INTRA- AND INTERSPECIFIC PHYLOGENY OF WILD FAGOPYRUM (POLYGONACEAE) SPECIES BASED ON

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1 American Journal of Botany 87(4): INTRA- AND INTERSPECIFIC PHYLOGENY OF WILD FAGOPYRUM (POLYGONACEAE) SPECIES BASED ON NUCLEOTIDE SEQUENCES OF NONCODING REGIONS IN CHLOROPLAST DNA TAKANORI OHSAKO AND OHMI OHNISHI Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University, Mozume-cho, Muko, Japan The intra- and interspecific phylogeny of Fagopyrum (Polygonaceae) species was studied using nucleotide sequence data from two noncoding regions in chloroplast DNA, the trnk (UUU) intron and the trnc (GCA)-rpoB spacer. Thirty-seven accessions of ten species and two unidentified samples in the urophyllum group of Fagopyrum were analyzed. Both of the studied regions showed high variability, including nucleotide substitutions, insertion/deletions, and inversions. Separate parsimony analyses of the two regions generated phylogenies that were largely consistent with each other. A single most parsimonious tree derived from the combined data of the two regions suggested that () either F. statice or F. leptopodum was derived from the ancestor more than once, () F. gracilipes, a tetraploid species, has recently been derived from diploid ancestor and rapidly spread out to its present distribution areas, and (3) F. pleioramosum, F. macrocarpum, and F. callianthum, three newly discovered species endemic to the upper Min River valley, differentiated from their common ancestral species in the present distribution area. Key words: species. Fagopyrum; intraspecific phylogeny; noncoding cpdna region; Polygonaceae; speciation; wild buckwheat The genus Fagopyrum (Polygonaceae) consists of 6 species, some of which have been discovered recently (Ohnishi, 998; Ohsako and Ohnishi, 998). Classifications of Fagopyrum have been proposed mainly in relation to the tribe Polygoneae (Meissner, 86; Gross, 93; Stewart, 930; Hedberg, 946; Haraldson, 978; Ronse Decraene and Akeroyd, 988). Most studies have concluded that Fagopyrum lies at the basal position of the tribe, and some authors have claimed that Fagopyrum is closely related to Fallopia (Gross, 93) or Persicaria (Ronse Decraene and Akeroyd, 988). Phylogenetic relationships among Fagopyrum species have recently been investigated using molecular data such as isozyme variation (Ohnishi and Matsuoka, 996), restricted fragment length polymorphism (RFLP) variation in cpdna (Ohnishi and Matsuoka, 996) and nucleotide sequence variation in cpdna and nuclear DNA (Yasui and Ohnishi, 998a, b). These studies indicated that Fagopyrum is divided into two major phylogenetic groups, the cymosum group and the urophyllum group. The cymosum group comprises two cultivated species, F. esculentum (common buckwheat) and F. tataricum (Tartary buckwheat), and two wild species. The urophyllum group includes ten wild species. Interspecific relationships among Fagopyrum species Manuscript received 3 May 999; revision accepted 9 August 999. The authors thank Dr. Yasuo Yasui for his kindness in providing the total DNA sample of F. cymosum and Prof. Michael J. Simmons, University of Minnesota, for reading the manuscript, correcting the English, and making numerous useful suggestions. This research was partially supported by JSPS Research Fellowships for Young Scientists to TO. Contribution from Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University Number 95. Author for correspondence. 573 have been clarified by these studies, but several issues on intraspecific differentiation remain to be resolved. Fagopyrum statice, an outcrossing perennial species, has been shown to be paraphyletic to an annual species, F. leptopodum, in a molecular phylogeny (Yasui and Ohnishi, 998a, b). The origin and intraspecific differentiation of F. gracilipes, a tetraploid self-fertilizing species, have not yet been clarified. Intra- and interspecific differentiation of three recently discovered species, F. pleioramosum, F. macrocarpum, and F. callianthum, is also an unsolved problem. These species were recently discovered in the upper Min River valley of Sichuan Province in China (Ohnishi, 998; Ohsako and Ohnishi, 998). In the present study, using multiple samples for each species, we investigated inter- and intraspecific phylogenetic relationships among the species of the urophyllum group based on nucleotide sequences of two noncoding regions in the cpdna, i.e., the trnk (UUU) gene intron and an intergenic spacer between the trnc (GCA) and rpob genes. The nucleotide substitution rate of plant cpdna is lower than that of nuclear DNA (Wolfe, Li, and Sharp, 987; Clegg, 993). However, nucleotide variation of noncoding regions in cpdna can be used for phylogenetic analyses at the intraspecific level because of their considerably higher evolutionary rate than gene-encoding regions (Dumolin-Lapégue et al., 997; Fujii et al., 997). The trnk intron consists of the matk gene coding region ( 550 base pairs [bp]) and two noncoding regions on both sides of the matk region (Neuhaus and Link, 987; Johnson and Soltis, 995). We investigated the entire 5 noncoding region and about one-fifth of the matk coding region from its 5 end. The trnc-rpob spacer includes the 5 flanking regions of the two genes on opposite strands. The 5 flanking region of trnc does not

2 574 AMERICAN JOURNAL OF BOTANY [Vol. 87 TABLE. Plant materials investigated in this study. Types of inversions found in the noncoding cpdna regions are indicated. Species Accession Fagopyrum leptopodum Diels C9077 C9080 C9466 C9467 C9553 C9554 C9767 C9768 C9776 C9780 F. statice (Lévl.) Gross C9469 C9470 C975 C9755 C9756 F. gracilipes Hemsl. C9096 C9453 C9575 F. capillatum Ohnishi F. rubifolium Ohsako et Ohnishi F. pleioramosum Ohnishi F. macrocarpum Ohsako et Ohnishi F. callianthum Ohnishi F. urophyllum (Bur. et. Franch.) Gross F. lineare Sam. Unidentified accessions C9798 C9700 C970 C949 C9589 C9566 C9567 C9565 C9586 C960 C956 C9564 C9443 C9444 C9759 C9758 C9706 C9707 Location (village, district, province) Heqin, Heqin, Yunnan Binchuan, Binchuan, Yunnan Muli, Muli, Sichuan Wushihe, Hanyuan, Sichuan Yongsheng, Yongsheng, Yunnan Shimen, Shimen, Sichuan Nishi, Zhongdian, Yunnan Luqian, Luqian, Yunnan Baoshan, Lijiang, Yunnan Dali, Dali, Yunnan Yuanmou, Yuanmou, Yunnan Chengjiang, Chengjiang, Sichuan Tonghai, Tonghai, Yunnan Kaiyuan, Kaiyuan, Yunnan Gejiu, Gejiu, Yunnan Kunming, Kunming, Yunnan Yanyuan, Yanyuan, Sichuan Zhongdian, Zhongdian, Yunnan Heqin, Heqin, Yunnan Laoyinxian, Baoshan, Yunnan Changchong, Wuding, Yunnan Jinan, Lijiang, Yunnan Maerkan, Maerkan, Sichuan Yanmen, Wenchuan, Sichuan Taoguanxian, Wenchuan, Sichuan Maowen, Maowen, Sichuan Putou, Lixian, Sichuan Maerkan, Maerkan, Sichuan Guanbao, Lixian, Sichuan Yanmen, Wenchuan, Sichuan Dali, Dali, Yunnan Kunming, Kunming, Yunnan Menning, Menning, Sichuan Binchuan, Binchuan, Yunnan Baoshan, Lijiang, Yunnan Benzilan, Zhongdian, Yunnan trnk intron Inversion type Outgroup F. cymosum Meisn. C9550 Ninglang, Ninglang, Yunnan trnc-rpob spacer include a promoter-like sequence, which is usually found in other trna genes (Wakasugi et al., 986). We will show that sequences of the two noncoding cpdna regions provide enough phylogenetic information to clarify the intraspecific differentiation of the Fagopyrum species. MATERIALS AND METHODS Plant materials Thirty-six accessions of ten species in the urophyllum group of Fagopyrum and an outgroup accession of F. cymosum Meisn. were used (Table ). The accessions were chosen so that they covered the known range (Ohnishi, 998) of each species (Fig. ). Voucher specimens of all used plant materials were deposited in the herbarium of the Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University. PCR amplification and direct sequencing Total DNA was extracted by the cetyl trimethyl ammonium bromide (CTAB) method (Zimmer, Rivin, and Walbot, 98). Polymerase chain reaction (PCR) amplifications of the two regions, the trnk intron and the intergenic spacer between trnc and rpob, were performed using a PJ000 Thermal Cycler (Perkin Elmer, Norwalk, Connecticut, USA) with standard protocol. The thermal conditions were as follows: 30 cycles of min at 96 C for denature, min at 50 C for annealing, and 3 min at 7 C for polymerization with a final extension of 7 min at 7 C. Primer pairs used for the PCR were trnk-394f dicot (5 -GGG GTT GCT AAC TCA ACG G-3 ) and trnk-r (5 -AAC TAG TCG GAT GGA GTA G-3 ) for the trnk intron (Johnson and Soltis, 995) and trnc5 -R (5 -TGC CTT ACC ACT CGG CCA T-3 ) and rpob5 -R (5 -GTA GAT ATT CCC TCA TTT CC-3 ) for the trnc-rpob spacer. PCR products were purified with the Geneclean II kit (BIO 0 Inc., La Jolla, California, USA) and used as the templates for subsequent cycle sequencing. Cycle sequencing was performed on a 373A DNA sequencer with the DiDeoxy Cycle Sequence kit (Applied Biosystems, Foster, California, USA) using primers situated at 300 bp intervals. DNA sequence analysis and phylogenetic analysis DNA sequences were aligned manually. Nucleotides involved in inversions found in both noncoding regions were substituted for their complementary sequences. Nucleotide substitutions within the inverted regions were included in the data matrix, and inversions were scored independently as phylogenetic characters. Potentially informative indels were scored and added to the data matrix. When informative nucleotide substitutions were within insertions, they were included in the data matrix; the character states of taxa with a deletion were scored as unknown. Phylogenetic analyses by most parsimonious method were performed using PAUP 3... (Swofford, 993) for the two cpdna regions separately and also for the combined data set. A heuristic search was performed for each data set, with RANDOM stepwise addition with 00

3 April 000] OHSAKO AND OHNISHI MOLECULAR PHYLOGENY OF FAGOPYRUM 575 Fig.. Sampling location of the accessions for each species in the urophyllum group. replications and TBR branch-swapping algorithm options. ACCTRAN optimization was selected. The COLLAPSE zero-length branches and MULPARS options were in effect for each search. For the combined tree, bootstrap analysis for the reliability of each branch (Felsenstein, 985) was performed with 000 replications by heuristic searches with SIMPLE stepwise addition and TBR branch-swapping options. Decay indices (DI) for relative branch support (Bremer, 988) were calculated by reconstructing trees up to four steps longer than most parsimonious trees by heuristic searches. To assess significant difference between trnk intron and trnc-rpob spacer phylogenies, Wilcoxon signed-ranks (WSR) test (Templeton, 983; Mason-Gamer and Kellogg, 996) was applied. The number of steps of each character under topological constraint was calculated with MacClade 3.07 (Maddison and Maddison, 993). All of the most parsimonious trees of one data set were used as the constraint to the other data set. RESULTS Sequence analyses The complete nucleotide sequences are deposited in DDBJ/EMBL/GenBank data-

4 576 AMERICAN JOURNAL OF BOTANY [Vol. 87 TABLE. Characterization of cpdna regions of the urophyllum group. Regions trnk intron noncoding region matk coding region total trnc-rpob spacer total Length No. of variable sites a 5 (3) 8 () 43 (4) 6 (30) 04 (54) No. of indels a (6) 0 (0) (6) 0 (4) 3 (0) a Number of potentially informative characters in parentheses. b Average of values for informative characters in the combined analysis including an outgroup. RI of substitutions b RI of indels and inversion b bases under the accession numbers GBAN-AB0699 to GBAN-AB06335 and GBAN-AB06736 to GBAN- AB0677. Sequence variability of the two cpdna regions is summarized in Table. The prefix GBAN- has been added to link the online version of American Journal of Botany to GenBank but is not part of the actual accession number. The length of the trnk intron region ranged from 037 to 03 bp for 37 accessions. Except for the outgroup F. cymosum (059 bp), this length variation is due to variation of the 5 noncoding subregion, ranging from 707 to 773 bp. The part of the matk coding region investigated had a constant length of 330 bp for all ingroup accessions except 37 bp for F. cymosum. In the trnk intron region 43 nucleotide substitutions were detected among the ingroup accessions, and 4 of which (55.8%) were potentially informative. Twelve indels were detected in the ingroup. Eight indels were duplications, i.e., direct tandem repeats of short sequences. Six out of indels were potentially informative. However, two of six informative indels were related to a structurally hypervariable region, and it is difficult to decide the order and directions among character states; hence they were not included in the data matrix. Consequently, four potentially informative indels were scored and added to the data matrix. In addition to indels, an inversion of nucleotides was detected in the noncoding region (Fig. ). The inversion type of each accession is shown in Table. The length of the inversion Fig.. Inversion types in the noncoding regions of cpdna. A representative sequence of each type is shown. Nucleotide substitutions and indels in the regions are omitted. Arrows under the nucleotides indicate the inverted repeats. The inversion types of each accession are shown in Table.

5 April 000] OHSAKO AND OHNISHI MOLECULAR PHYLOGENY OF FAGOPYRUM 577 varied from 3 to 78 bp among the accessions. This variation is mostly due to duplications. The inversion was bordered by a pair of inverted repeat sequences 9 bp long. Two inversion types were segregating within a species in five out of seven species. The length of nucleotide sequences in the trnc-rpob spacer ranged from 03 to 30 bp for 36 ingroup accessions. The sequence of the outgroup (F. cymosum) is 8 bp, longer than those of the ingroup accessions. Sixty-one nucleotide substitutions were detected among the ingroup in the trnc-rpob spacer. Of these substitutions, 30 (49.3%) were potentially informative. Twenty indels were found in the ingroup and 3 were duplications. Fourteen out of 0 indels were potentially informative. Two of them were mutations in the number of poly-a and poly-t nucleotides, respectively. Because they were difficult to score, they were not included in the data matrix. An inversion was also found in the trncrpob spacer (Fig. ). The length of the inverted nucleotide sequence was bp, which was much longer than that found in the trnk intron. The inverted sequences were situated between inverted repeat sequences 7 or 0 bp long. Phylogenetic analyses For the phylogenetic analysis of the trnk intron, the inversion was excluded from the data matrix because it shows higher homoplasy (six independent changes) than any other character (no homoplasy within the ingroup) in the most parsimonious trees. The analysis of the trnk intron data without the inversion resulted in two most parsimonious trees with a consistency index (CI: Kluge and Farris, 969) of and retention index (RI: Farris, 989) of 0.970, one of which is shown in Fig. 3. The topology of the two trees is consistent with the strict consensus of ten trees produced with the inversion in the data set, except that the former did not support a clade of two F. gracilipes accessions and F. capillatum. Twenty-seven most parsimonious trees with CI 0.90 and RI were obtained by phylogenetic analysis of the trnc-rpob spacer sequences. Fig. 4 shows one of them. The trnk intron phylogeny and the trnc-rpob spacer phylogeny are mostly consistent. In both phylogenies, three groups, each comprising two or three species, were recognized. They were the F. leptopodum-f. statice group, the F. gracilipes-f. capillatum-f. rubifolium group, and the F. pleioramosum-f. macrocarpum-f. callianthum group. The most remarkable difference between the two phylogenies lies in the relationships among F. urophyllum, F. lineare, and other species. Fagopyrum urophyllum and F. lineare form a clade with F. pleioramosum, F. macrocarpum, F. callianthum, and C9707 in the trnk intron phylogeny. In the trnc-rpob spacer phylogeny, the clade consisting of the two F. urophyllum accessions and F. lineare is a sister of a large clade of five species, F. leptopodum, F. statice, F. gracilipes, F. capillatum, and F. rubifolium. This difference is due to a shift in branching at the base of the tree. To assess the topological difference between the phylogeny from the two separate data statistically, the WSR test was performed. For all 08 comparisons between the most parsimonious and constraint trees, two-tailed WSR tests detect no significant difference at the 5% level. To obtain greater resolution, a phylogenetic analysis based on the combined data of the two regions was performed. The inversion in the trnk intron was excluded from the data set. A single most parsimonious tree (CI and RI 0.95) was derived from the combined analysis (Fig. 5). The phylogeny of the combined data is essentially the same as those of the separate data. Moreover, some unresolved polytomies in the separate analyses were resolved: for example, the intraspecific relationships in F. leptopodum and monophyly of F. rubifolium (C9589) and an unidentified accession C9706. The rooting of the tree is the same as that in trnc-rpob spacer trees, i.e., a clade consisting of two F. urophyllum accessions and F. lineare is a sister group to a clade consisting of F. leptopodum, F. statice, F. gracilipes, F. capillatum, and F. rubifolium. Significant intraspecific differentiation was observed in three species, F. leptopodum, F. statice, and F. urophyllum (Fig. 5). In F. leptopodum, two major phylogenetic groups were recognized. One consists of two accessions, C9466 and C9767, and the other is a group of the remaining eight accessions. Among five F. statice accessions, C9469 (Yuanmou, Yunnan) was included in a clade with F. leptopodum. The other four accessions form another clade sister to F. leptopodum. Three accessions of F. urophyllum were separated into two groups, C9759-C9443 and C9444, which were highly differentiated and polyphyletic. Accession C9444 was sister to all the other accessions of the ingroup, although this relationship was poorly supported by bootstrap (37%) and decay () values. DISCUSSION Nucleotide sequence diversity in noncoding regions of cpdna In total 04 nucleotide substitutions were found in the two noncoding cpdna regions (combined length.3 kbp) that were analyzed. Among these variable nucleotides, 54 were phylogenetically informative. Thirtyfour structural mutations were also found within the regions studied and of them were potentially phylogenetically informative. The retention index for indels was as high as that for nucleotide substitutions (Table ), indicating that indels in noncoding regions of cpdna are as useful as nucleotide substitutions in phylogenetic analyses. Both of two inversions found in the noncoding regions were bordered by inverted repeat sequences (Fig. ). This finding suggests that formation of stem-loop structures and recombination in the stems are responsible for the inversions (Sang, Crawford, and Stuessy, 997). The change in inversion types in the trnk intron was much more homoplastic (CI 0.66) than other characters. This result is consistent with previous reports that inversions in noncoding regions of cpdna in other plants are more variable than nucleotide substitutions (e.g., rpl6 intron of bamboo: Kelchner and Wendel, 996; psbatrnh spacer of peony: Sang, Crawford, and Stuessy, 997). In contrast, phylogenetic relationships in Figs. 4 and 5 required only a single occurrence of the inversion in the trnc-rpob spacer (Table ). This inversion seems to be correlated with length change (7 0 bp) in the inverted repeats (Fig. ).

6 578 AMERICAN JOURNAL OF BOTANY [Vol. 87 Fig. 3. One of the two most parsimonious trees reconstructed by the nucleotide sequence data of the trnk intron. Length 5 steps; CI 0.960; RI The length of each branch is shown above the branch. The branch with broken line indicates the clade that collapses in the strict consensus of two trees. Interspecific phylogenetic relationships Because the combined phylogeny has highest resolvability and is most reliable, it provides the best information to discuss interspecific relationships, speciation, and geographical differentiation. Three groups were recognized in the urophyllum group of Fagopyrum: the F. leptopodum-f. statice group, the F. gracilipes-f. capillatum-f. rubifolium group, and the F. pleioramosum-f. macrocarpum-f. callianthum group. Fagopyrum urophyllum was basal to all other species. These relationships are consistent with the results of previous molecular systematic studies by Ohnishi and Matsuoka (996), Yasui and Ohnishi (998a, b), and Ohsako and Ohnishi (998). Members of each of the three groups share several morphological characters. Lustrous hairless leaf surface is a synapomorphy of F. leptopodum and F. statice. These two species also share characters such as wax on the stem, leafless flower-bearing branches, and equal size of upper and lower perianths, although they are not synapomorphies because these characters have evolved in parallel in other species. The character shared by F. gracilipes, F. capillatum, F. rubifolium, and C9706 is heavy pubescence on the stems and stipules (Ohnishi and Matsuoka, 996; Ohsako and Ohnishi, 998). Fagopyrum pleioramosum, F. macrocarpum, and F. callianthum share heterostylous selfcompatibility and larger achenes than the other two groups. However, the achenes of F. urophyllum are as large as those of F. macrocarpum and F. callianthum;

7 April 000] OHSAKO AND OHNISHI MOLECULAR PHYLOGENY OF FAGOPYRUM 579 Fig. 4. One of the 7 most parsimonious trees reconstructed by the nucleotide sequence data of the trnc-rpob spacer. Length 87 steps; CI 0.90; RI The length of each branch is shown above the branch. The branches with broken lines indicate the clades that collapse in the strict consensus of 7 trees. thus, large achenes might be plesiomorphic in the urophyllum group. Fagopyrum lineare is very close to F. urophyllum in the molecular phylogeny, but these two species are morphologically quite different. Fagopyrum lineare is rather similar to F. leptopodum in such characters as slender branches, small white flowers, and small achenes. The apparent resemblance between F. lineare and F. leptopodum might be due to parallelism. Indeed, the parsimonious analysis of the combined data with the constraint of the monophyly of F. leptopodum, F. statice, and F. lineare resulted in six steps excess of the tree length (39 steps) over that with no constraint (33 steps), indicating the distant relationship between the F. leptopodum-f. statice group and F. lineare. Fagopyrum lineare might have accumulated autoapomorphic characters at both the morphological and molecular levels since divergence from its ancestor. Speciation pattern and geographic differentiation Accessions of two closely related species, F. leptopodum and F. statice, formed a monophyletic group. Unless hybridization between the two species is assumed, multiple divergence of one species from the ancestral species must be considered because both species are nonmonophyletic (Fig. 5). An hypothesis of the multiple speciation of F. statice is schematically shown in Fig. 6 by arrows. This hypothesis is supported by a clear morphological difference between the primary lineage (the clade of C9470, C975, C9755, and C9756; cordate leaf blade, long pet-

8 580 AMERICAN JOURNAL OF BOTANY [Vol. 87 Fig. 5. A single most parsimonious tree reconstructed by the combined data of the two nucleotide sequences. Length 33 steps; CI 0.933; RI The length of each branch is shown above the branch. Bootstrap and decay values are shown below the branch. iole of cotyledons) and the secondarily differentiated lineage (C9469; sagittate leaf blade, short petiole of cotyledons). The morphological and geographical discrimination of the accession C9469 from other F. statice accessions suggests another hypothesis that the accession C9469 is a hybrid of the two species. If this is true, only a single step of speciation is required. This issue might be clarified by comparing the cpdna phylogeny with a nuclear DNA phylogeny, which is now under investigation. Phylogenetic differentiation of F. leptopodum is not completely associated with geographical distribution. The northernmost two accessions (C9467 and C9554 from southwestern Sichuan) and the easternmost accession (9768 from Luqian) formed a clade with the accession from the central part of the distribution (C9553 from Yongsheng). This is probably due to a recent long-distance dispersal from Yongsheng to southwestern Sichuan and to Luqian (Fig. 6). Except for these accessions, geographical structuring is clear, with northern (small) and southern (large) lineages as shown in Fig. 6. The center of the distribution of F. leptopodum is the northwestern part of Yunnan Province. The initial differentiation of northern and southern lineages (indicated by circles in Fig. 6) at the center was followed by further differentiation within each of the two lineages. Compared to F. leptopodum, intraspecific differentiation of F. statice was less obvious. The separation of the northern accession C9469 from other southern accessions might not be due to geographical differentiation but to a different origin as discussed above. Fagopyrum gracilipes, F. capillatum, F. rubifolium, and an unidentified accession C9706 formed a clade in the combined tree (Fig. 5). No sequence variation except for an inversion in the trnk intron was detected among the six accessions of F. gracilipes in spite of sampling from wide geographical area (see Table and Fig. ). Fagopyrum gracilipes, a tetraploid self-fertilizing species, is sister to F. capillatum, a diploid outcrossing species, and only two nucleotide substitutions have occurred in the regions studied since the separation of F. gracilipes from its hypothetical ancestor (Fig. 5). This result might indicate that F. gracilipes has recently originated from the diploid outcrossing ancestor. Fagopyrum gracilipes has a weedy habit and flourishes in disturbed environments such as farm fields. These characteristics might

9 April 000] OHSAKO AND OHNISHI MOLECULAR PHYLOGENY OF FAGOPYRUM 58 Fig. 6. Geographic differentiation of F. leptopodum and F. statice suggested by cpdna phylogeny. Circles indicate three major lineages and arrows indicate the speciation events. contribute to the rapid dispersal of F. gracilipes over a wide range. A F. gracilipes-like accession C9706 that is heterostylous and outcrossing was distantly related to F. gracilipes and showed a sister relationship to another self-fertilizing species, F. rubifolium. This result suggests that the accession C9706 should be separated from F. gracilipes as a new species. This is an issue that will be discussed in more detail elsewhere. The three species distributed in the upper Min River valley formed a robust clade (Fig. 5). Little variation was seen among cpdna sequences of the five accessions of F. pleioramosum and F. macrocarpum, which is consistent with the high genetic similarity between these species revealed by isozyme analysis (Ohsako and Ohnishi, 998). Intraspecific variation of F. callianthum was also low. The lack of intraspecific variation in these species might be due to the restricted distribution to a narrow area and the small population sizes. The F. pleioramosum-f. macrocarpum complex and F. callianthum are monophyletic but well differentiated from each other and have a limited common distribution area. This phylogeographical pattern suggests that these species have differentiated in the present distribution area and have remained there without expanding their distribution. The sister of the F. pleioramosum-f. macrocarpum-f. callianthum clade is an unknown accession, C9707; however this accession is too far differentiated from the clade to be a direct ancestor of the three species and their sister relationship is weakly supported with a low decay value of DI. Based on the combined tree, the F. pleioramosum-f. macrocarpum-f. callianthum clade and C9707 seem to have been derived during the early differentiation of the urophyllum group. Fagopyrum urophyllum was polyphyletic in all phylogenies (Figs. 3 5). In the combined tree (Fig. 5), F. urophyllum is sister to the entire urophyllum group, whereas F. urophyllum is the sister of the group consisting of F. pleioramosum, F. macrocarpum, F. callianthum, and C9707 in the trnk intron trees (Fig. 3). Polyphyly of F. urophyllum is probably due to ancestral polymorphism (Knox and Palmer, 995). Fagopyrum lineare is a sister to a small clade with two F. urophyllum accessions, though it has a number of autoapomorphies 0 nucleotide substitutions in the both regions. A previous phylogenetic study using nucleotide sequences in another cpdna region (Yasui and Ohnishi, 998a) has also shown the sister relationship between F. lineare and the F. urophyllum accession from Dali. To confirm the origin of F. lineare, more samples of F. lineare and F. urophyllum must be collected and analyzed. The preceding discussion depends on the accuracy of the phylogeny inferred from cpdna sequence variability (Figs. 3 5). However, we must note that gene trees often differ from species trees or population trees because of various factors such as lineage sorting (Takahata, 989) and hybridization. In the present study two different data sets derived from the same genome were mostly consistent, and they were combined for higher resolution. A more conservative and reliable approach would be to obtain the consensus of two separate analyses, but this method gives very limited phylogenetic resolution. Additional data from the nuclear genome might help to resolve the intraspecific phylogeny and to reduce the discordance between gene trees and species/population trees. LITERATURE CITED BREMER, K The limits of amino acid sequence data in angiosperm phylogenetic reconstruction. Evolution 4: CLEGG, M. T Chloroplast gene sequences and the study of plant evolution. Proceedings of the National Academy of Sciences, USA 90: DUMOLIN-LAPÉGUE, S., B. DEMESURE, S.FINESCHI, V.LE CORRE, AND R. J. PETIT Phylogeographic structure of white oaks throughout the European continent. Genetics 46: FARRIS, J. S The retention index and the rescaled consistency index. Cladistics 5: FELSENSTEIN, J Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: FUJII, N., K. UEDA, Y. WATANO, AND T. SHIMIZU Intraspecific sequence variation of chloroplast DNA in Pedicularis chamissonis Steven (Scrophulariaceae) and geographic structuring of the Japanese Alpine plants. Journal of Plant Research 0: GROSS, H. 93. Remarques sur les Polygonées de l Asie Orientale. Bulletin de Géographie Botanique 3: 7 3. HARALDSON, K Anatomy and taxonomy in Polygonaceae subfam. Polygonoideae Meissn. emend. Jaretzky. Symbolae Botanicae Upsalienses : 95. HEDBERG, O Pollen morphology in the genus Polygonum L. s.

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