI-1: 3x; A2*B1, I ; * ; H. Tsuji ; Shimokawachi, Kusu-machi, Oita Pref. OK-1a: 2x; A2A17, I; ; Ebihara ; Urauchi River, Iriomote Isl., Okinawa Pref. OK-1b: 2x; A10A16, I; ; Ebihara ;.OK-1c: 4x; A8**C16, III; ** ; S. Fujimoto s.n.;. OK-2a: 3x; A2*C15, I; * ; Ebihara ; Hinai Falls, Iriomote Isl., Okinawa Pref. OK-2b: 2x; A2A20, I; ; Ebihara ;.OK- 2c: 2x; A15A21, I; ; M. Hasebe ;.OK-3: 3x; A1*C1, I; * ; T. Takara s.n.; Mt. Hitotsu-dake, Nago-shi, Okinawa Pref. OK-4: 4x; C1**D1, IV; ** ; T. Takara ; Mt. Nishime, Kunigami-son, Okinawa Pref. OK-5a: 4x; C6**D1, IV; ** ; T. Takara ; Mt. Kuhru, Naha-shi, Okinawa Pref. OK-5b: 3x; A2A*A10, I; ; T. Takara ;.OK-6a: NA; [A1/A18/C3/C8], III; [ / / / ]; T. Kuramata ; Mt. Yonaha-dake, Kunigami-son, Okinawa Pref. OK-6b: 2x; C3C17, III; ; Y. Inoue ;.OK-7: 2x; C14C14, III; ; T. Takara ; Mt. Tano-dake, Nago-shi, Okinawa Pref. OK-8a: NA; [A2/A15/A16], I; [ / / ]; T. Takara ; Mt. Iyu-dake, Higashi-son, Okinawa Pref. OK-8b: 3x; A1A*A15, I; ; T. Takara ;. OS-1: 2x; A1A1, I; ; K. Tsujii ; Ohtani Forest Road, Izumishi, Osaka Pref. OS-2: 3x; A1*B1, I ; * ; K. Tsujii ; Negorodani, Izumi-shi, Osaka Pref. OS-3: 3x; A1*B1, I; * ; K. Tsujii ; Kagata, Kawachi-nagaono-shi, Osaka Pref. OS-4: 3x; A1*B1, II; * ; K. Tsujii ; Mt. Iwawaki, Kawachi-nagaono-shi, Osaka Pref. OS- 5: 3x; A1*B2, I; * ; K. Tsujii ; Nagaredani, Kawachi-nagaonoshi, Osaka Pref. OS-6: 3x; A2*B1, I; * ; K. Tsujii ; Hirokawa, Kanan-cho, Osaka Pref. OS-7: 4x; A1**C3, III; ** ; K. Tsujii ; Nakanotani, Chihaya-akasaka-mura, Osaka Pref. OS-8: 2x; A1A14, I; ; K. Tsujii ; Makio-san, Izumi-shi, Osaka Pref. OS-9: 3x; A1A2B1, I; ; K. Tsujii ; Sobakawatani, Izumi-shi, Osaka Pref. OS-10: 3x; A1A2B1, I ; ; K. Tsujii ; Warabikasu-tani, Izumishi, Osaka Pref. OS-11: 3x; A1A2B1, I; ; K. Tsujii ; Takihata, Kawachi-nagaono-shi, Osaka Pref. OS-12a: 4x; A6**C12, III; ** ; K. Tsujii ; Inunaki, Izumisano-shi, Osaka Pref. OS-12b: 3x; A1*B3, II; * ; K. Tsujii ;. OS-12c: 3x; A1*B1, I; * ; K. Tsujii ;.OS-12d: 3x; A1*B1, I; * ; K. Tsujii ;.OS- 13: 3x; A1*B1, II; * ; K. Tsujii ; Chichioni River, Izumi-shi, Osaka Pref. OS-14a: 3x; A1A2B1, I; ; K. Tsujii ; Sengokudani, Kawachi-nagaono-shi, Osaka Pref. OS-14b: 3x; A1*B1, I; * ; K. Tsujii ;.OY-1: 3x; A1B1B3, II; ; K. Mizote ; Sugo, Niimi-shi, Okayama Pref. OY-2: 3x; A1*B3, I; * ; R. Ikehata 6276; Kakyojino-taki Falls, Kume-cho, Okayama Pref. OY-3: 3x; A1*B3, I; * ; R. Ikehata 6277; Kashiwagatani, Kagamino-cho, Okayama Pref. OY-4: 3x; A1*B1, II; * ; R. Ikehata 6278; Amagi, Okutsu-cho, Okayama Pref. OY- 5: 3x; A1A2B1, I; ; R. Ikehata 6279; Ohtsuri, Okutsu-cho, Okayama Pref. OY-6a: 4x; A1*B1B3, I; * ; K. Mizote ; Okutsugawa Valley, Shoboku-cho, Okayama Pref. OY-6b: 3x; A1*B1, II; * ; K. Mizote ;.OY-6c: 4x; A1*B1B2, II; * ; K. Mizote ;. OY-7: 3x; A2B1B2, I ; ; R. Ikehata 6334; Yamanori Fudo Falls, Chukason, Okayama Pref. OY-8: 3x; A1B1B3, I; ; K. Mizote ; Kogura, Mitsu-cho, Okayama Pref. OY-9: NA; [A2/B1/B3], II; [ / / ]; K. Mizote ; Iwai Falls, Kamisaibara-son, Okayama Pref. OY-10: 3x; A1B1B2, II; ; R. Ikehata ; Yubara Dam, Yubara-cho, Okayama Pref. SG-1a: 3x; A1*C13, I; * ; Takamiya ; Kishiyama, Kitahata-mura, Saga Pref. SG-1b: 3x; A1*C13, I ; * ; Takamiya ;.SG-1c: 3x; A1*B1, I; * ; Takamiya ;.SG- 2: 3x; A1A2B1, II; ; S. Tsutsui ; Ogawachi, Higashi-sefurison, Saga Pref. SM-1: 3x; A2A*A4, I ; ; Ebihara ; Miyauchi, Yakumo-mura, Shimane Pref. SM-2: 4x; A1A3*B1, I ; * ; Y. Sugimura ; Kiyomizu-daishi Temple, Yunotsu-cho, Shimane Pref. SM-3: NA; [A1/B3], I; [ / ]; Y. Sugimura ; Takagi, Hirose-machi, Shi-