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Systematics and Phytogeography |
2Department of System Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan; 3Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan; 4Tsukuba Botanical Garden, National Science Museum, 4-1-1 Amakubo, Tsukuba 305-0005, Japan; 5Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue-shi, Shimane 690-8504, Japan; 6The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba 261-8586, Japan; 7Graduate School of Science and Technology, 2-39-1 Kurokami, Kumamoto 860-8555, Japan; 8Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Received for publication January 13, 2005. Accepted for publication May 13, 2005.
ABSTRACT
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 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. radicans, 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 large-sized plants are more often found in the areas under the influence of warm currents (Pacific side of Honshu southward).
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, 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.
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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 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 mm2) 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 Na2SO3, 50 mM Tris-HCl [pH 7.5], 25 µg/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 mm2) for internal standard. The crushed tissue was placed on ice for 5 min and filtered through 30-µm 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 001118-01, 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 (http:// www.ncbi.nlm.nih.gov). 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'-CAAGCGGCAGCCRAYTCAG-3'), 35 cycles: 94°C (45 s), 52°C (45 s), 72°C (75 s) using diluted total DNA in 10 ng/µL 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 single-copy 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'-GCTCCAATGTTTGTAATGGG-3', 6FB: 5'-GCTCCAATGTTTGTCATGGG-3', 6FC: 5'-GCTCCAATGTTTGTGATGGG-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'-GAGGGTTTGATGACCACAGT-3') and GapC-CR-2 (5'-CTTTTCCACTCGACAAGTCAG-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 0.5x 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 in 250 µL 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 µL 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 1 000 000 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. 23.4 pg (Narayan, 1987
), genome size of this diploid material is estimated as ca. 73.2 pg. For other diploids, that of the "medium" form is ca. 77.7 pg on an average (N = 16), that of the "small" form is ca. 77.6 pg (N = 8), and that of the "large" form is ca. 92.3 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).
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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).
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, 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.
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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 "ß"), 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 , ß, 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 ß genome in the rbcL sequence is 20 bp, which is too large 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 ß is sterile (both irregular meiosis and spores observed), and therefore reproductive isolation has been established, even between the genome and the ß genome.
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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, darker-colored 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.
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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 ß genome (genomic formula: *ß), 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: ß) 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.
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Geographic distribution of sequences
The , ß, 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 autotriploid. The ß 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.
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 ßß, ß, *, and ß* 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 **ß. 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 ß 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 , ß, 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.
APPENDIX 1.
Voucher specimens examined in this study. 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
*ß; Ebihara 031207-01; Kuragari Valley, Nukata-cho, Aichi Pref.AC-2a: 4x; A1**B3, II; **ß; Ebihara 031207-03; Natsuyama, Nukata-cho, Aichi Pref.AC-2b: 3x; A1A2B3, I; ß; Ebihara 031207-04; —.AC-2c: 3x; A1*B3, I; *ß; Ebihara 031207-05; —.AK-1: 3x; A1*B1, I; *ß; T. Kikuchi 040516-01; Mt. Nanakura, Futatsui-machi, Akita Pref.AK-2: 3x; A1A2B1, II; ß; T. Kikuchi 041017-01; Ohkuzo, Hinai-cho, Akita Pref.AK-3a: 2x; B1B1, II; ßß; Y. Horii 041030-01; Takinogashira, Oga-shi, Akita Pref.AK-3b: 3x; B1B1B1, II; BBB; Y. Horii 041030-02; —.AK-3c: 3x; B1B*B2, II; ßßß; Y. Horii 041030-03; —.AK-3d: 2x; B2B3, II; BB; Y. Horii 041030-04; —.AK-4: 3x; A1B1B2, I; ßß; Y. Horii 041031-01; Ishizawa-ohtaki Fall, Honjo-shi, Akita Pref.AM-1: 2x; B1B2, II; ßß; Ebihara 030627-01; Sugenuma, Towadako-machi, Aomori Pref.AM-2: 3x; B1B*B2, II; ßßß; Ebihara 030628-03; Oirase, Towadako-machi, Aomori Pref.AM-3a: 2x; B1B4, II; ßß; T. Oka 040704-01; Komagome, Aomori-shi, Aomori Pref.AM-3b: 3x; B1B*B2, II; ßßß; T. Oka 040704-02; —.AM-3c: 2x; B1B4, II; ßß; T. Oka 040704-03; —. AM-3d: 2x; B1B1, II; ßß; T. Oka 040704-04; —.AM-3e: 2x; B1B1, II; ßß; T. Oka 040704-05; —.CB-1a: NA; ;obB1/B2;cb, II; ;obß/ß;cb; Ebihara 000507-01; Hachiro-zuka, Kimitsu-shi, Chiba Pref.CB-1b: 3x; B1B1B1, II; ßßß; Ebihara 030225-02; —.CB-2: 3x; B1B2C1, II; ßß; Ebihara 030225-01; Mt. Mitsuishi, Kimitsu-shi, Chiba Pref.CB-3a: 4x; A13B1C4*, III; ß*; Ebihara 030225-03; Uchiura, Amatsu-kominato-machi, Chiba Pref.CB-3b: 4x; A1A5B1C2, I'; ß; Ebihara 030225-04; —.EH-1: 3x; A3A*A12, I'; ; M. Hyodo 11415; Tomidani, Ohzu-shi, Ehime Pref.EH-2: 3x; A1*B1, I; *ß; M. Hyodo 040307-01; Kusukubo, Tanbara-cho, Ehime Pref.FI-1a: 4x; A1A2B1B3, I; ßß; K. Akai 040506-01; Mt. Monju, Fukui-shi, Fukui Pref.FI-1b: 4x; A1A2*B3, II; *ß; K. Akai 040506-02; —.FI-2a: 3x; A2B1B3, II; ßß; Y. Saito 040616-02; Iwaya, Katsuyama-shi, Fukui Pref.FI-2b: 3x; A2*B1, II; *ß; Y. Saito 040616-03; —.FI-2c: 3x; A2*B1, II; *ß; Y. Saito 040616-04; —.FI-2d: 3x; A2*B3, I; *ß; Y. Saito 040616-05; —.FI-3a: 4x; A2**B3, II; **ß; Y. Saito 040616-06; Komyoji, Eiheiji-cho, Fukui Pref.FI-3b: 4x; A2**B3, I; **ß; Y. Saito 040616-07; —.FI-3c: 4x; A2**B3, I; **ß; Y. Saito 040704-08; —.FI-3d: 4x; A2**B3, II; **ß; Y. Saito 040704-09; —.FI-3e: 4x; A2**B3, II; **ß; Y. Saito 040704-10; —.FI-3f: 4x; A2**B3, II; **ß; Y. Saito 040704-11; —.FO-1a: 3x; A2*B1, I; *ß; S. Tsutsui 040724-01; Narutake, Nakagawa-machi, Fukuoka Pref.FO-1b: 3x; A3*C2, I; *; S. Tsutsui 040724-02; —.FO-2: 3x; A1A*A6, I'; ; S. Tsutsui 040724-03; Saruyamadani, Nakagawa-machi, Fukuoka Pref.FO-3: 3x; A1*B1, I; *ß; Takamiya 040919-01; Iharayama, Maebaru-shi, Fukuoka Pref.FO-4: 3x; A1A2C1, I'; ; S. Tsutsui 040909-01; Kuwanokochi, Nakagawa-machi, Fukuoka Pref.GF-1: 3x; A1*B3, II; *ß; Ebihara 040515-01; Momijigataki Fall, Mugegawa-cho, Gifu Pref.GF-2: 3x; A1B1B3, II; ßß; Ebihara 040515-03; Iwakado, Horado-mura, Gifu Pref.GF-3: 3x; A5A7B1, I'; ß; Ebihara 040515-04; Kamagataki Falls, Gujo-shi, Gifu Pref.HG-1: 3x; A3B1B3, II; ßß; T. Suzuki 030727-01; Kutani, Hamasaka-cho, Hyogo Pref.HG-2: 3x; A1*B1, I'; *ß; T. Suzuki 030727-02; Yodo, Hamasaka-cho, Hyogo Pref.HG-3: 3x; A1*B3, I; *ß; T. Suzuki 030930-01; Mt. Koganegadake, Sasayama-shi, Hyogo Pref.HG-4: 4x; A1**B1, II; **ß; Ebihara 031014-01; Yamagai, Aogaki-cho, Hyogo Pref.HG-5a: 3x; A1*B1, II; *ß; Ebihara 031014-02; Torogawa-inari, Muraoka-cho, Hyogo Pref.HG-5b: 3x; A2B1B3, II; ßß; Ebihara 031014-05; —.HR-1: 4x; A1A2*B2, II; *ß; N. Hamada 040500-01; Mt. Ohyorogi, Takano-cho, Hiroshima Pref.HR-2: 3x; A1B1B2, II; ßß; N. Hamada 040500-02; Mendaki Falls, Takano-cho, Hiroshima Pref.KC-1: 3x; A1*B1, I'; *ß; K. Yamaoka 030608-01; Kirimigawa, Ochi-cho, Kochi Pref.KC-2: NA; ;obA1;cb, I'; ;ob;cb; K. Yamaoka 0306; Yasui Valley, Ikegawa-cho, Kochi Pref.KC-3a: 3x; A2*C6, I; *; Takamiya 030720-01; Kubotsu, Tosa-shimizu-shi, Kochi Pref.KC-3b: 2x; A1A1, I; ; Takamiya 030720-02; —.KC-4: 3x; A1*B1, II; *ß; K. Yamaoka 030915-01; Mt. Imano, Tosa-shimizu-shi, Kochi Pref.KC-5: 3x; A2*C2, III; *; A. Narita 040222-01; None River, Toyo-cho, Kochi Pref.KC-6: 3x; A1A2C2, I; ; H. Sada 041029-01; Kyoho, Sukumo-shi, Kochi Pref.KG-1a: NA; ;obC3/C6;cb, III; ;ob/;cb; Takamiya s.n.; Takinoshita River, Kagoshima-shi, Kagoshima Pref.KG-1b: 3x; A1C3C6, I; ; T. Maruno 031214-01; —.KG-1c: 3x; A1*B1, I; *ß; T. Maruno 031214-03; —.KG-2a: 4x; A3**C2, I; **; Takamiya 030704-01; Jyusso, Ohkuchi-shi, Kagoshima Pref.KG-2b: 3x; A3*C2, I; *; Takamiya 030704-02; —.KG-3a: 2x; A1A1, I; ; Takamiya 031102-02; Hana-age River, Yaku Isl., Kagoshima Pref.KG-3b: 3x; A6A7C2, I; ; Takamiya 031102-01; —.KG-4a: 2x; A1A1, I; ; Takamiya 041028-01; Suzu River, Yaku Isl., Kagoshima Pref.KG-5a: 2x; A2A2, I; ; Takamiya 040508-01; Koseda, Yaku Isl., Kagoshima Pref.KG-5b: 2x; A2A2, I; ; Takamiya 040508-02; —.KG-6a: 3x; A2A*A6, I'; ; Takamiya 040510-02; Nagata, Yaku Isl., Kagoshima Pref.KG-6b: 3x; A2A*A6, I; ; Takamiya 040510-03; —.KG-7: 3x; A2A2A2, I; ; Takamiya 040510-05; Hanayama Virgin Forest, Yaku Isl., Kagoshima Pref.KG-8a: 2x; A2A2, I; ; Takamiya 040511-01; Anbo—Arakawa Dam, Yaku Isl., Kagoshima Pref.KG-8b: 2x; A2A2, I; ; Takamiya 040511-02; —.KG-8c: 2x; A2A2, I; ; Takamiya 040511-03; —.KG-9a: 2x; A1A1, I'; ; Takamiya 040725-01; Shiratani Unsuikyo—Tsuji Pass, Yaku Isl., Kagoshima Pref.KG-9b: 3x; A1A*A6, I'; ; Takamiya 040725-02; —.KG-9c: 2x; A2A2, I'; ; Takamiya 040725-03; —.KG-10: 3x; A2*B1, I; *ß; Takamiya 040802-01; Fuke, Ohkuchi-shi, Kagoshima Pref.KG-11: 2x; A1A1, I; ; Takamiya 041029-01; Mt. Motchomu, Yaku Isl., Kagoshima Pref.KM-1: 3x; A1A*A15, I'; ; Takamiya 030710-01; Nagaso, Kahoku-machi, Kumamoto Pref.KM-2: 3x; A1A1A1, I; ; Takamiya 031127-01; Oritachi, Itsuki-mura, Kumamoto Pref.KM-3: 3x; A1*C2, I; *; Takamiya 040606-01; Fukuregi, Amakusa-machi, Kumamoto Pref.KM-4a: 3x; A1*B1, I; *ß; Takamiya 040610-01; Mae, Yamae-mura, Kumamoto Pref.KM-4b: 3x; A3A*A9, I'; ; Takamiya 040610-02; —.KM-4c: 3x; A1*B1, I'; *ß; Takamiya 040610-03; —.KM-5: 3x; A1A4B1, I; ß; Takamiya 040823-01; Kanaji, Yabe-machi, Kumamoto Pref.KN-1a: 3x; A2B1B3, II; ßß; Ebihara 030725-01; Jinmuji, Zushi-shi, Kanagawa Pref.KN-1b: 4x; A2B1C4*, III; ß*; Ebihara 030725-02; —.KN-1c: 3x; A1B1C1, III; ß; Ebihara 030725-03; —.KN-1d: 3x; A1B1C1, II; ß; Ebihara 030725-04; —.KN-1e: 3x; A3*C1, III; *; Ebihara 030725-05; —.KN-1f: 3x; A1*B1, I'; *ß; Ebihara 030725-06; —.KN-1g: 4x; A1B1C2*, III; ß*; Ebihara 030725-07; —.KN-2: 3x; B1*C1, III; ß*; T. Oka 041106-01; Asahina-cho, Yokohama-shi, Kanagawa Pref.KN-3: 4x; A2B1C1*, III; ß*; T. Oka 041103-02; Kawana, Fujisawa-shi, Kanagawa Pref.KR-1: 3x; A1A4B1, I'; ß; Matsumoto SM0306-55; Hyo-dong River, Cheju Isl., South Korea.KR-2: 3x; A1*B1, I'; *ß; Matsumoto SM0306-48; Donneko Valley, Cheju Isl., South Korea.KT-1: 4x; A1*B1B3, II; ßß; F. Kasetani 040627-01; Izuriha-cho, Kyoto-shi, Kyoto Pref.ME-1: 3x; A2A6C2, I'; ; Ebihara 991121-02; Sebara, Kiho-cho, Mie Pref.ME-2: 2x; A1A1, I; ; Ebihara 991121-03 ;obTI;cb; Sebara, Kiho-cho, Mie Pref.ME-3: 3x; A2A6C2, I'; ; K. Ohora 030706-01; Asari, Kiho-cho, Mie Pref.ME-4: 3x; A1A5B1, I'; ß; F. Kasetani 040321-03; Kisohara, Miyagawa-mura, Mie Pref.ME-5: 4x; A1A2*B1, I; *ß; K. Seto 61750; Miyanotani, Iitaka-cho, Mie Pref.MZ-1: 4x; A1**C2, III; *; Takamiya 031120-01; Daimaru River, Miyazaki-shi, Miyazaki Pref.MZ-2: 4x; A1*C1C2, III; *; Takamiya 031120-02; Uchiumi River, Miyazaki-shi, Miyazaki Pref.MZ-3: 3x; A1A1A1, I'; ; Takamiya 041010-01; Kuruson Valley, Ebino-shi, Miyazaki Pref.MZ-4a: 3x; A1*C3, III; *; S. Fujimoto 041111-01; Kobuse Fall, Nichinan-shi, Miyazaki Pref.MZ-4b: 3x; A1A1A1, I; ; S. Fujimoto 041111-02; —.MZ-4c: 3x; A13A*A22, I'; ; S. Fujimoto 041111-03; —.NG-1: 4x; A3*B1B3, II; *ßß; Ebihara 000906-1; Mt. Awagatake, Kamo-shi, Niigata Pref.NG-2a: 3x; B1B*B3, II; ßßß; S. Sato 040614-01; Minamisawa River, Yuzawa-machi, Niigata Pref.NG-2b: 3x; A1*B1, II; *ß; S. Sato 040614-02; —.NG-2c: 3x; A1*B1, I; *ß; S. Sato 040614-03; —.NG-3a: 4x; A1**B3, I; **ß; S. Ishizawa 040617-01; Shiratama-no-taki Falls, Niitsu-shi, Niigata Pref.NG-3b: 3x; A1*B3, I; *ß; S. Ishizawa 040617-02; —.NG-4: 3x; B1B*B2, II; ßßß; H. Kariya 040800-01; Mt. Yahiko, Yahiko-mura, Niigata Pref.NG-5: 3x; A1*B3, II; *ß; S. Ishizawa 040821-01; Tsukimizu-no-ike Pond, Itoigawa-shi, Niigata Pref.NG-6: 3x; A2B3B4, II; ßß; Y. Tosaka 040808-01; Kirinzan, Kanose-machi, Niigata Pref.NG-7a: 3x; A1B1B5, II; ßß; H. Kariya 040828-01; Jyujikyo Valley, Muika-machi, Niigata Pref.NG-7b: 3x; A1B1B2, II; ßß; S. Ishizawa 040829-01; —.NG-8a: 2x; B2B4, II; ßß; S. Nishiyama 041000-01; Suyoshi, Nagaoka-shi, Niigata Pref.NG-8b: NA; ;obA1/A2/B1;cb, II; ;ob//ß;cb; S. Nishiyama 041000-01; —.NG-9: 2x; ;obA2/B2;cb, II; ;ob/ß;cb; M. Sasagawa 041017-01; Sugigawa, Muramatsu-machi, Niigata Pref. NG-10: 2x; B1*B2, II; ßß; H. Kariya 001121-01; Mt. Kakuda, Maki-machi, Niigata Pref.NN-1: 3x; A2*B1, II; *ß; S. Ishizawa 041114-01; O-irisawa, Sakae-mura, Nagano Pref.NR-1: 3x; A1*B1, I'; *ß; F. Kasetani 031026-01; Mt. Kasuga, Nara-shi, Nara Pref.NR-2: 3x; A2*B1, I; *ß; F. Kasetani 031122-01; Nishitani, Murou-mura, Nara Pref.NR-3: 3x; A4A7B3, II; ß; F. Kasetani 031122-02; Kisadani, Yoshino-cho, Nara Pref.NR-4: 3x; A2*B1, I; *ß; K. Ohora 031123-01; Kotochi, Kamikitayama-mura, Nara Pref.NR-5: 3x; A4A*A1, I'; ; F. Kasetani 031203-01; Nishigawa, Kawakami-mura, Nara Pref.NR-6: 3x; A1*B1, I'; *ß; F. Kasetani 031214-01; Ohmata, Higashiyoshino-mura.NR-7: 4x; A1**B3, I; **ß; F. Kasetani 040110-01; Hikawase River, Nishiyoshino-mura, Nara Pref.NR-8: 3x; A1A6B2, I; ß; F. Kasetani 040328-01; Kamitako River, Kawakami-mura, Nara Pref.NR-9: 3x; A2*B1, I'; *ß; K. Seto 040719-01; Sannokoh, Kawakami-mura, Nara Pref.NR-10: 4x; A2*B1B3, I; *ßß; F. Kasetani 040801-01; Takimoto-cho, Tenri-shi, Nara Pref.NR-11: 3x; A1A2B1, I'; ß; K. Seto 040809-01; Tanigaito, Totsukawa-mura, Nara Pref.NR-12: 3x; A1*B1, II; *ß; K. Seto 040827-01; Murouji Temple, Murou-mura, Nara Pref.NR-13: 3x; A1*B1, I'; *ß; F. Kasetani 040918-01; Ryuchin Valley, Haibara-cho, Nara Pref.OI-1: 3x; A2*B1, I'; *ß; H. Tsuji 040718-01; Shimokawachi, Kusu-machi, Oita Pref.OK-1a: 2x; A2A17, I; ; Ebihara 001118-01; Urauchi River, Iriomote Isl., Okinawa Pref.OK-1b: 2x; A10A16, I; ; Ebihara 001118-03; —.OK-1c: 4x; A8**C16, III; **; S. Fujimoto s.n.; —.OK-2a: 3x; A2*C15, I; *; Ebihara 001121-01; Hinai Falls, Iriomote Isl., Okinawa Pref.OK-2b: 2x; A2A20, I; ; Ebihara 001121-03; —.OK-2c: 2x; A15A21, I; ; M. Hasebe 031217-01; —.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 040718-01; Mt. Nishime, Kunigami-son, Okinawa Pref.OK-5a: 4x; C6**D1, IV; **; T. Takara 040821-01; Mt. Kuhru, Naha-shi, Okinawa Pref.OK-5b: 3x; A2A*A10, I; ; T. Takara 040821-02; —.OK-6a: NA; ;obA1/A18/C3/C8;cb, III; ;ob///;cb; T. Kuramata 030228-01; Mt. Yonaha-dake, Kunigami-son, Okinawa Pref.OK-6b: 2x; C3C17, III; ; Y. Inoue 041113-01; —.OK-7: 2x; C14C14, III; ; T. Takara 041006-01; Mt. Tano-dake, Nago-shi, Okinawa Pref.OK-8a: NA; ;obA2/A15/A16;cb, I; ;ob//;cb; T. Takara 041103-02; Mt. Iyu-dake, Higashi-son, Okinawa Pref.OK-8b: 3x; A1A*A15, I; ; T. Takara 041103-03; —.OS-1: 2x; A1A1, I; ; K. Tsujii 040400-01; Ohtani Forest Road, Izumi-shi, Osaka Pref.OS-2: 3x; A1*B1, I'; *ß; K. Tsujii 040400-02; Negorodani, Izumi-shi, Osaka Pref.OS-3: 3x; A1*B1, I; *ß; K. Tsujii 040400-03; Kagata, Kawachi-nagaono-shi, Osaka Pref.OS-4: 3x; A1*B1, II; *ß; K. Tsujii 040400-04; Mt. Iwawaki, Kawachi-nagaono-shi, Osaka Pref.OS-5: 3x; A1*B2, I; *ß; K. Tsujii 040400-05; Nagaredani, Kawachi-nagaono-shi, Osaka Pref.OS-6: 3x; A2*B1, I; *ß; K. Tsujii 040500-01; Hirokawa, Kanan-cho, Osaka Pref.OS-7: 4x; A1**C3, III; **; K. Tsujii 040500-02; Nakanotani, Chihaya-akasaka-mura, Osaka Pref.OS-8: 2x; A1A14, I; ; K. Tsujii 040500-03; Makio-san, Izumi-shi, Osaka Pref.OS-9: 3x; A1A2B1, I; ß; K. Tsujii 040500-04; Sobakawatani, Izumi-shi, Osaka Pref.OS-10: 3x; A1A2B1, I'; ß; K. Tsujii 040500-05; Warabikasu-tani, Izumi-shi, Osaka Pref.OS-11: 3x; A1A2B1, I; ß; K. Tsujii 040500-06; Takihata, Kawachi-nagaono-shi, Osaka Pref.OS-12a: 4x; A6**C12, III; **; K. Tsujii 040500-07; Inunaki, Izumisano-shi, Osaka Pref.OS-12b: 3x; A1*B3, II; *ß; K. Tsujii 040500-08; —.OS-12c: 3x; A1*B1, I; *ß; K. Tsujii 040500-09; —.OS-12d: 3x; A1*B1, I; *ß; K. Tsujii 040500-10; —.OS-13: 3x; A1*B1, II; *ß; K. Tsujii 040600-01; Chichioni River, Izumi-shi, Osaka Pref.OS-14a: 3x; A1A2B1, I; ß; K. Tsujii 040600-02; Sengoku-dani, Kawachi-nagaono-shi, Osaka Pref.OS-14b: 3x; A1*B1, I; *ß; K. Tsujii 040600-03; —.OY-1: 3x; A1B1B3, II; ßß; K. Mizote 040509-01; Sugo, Niimi-shi, Okayama Pref.OY-2: 3x; A1*B3, I; *ß; R. Ikehata 6276; Kakyojino-taki Falls, Kume-cho, Okayama Pref.OY-3: 3x; A1*B3, I; *ß; R. Ikehata 6277; Kashiwagatani, Kagamino-cho, Okayama Pref.OY-4: 3x; A1*B1, II; *ß; R. Ikehata 6278; Amagi, Okutsu-cho, Okayama Pref.OY-5: 3x; A1A2B1, I; ß; R. Ikehata 6279; Ohtsuri, Okutsu-cho, Okayama Pref.OY-6a: 4x; A1*B1B3, I; *ßß; K. Mizote 040626-01; Okutsugawa Valley, Shoboku-cho, Okayama Pref.OY-6b: 3x; A1*B1, II; *ß; K. Mizote 040918-01; —.OY-6c: 4x; A1*B1B2, II; *ßß; K. Mizote 040918-02; —.OY-7: 3x; A2B1B2, I'; ßß; R. Ikehata 6334; Yamanori Fudo Falls, Chuka-son, Okayama Pref.OY-8: 3x; A1B1B3, I; ßß; K. Mizote 040829-01; Kogura, Mitsu-cho, Okayama Pref.OY-9: NA; ;obA2/B1/B3;cb, II; ;ob/ß/ß;cb; K. Mizote 040919-01; Iwai Falls, Kamisaibara-son, Okayama Pref.OY-10: 3x; A1B1B2, II; ßß; R. Ikehata 041007-01; Yubara Dam, Yubara-cho, Okayama Pref.SG-1a: 3x; A1*C13, I; *; Takamiya 040612-01; Kishiyama, Kitahata-mura, Saga Pref.SG-1b: 3x; A1*C13, I'; *; Takamiya 040612-02; —.SG-1c: 3x; A1*B1, I; *ß; Takamiya 040612-03; —.SG-2: 3x; A1A2B1, II; ß; S. Tsutsui 041018-01; Ogawachi, Higashi-sefuri-son, Saga Pref.SM-1: 3x; A2A*A4, I'; ; Ebihara 030423-01; Miyauchi, Yakumo-mura, Shimane Pref.SM-2: 4x; A1A3*B1, I'; *ß; Y. Sugimura 030312-01; Kiyomizu-daishi Temple, Yunotsu-cho, Shimane Pref.SM-3: NA; ;obA1/B3;cb, I; ;ob/ß;cb; Y. Sugimura 021220-01; Takagi, Hirose-machi, Shimane Pref.SM-4: 4x; A1**C3, III; **; Lin 030613-01; Nonami, Shimane-cho, Shimane Pref.SM-5: 3x; A1A2B1, I'; ß; Y. Sugimura 030930-01; Kitadani, Oki Isl., Shimane Pref.SM-6: 3x; A1*B1, I'; *ß; Y. Sugimura 030825-01; Fube, Hirose-machi, Shimane Pref.ST-1: 3x; A1*B1, II; *ß; S. Fujimoto 030419; Ushikubi Pass, Ogano-machi, Saitama Pref.ST-2: 4x; A1A6B1B3, I; ßß; Ebihara 040404-01; Han-nya, Ogano-machi, Saitama Pref.SZ-1: NA; ;obA1/B1;cb, I; ;ob/ß;cb; Ebihara 010604-02; Kawazu-nanadaru Falls, Kawazu-cho, Shizuoka Pref.SZ-2: 3x; A1A5B1, I'; ß; Matsumoto 990731-9; Kamifunabara, Izu-shi, Shizuoka Pref.SZ-3a: 4x; A1**C4, III; **; Matsumoto 030412-1; Shikine, Shimoda-shi, Shizuoka Pref.SZ-3b: 4x; A6**B2, II; **ß; Matsumoto 030412-2; —.SZ-3c: 4x; A1B1C1, III; ß; Matsumoto 030412-3; —.SZ-4a: 4x; A1*C4C5, III; *; Matsumoto SM030727-1; Banjo-no-taki Falls, Izu-shi, Shizuoka Pref.SZ-4b: 4x; A2A5B1C1, III; ß; Matsumoto SM030727-2; —.SZ-5: 4x; A1**C2, III; **; S. Fujimoto s.n.; Futo, Ito-shi, Shizuoka Pref.SZ-6a: NA; ;obC8;cb, III; ;ob;cb; Matsumoto 031206-9; Shirokawa, Nishi-izu-cho, Shizuoka Pref.SZ-6b: 3x; A1*C4, I; *; Matsumoto 031206-10; —.SZ-6c: 3x; A3*C2, III; *; Matsumoto 031206-11; —.SZ-6d: 3x; A1A5B1, I'; ß; Ebihara 040214-08; —.SZ-6e: 3x; A1A1A1, I; ; Ebihara 040214-09; —.SZ-6f: 3x; A3*C2, III; *; Matsumoto 031206-12; —.SZ-7a: 3x; A1*B1, I; *ß; M. Tanaka 031128-01; Takouma, Shimoda-shi, Shizuoka Pref.SZ-7b: 3x; A1*B1, I; *ß; Matsumoto 040202-15; —.SZ-8a: 2x; A1B2, II; ß; Ebihara 040214-01; Shiroyama Park, Shimoda-shi, Shizuoka Pref.SZ-8b: 3x; A1*C1, I'; *; Ebihara 040214-02; —.SZ-8c: 4x; A1**C1, III; *; Ebihara 040214-03; —.SZ-9: 3x; A6A11C4, I'; ; Matsumoto 040202-14; Kochi, Shmoda-shi, Shizuoka Pref.SZ-10a: 3x; A1*C1, I; *; M. Tanaka 040130-01; Ohnabe, Kawazu-cho, Shizuoka Pref.SZ-10b: 4x; A1**C1, I; **; Y. Sonehara 040314-01; —.SZ-11a: 3x; A1*C2, I; *; M. Tanaka 040403-01; Rendaiji, Shimoda-shi, Shizuoka Pref.SZ-11b: 4x; A1B1C2*, I'; ß*; M. Tanaka 040403-02; —.TK-1a: 2x; A2A2, I; ; Ebihara 011128-05; Higashidaira, Chichijima Isl. (Bonin), Tokyo Pref. TK-1b: 2x; A8A8, I; ; T. Yasui s.n.; —.TK-1c: 2x; A2A2, I; ; T. Yasui s.n.; —.TK-2: NA; ;obA/C?;cb, III; —; Ebihara 011123-03; Mt. Sekimon, Hahajima Isl. (Bonin), Tokyo Pref. TK-3: NA; ;obA1/B1/C1;cb, I'; ;ob/ß/;cb; T. Okamoto s.n.; Yasundo-go, Aogashima Isl., Tokyo Pref.TK-4: 4x; A1*C3C7, III; *; T. Oka 030719-01; Ikenosawa, Aogashima Isl., Tokyo Pref.TK-5a: 4x; A2**C2, III; **; S. Fujimoto 030928-01; Kamogawa Forest Rd., Hachijyo Isl., Tokyo Pref.TK-5b: 3x; A2*C2, I; *; S. Fujimoto 030928-02; —.TK-5c: 4x; A2**C2, III; **; S. Fujimoto 030928-03; —.TK-6: 4x; A4**C7, III; **; S. Fujimoto 030928-04; Mt. Hachijyo-fuji, Hachijyo Isl., Tokyo Pref.TK-7: 4x; A2**C2, III; **; S. Fujimoto 030927-01; Hotaru Waterway, Hachijyo Isl., Tokyo Pref.TK-8: 2x; A19B1, II; ß; S. Fujimoto 031117-01; Jataki Falls, Hachioji-shi, Tokyo Pref.TS-1: NA; ;obC5/C11;cb, III; ;ob/;cb; A. Narita 040207-01; Tamagasa, Kainan-cho, Tokushima Pref.TS-2a: 3x; A2*C2, I'; *; A. Narita 040214-01; Kaitsu, Kainan-cho, Tokushima Pref.TS-2b: 3x; A2A2A2, I; ; A. Narita 040214-02; —.TS-2c: 3x; A2A2A2, I; ; A. Narita 040214-03; —.TS-2d: 2x; A2A2, I; ; A. Narita 040214-04; —.TS-3: 3x; A2A2A2, I'; ; A. Narita 040221-01; Ohbi, Kainan-cho, Tokushima Pref.TS-4: 3x; A1*B1, II; *ß; A. Narita 040306-01; Furuya, Kaminaka-cho, Tokushima Pref.TS-5: 3x; A1A*A5, I'; ; A. Narita 040307-01; Kitogatani, Kainan-cho, Tokushima Pref.TS-6: 2x; A1A1, I; ; A. Narita 040320-01; Hohno, Aioi-cho, Tokushima Pref.TT-1: 3x; A1*B3, I'; *ß; Ebihara 041007-01; Ikkon-naru, Kotoura-cho, Tottori Pref.TT-2: 4x; A3B1C7*, III; ß*; Ebihara 041007-02; Imadaki Falls, Yurihama-cho, Tottori Pref.TT-3a: 4x; A3**C7, I; **; Ebihara 041007-03; Fudo Falls, Yurihama-cho, Tottori Pref.TT-3b: 4x; A3B1C7*, III; ß*; Ebihara 041007-04; —.TT-4: 3x; A1*B1, I'; *ß; Ebihara 041007-05; Inugami Shrine, Tottori-shi, Tottori Pref.TW-1: NA; C3C7, III; ;ob/;cb; S. Fujimoto 020908-01; Neidong Forest Area, Wulai, Taipei Co., TaiwanTW-2: 2x; C6C6, III; ; Ebihara 031116-09; Hunghoku Valley, Wulai, Taipei Co., Taiwan.TW-3: 2x; C3C10, III; ; Ebihara 031126-05; Mt. Laofo-shan, Pingtung Co., Taiwan.TW-4: 3x; A2A2A2, I'; ; Matsumoto T03-149; Kunlun Medical Plant Garden, Taoyuan Co., Taiwan.TW-5: NA; ;obC8/C9;cb, III; ;ob/;cb; T.A. Ranker 2065 ;obCOLO;cb; Yunsen waterfall trail, San Hsia, Taipei Co., Taiwan.TY-1: 3x; A2B1B3, II; ßß; M. Ohta 030722-01; Sakaue, Toga-mura, Toyama Pref.TY-2a: 2x; B2B2, II; ßß; M. Ohta 040824-01; Ohiwa, Kami-ichi-machi, Toyama Pref.TY-2b: 4x; A1**B3, II; *ß; M. Ohta 040929-2; —.TY-2c: 3x; A1*B2, II; *ß; M. Ohta 040929-3; —.TY-2d: 3x; A1*B3, I; *ß; M. Ohta 040929-4; —.WK-1: 3x; A1*C1, I'; *; M. Tanaka 040229-01; Taki, Hikigawa-cho, Wakayama Pref.WK-2a: 3x; A1A*A6, I; ; S. Fujimoto s.n.; Kuwanoki-tani, Shingu-shi, Wakayama Pref.WK-2b: 2x; A1A6, I'; ; S. Fujimoto s.n.; —.WK-3: 3x; A2A6C2, I; ; M. Tanaka 040620-01; Koyazaka, Shingu-shi, Wakayama Pref.YG-1a: 3x; A2*B1, II; *ß; Ebihara 040316-01; Hosenji, Yamaguchi-shi, Yamaguchi Pref.YG-1b: 4x; A2*B1B3, II; *ßß; Ebihara 040316-02; —.YG-1c: 3x; A2*B1, I'; *ß; Ebihara 040316-03; —.YG-1d: 3x; A2*B3, II; *ß; Ebihara 040316-04; —. APPENDIX 2.
GenBank accession numbers of rbcL and GapCp sequences
FOOTNOTES
1 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 (e-mail: ebiharak{at}dolphin.c.u-tokyo.ac.jp
)
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