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0 Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University, Mozume-cho, Muko, 617-0001 Japan
Received for publication May 13, 1999. Accepted for publication August 9, 1999.
ABSTRACT
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 (1) either F. statice or F. leptopodum was derived from the ancestor more than once, (2) 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: Fagopyrum • intraspecific phylogeny • noncoding cpDNA region • Polygonaceae • speciation • wild buckwheat species
The genus Fagopyrum (Polygonaceae) consists of ~16 species, some of which have been discovered recently (Ohnishi, 1998
; Ohsako and Ohnishi, 1998
). Classifications of Fagopyrum have been proposed mainly in relation to the tribe Polygoneae (Meissner, 1826
; Gross, 1913
; Stewart, 1930
; Hedberg, 1946
; Haraldson, 1978
; Ronse Decraene and Akeroyd, 1988
). 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, 1913
) or Persicaria (Ronse Decraene and Akeroyd, 1988
).
Phylogenetic relationships among Fagopyrum species have recently been investigated using molecular data such as isozyme variation (Ohnishi and Matsuoka, 1996
), restricted fragment length polymorphism (RFLP) variation in cpDNA (Ohnishi and Matsuoka, 1996
) and nucleotide sequence variation in cpDNA and nuclear DNA (Yasui and Ohnishi, 1998a, 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 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, 1998a, 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, 1998
; Ohsako and Ohnishi, 1998
).
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, 1987
; Clegg, 1993
). 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., 1997
; Fujii et al., 1997
). The trnK intron consists of the matK gene coding region (~1550 base pairs [bp]) and two noncoding regions on both sides of the matK region (Neuhaus and Link, 1987
; Johnson and Soltis, 1995
). 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 include a promoter-like sequence, which is usually found in other tRNA genes (Wakasugi et al., 1986
).
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 1). The accessions were chosen so that they covered the known range (Ohnishi, 1998
) of each species (Fig. 1). Voucher specimens of all used plant materials were deposited in the herbarium of the Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University.
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). Polymerase chain reaction (PCR) amplifications of the two regions, the trnK intron and the intergenic spacer between trnC and rpoB, were performed using a PJ2000 Thermal Cycler (Perkin Elmer, Norwalk, Connecticut, USA) with standard protocol. The thermal conditions were as follows: 30 cycles of 1 min at 96°C for denature, 2 min at 50°C for annealing, and 3 min at 72°C for polymerization with a final extension of 7 min at 72°C. Primer pairs used for the PCR were trnK-3914F dicot (5'-GGG GTT GCT AAC TCA ACG G-3') and trnK-2R (5'-AAC TAG TCG GAT GGA GTA G-3') for the trnK intron (Johnson and Soltis, 1995
) 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 101 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 3.1.1. (Swofford, 1993
) 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 100 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, 1985
) was performed with 1000 replications by heuristic searches with SIMPLE stepwise addition and TBR branch-swapping options. Decay indices (DI) for relative branch support (Bremer, 1988
) 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, 1983
; Mason-Gamer and Kellogg, 1996
) was applied. The number of steps of each character under topological constraint was calculated with MacClade 3.07 (Maddison and Maddison, 1993
). 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 databases under the accession numbers GBAN-AB026299 to GBAN-AB026335 and GBAN-AB026736 to GBAN-AB026772. Sequence variability of the two cpDNA regions is summarized in Table 2. 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.
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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, 1969
) of 0.960 and retention index (RI: Farris, 1989
) 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 = 0.920 and RI = 0.945 were obtained by phylogenetic analysis of the trnC-rpoB spacer sequences. Fig. 4 shows one of them.
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To assess the topological difference between the phylogeny from the two separate data statistically, the WSR test was performed. For all 108 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 = 0.933 and RI = 0.952) 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 C97106. 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.
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DISCUSSION
Nucleotide sequence diversity in noncoding regions of cpDNA
In total 104 nucleotide substitutions were found in the two noncoding cpDNA regions (combined length 2.3 kbp) that were analyzed. Among these variable nucleotides, 54 were phylogenetically informative. Thirty-four structural mutations were also found within the regions studied and 22 of them were potentially phylogenetically informative. The retention index for indels was as high as that for nucleotide substitutions (Table 2), 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. 2). This finding suggests that formation of stem-loop structures and recombination in the stems are responsible for the inversions (Sang, Crawford, and Stuessy, 1997
). The change in inversion types in the trnK intron was much more homoplastic (CI = 0.166) 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., rpl16 intron of bamboo: Kelchner and Wendel, 1996
; psbA-trnH spacer of peony: Sang, Crawford, and Stuessy, 1997
). In contrast, phylogenetic relationships in Figs. 4 and 5 required only a single occurrence of the inversion in the trnC-rpoB spacer (Table 1). This inversion seems to be correlated with length change (7–10 bp) in the inverted repeats (Fig. 2).
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 (1996)
, Yasui and Ohnishi (1998a, b)
, and Ohsako and Ohnishi (1998)
. 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 C97106 is heavy pubescence on the stems and stipules (Ohnishi and Matsuoka, 1996
; Ohsako and Ohnishi, 1998
). Fagopyrum pleioramosum, F. macrocarpum, and F. callianthum share heterostylous self-compatibility 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; 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 (319 steps) over that with no constraint (313 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, C9752, C9755, and C9756; cordate leaf blade, long petiole 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.
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Fagopyrum gracilipes, F. capillatum, F. rubifolium, and an unidentified accession C97106 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 1 and Fig. 1). 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 contribute to the rapid dispersal of F. gracilipes over a wide range. A F. gracilipes-like accession C97106 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 C97106 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, 1998
). 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, C97107; 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 = 1. Based on the combined tree, the F. pleioramosum-F. macrocarpum-F. callianthum clade and C97107 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 C97107 in the trnK intron trees (Fig. 3). Polyphyly of F. urophyllum is probably due to ancestral polymorphism (Knox and Palmer, 1995
).
Fagopyrum lineare is a sister to a small clade with two F. urophyllum accessions, though it has a number of autoapomorphies—20 nucleotide substitutions in the both regions. A previous phylogenetic study using nucleotide sequences in another cpDNA region (Yasui and Ohnishi, 1998a
) 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, 1989
) 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.
FOOTNOTES
1 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. 
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