Genes Genet. Syst. (2002) 77, p. 399–408 Two new Fagopyrum () species, F. gracilipedoides and F. jinshaense from Yunnan, China

Takanori Ohsako1, Kyoko Yamane2, and Ohmi Ohnishi2* 1Faculty of Engineering, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto 603-8555, Japan 2Plant Germ-Plasm Institute, Graduate School of Agriculture, Kyoto University, Nakajo 1, Mozume-cho, Muko city 617-0001, Japan

(Received 9 September 2002, accepted 29 October 2002)

Two new Fagopyrum (Polygonaceae) species, F. gracilipedoides and F. jin- shaense, are described. Fagopyrum gracilipedoides was only found at Baoshan village, Lijiang district, Yunnan province, China. This species resembles the weedy species, F. gracilipes in morphology but they differ in both mating system and ploidy level; F. gracilipedoides is a heterostylous self-incompatible diploid species (2n = 16), whereas F. gracilipes is a self-compatible tetraploid species (2n = 32). Fagopyrum jinshaense was found along the Jinsha River valley near the border of Sichuan, Yunnan, and Tibet in China. Fagopyrum jinshaense is morphologically similar to F. gilesii, but they differ in the morphology of their . Fagopyrum jinshaense has long spike-like inflorescences, whereas F. gilesii has a compact head-like one. Both F. jinshaense and F. gilesii are self- incompatible with heterostylous flowers and are diploid. Interspecific crosses between the two new species and already known species in the urophyllum group of Fagopyrum resulted in the production of self- and cross-sterile hybrids in the species combinations attempted. These results indicated that both new species are reproductively isolated from other Fagopyrum species. Molecular phyloge- netic analysis based on the nucleotide sequence of the trnK gene intron in chloro- plast DNA revealed the following points: 1) F. gracilipedoides, F. rubifolium and F. gracilipes / F. capillatum showed a trifurcating relationship and 2) F. jinshaense formed a clade with F. pleioramosum, F. callanthum, and F. macrocarpum, and was only distantly related to F. gilesii. In the clade of F. capillatum, F. gracilipes, F. gracilipedoides and F. rubifolium, the breakdown of self-incompatibility accom- panying genome duplication seems to have occurred twice independently.

b). Phylogenetic relationships in the Fagopyrum, INTRODUCTION including these new species, have been investigated Wild species of the genus Fagopyrum, family Polygo- based on allozyme (Ohnishi and Matsuoka, 1996), nuclear naceae, have been found growing in the Sichuan and Yun- DNA (Yasui and Ohnishi, 1998a) and chloroplast DNA nan provinces of China and in the areas around these (cpDNA) (Ohnishi and Matsuoka, 1996; Yasui and provinces. Seventeen wild species are known so far, in Ohnishi, 1998b; Ohsako and Ohnishi, 2000; Ohsako et al., addition to the two cultivated species, common buck- 2001) sequences. Yasui and Ohnishi (1998a, b) clarified wheat F. esculentum and Tartary F. tatari- that members of the genus are divided into two mono- cum. Among them, six wild species and the wild ancestor phyletic groups; the cymosum group and the urophyllum of cultivated common buckwheat were newly found and group. described in the last decade: F. homotropicum, F. capilla- In the present study two additional new species, F. gra- tum, F. pleioramosum, F. callianthum (Ohnishi 1998a), F. cilipedoides and F. jinshaense, are described. These new rubifolium, F. macrocarpum (Ohsako and Ohnishi, 1998), species were found in the northwestern part of Yunnan and F. esculentum ssp. ancestrale (Ohnishi, 1991, 1998a, province, China. Both new species have morphologically similar species in the urophyllum group. Differences in Edited by Yoshio Sano morphological characters, chromosome number and mat- * Corresponding author. E-mail: [email protected] ing systems were used as the criteria for judging the new

400 T. OHSAKO et al. collections to be actually distinct species. In addition, Observation of morphology and chromosome. interspecific crossing experiments were performed for morphological observation were grown in between the new species and already known Fagopyrum ceramic pots (a single in each pot) in a greenhouse species to confirm the validity for our definition of them of the Plant Germ-Plasm Institute, Graduate School of as new species. Phylogenetic relationships among these Agriculture, Kyoto University at Mozume-cho, Muko city, new species and already known Fagopyrum species were Japan. In addition to the two new species described surveyed using the nucleotide sequence of the intron of herein, three additional species, F. rubifolium, F. macro- the chloroplast trnK (UUU) gene that includes a part of carpum, and F. gilesii, were newly surveyed for their the matK gene. As shown below, phylogenetic relation- chromosome number in the present study. Their ships among new species, F. gracilipedoides and F. jin- achenes were soaked on wet filter paper in petri dishes shaense, and other species in the urophyllum group are and incubated at 25°C for germination. Root tips now established in the molecular phylogeny. (~10mm long) were removed from roots and were pre- treated in 0.002M 8-hydroxyquinoline for 3 hrs at 18°C. The tips were fixed in 3:1 ethanol-glacial acetic MATERIALS AND METHODS acid for 48 hours at room temperature, and then rinsed Plant Materials. Plant accessions used for observation well in distilled water. The fixed root tips were trans- of morphological characters, cross experiments, chromo- ferred to an enzymatic mixture (2% cellulase Onozuka some observation, and molecular phylogenetic analyses RS, Yakult, Tokyo, 1.5% macerozyme R200, Yakult, 0.3% are shown in Table 1. pectolyase Y23, Seishin Pharm. Co., Tokyo, 1mM EDTA,

Table 1. Plant materials used in the present study.

Species (Abbreviation) Accession Location Used for (village or town, Morphological Count of Cross Molecular district, province) observation chromosomes experiment systematics Fagopyrum urophyllum (Bur. et French) Gross (uro) C9443 Xiaguan, Dali, Yunnan ○○ C9444 Kunming, Kunming, Yunnan ○○ F. leptopodum Diels (lep) C9077 Heqing, Heqing, Yunnan ○○ C9080 Binchuan, Binchuan, Yunnan ○ C9466 Muli, Muli, Sichuan ○○ C9767 Nii, Zhongdian, Yunnan ○○ C9776 Baoshan, Lijiang, Yunnan ○○ C9780 Dali, Dali, Yunnan ○○ F. statice (Levl.) Gross (sta) C9469 Yuanmou, Yuanmou, Yunnan ○○○ C9470 Chengjian, Chengjian, Yunnan ○○○ F. lineare Sam. (lin) C9758 Binchuan, Binchuan, Yunnan ○○ C9463 Xianyun, Xianyun, Yunnan ○○ C9666 Qiaodian, Binchuan, Yunnan ○○ F. gracilipes Dammer (gra) C9096 Kunming, Kunming, Yunnan ○○ C9453 Yanyuan, Yanyuan, Yunnan ○○ C9820 Fengu, Lijiang, Yunnan ○○ F. capillatum Ohnishi (cap) C9149 Jinan, Lijiang, Yunnan ○○○ C9462 Guanmei, Yongsheng, Yunnan ○ F. rubifolium Ohsako et Ohnishi (rub) C9589 Maerkan, Maerkan, Sichuan ○○○○ F. pleioramosum Ohnishi (ple) C9567 Taoguanxian, Wenchuan, Sichuan ○○ F. macrocarpum Ohsako et Ohnishi (mac) C9565 Maowen, Maowen, Sichuan ○○○○ F. callianthum Ohnishi (cal) C9562 Guanbao, Lixian, Sichuan ○○ C9564 Yanmen, Wenchuan, Sichuan ○○ F. gilesii Hemsl. (gil) C9836 Foshan, Deqin, Yunnan ○○○○ F. cymosum Meisn. C9550 Ninglang, Ninglang, Yunnan ○ F. gracilipedoides (new species) C97106 Baoshan, Lijiang, Yunnan ○○○○ F. jinshaense (new species) C97107 Benzilan, Deqin, Yunnan ○○○○ New Fagopyrum species from Yunnan, China 401

Table 2. Percentage of seed set after crosses and germination rate of hybrid seeds. Cross combination Female parent Male parent Seed set1 Germination rate2 C97106 staC9469 0.0 (20) C97106 graC9453 63.6 (22) 7.1 (14) C97106 capC9149 20.0 (30) 83.3 (6) C97106 rubC9589 61.5 (26) 93.8 (16) C97106 macC9565 0.0 (25) C97106 calC9562 0.0 (34) C97106 linC9463 44.4 (18) 0.0 (8) C97106 gilC9836 0.0 (46) lepC9776 C97106 0.0 (23) staC9470 C97106 60.0 (30) 16.7 (18) graC9453 C97106 14.3 (7) 100.0 (1) capC9149 C97106 37.9 (29) 72.7 (11) macC9565 C97106 0.0 (9) calC9562 C97106 0.0 (19) C97107 gilC9836 19.2 (26) 0.0 (5) C97107 gilC9836 – 3.8 (26) C97107 gilC9836 – 25.0 (8) C97107 lepC9780 0.0 (22) C97107 staC9469 0.0 (20) C97107 staC9470 0.0 (22) C97107 graC9453 0.0 (20) C97107 capC9149 15.0 (20) 66.7 (3) C97107 macC9565 0.0 (19) C97107 calC9562 0.0 (21) C97107 linC9463 0.0 (20) gilC9836 C97107 – 0.0 (23) lepC9780 C97107 0.0 (22) lepC9776 C97107 0.0 (28) staC9470 C97107 27.3 (22) 0.0 (6) graC9453 C97107 0.0 (5) capC9149 C97107 90.5 (21) 0.0 (19) rubC9589 C97107 50.0 (8) 100.0 (4) macC9565 C97107 0.0 (20) calC9562 C97107 0.0 (22) linC9666 C97107 0.0 (18) gilC9836 lepC9780 0.0 (32) gilC9836 lepC9767 0.0 (20) gilC9836 staC9469 0.0 (33) gilC9836 linC9666 0.0 (33) lepC9780 gilC9836 0.0 (25) staC9470 gilC9836 0.0 (21) graC9820 gilC9836 0.0 (25) rubC9589 gilC9836 0.0 (15) macC9565 gilC9836 0.0 (29) linC9666 gilC9836 0.0 (15) 1 The number of flowers pollinated is shown in parentheses.

2 The number of seeds examined is shown in parentheses. No cross- and /or self- fertile hybrid was obtained in the present study. 402 T. OHSAKO et al. pH 4.2) at 37°C for 2 hrs for tissue softening. The root branch with 2000 replications by heuristic searches of the tips were then stained with 3% Giemsa solution for 10 MP analysis. Bremer support index (Bremer, 1988) was min. Chromosome numbers were counted on at least ten also calculated for each branch by reconstructing trees up individuals for each species. to three steps longer than most parsimonious trees (117 steps) using heuristic searches. Cross experiment. Each of the two new species F. gra- cilipedoides and F. jinshaense was reciprocally crossed RESULTS between most of the species in the urophyllum group. The combinations that were attempted are shown in Description of new species. Table 2. When the self-compatible species were used as Fagopyrum gracilipedoides Ohsako et Ohnishi, spec. nova the female parent, the flower buds were emasculated one Fagopyro gracilipedi Hermsl. et F. capillato Ohnishi sim- day before pollination or in the early morning prior to pol- ile, sed differt a priore autoincompatibilitate heterostyl- lination and the was covered with a paper ica, a secundo foliis, floribus et acheniis minoribus. bag. In combinations involving self-incompatible species Planta annua, heterostyliciter multiplicabilis, 20–50 cm. as the female parent, long-styled (pin) individuals were alta, a simili F. capillato 60–150 cm. alto humilior. pollinated with pollen from short-styled (thrum) or homo- Flores numerosi, parvi, perigonio albo. Lamina sagit- stylous individuals after emasculation of the female tata vel ovata. Caules, ochreae et folia, ut in F. gracili- parent. For the cross combination between F. gilesii and pedi, valde pubescentes. Achenia circa 3 mm. longa. F. jinshaense, four pairs of individuals were selected and Numerus chromosomatum: 2n = 16. crossed with each other. One pair was used to assess the Distributio: China, intra districtum Lijiang provinciae percentage of seed set. Cross-pollination was performed Yunnan occurrens. on the other three pairs only to obtain hybrid seeds with- Holotypus: in herbario Institutum Plant Generativum out counting the number of pollinated flowers. In addi- Plasma, Universitas Kyotoensis conservatur, Ohsako tion to the two new species, F. gilesii was also surveyed #97-55. for crossability to other species in the urophyllum group, to increase our knowledge about the crossability of this Fagopyrum gracilipedoides Ohsako et Ohnishi recently collected species. This new species is morphologically similar to F. gracil- ipes Dammer and F. capillatum Ohnishi, but can be dis- Phylogenetic analyses. PCR amplification and direct tinguished from F. gracilipes by having heterostylous sequencing of the chloroplast trnK (UUU) gene intron of self-incompatibility and from F. capillatum by having a F. gilesii was performed as described in Ohsako and smaller leaf, flower and achene. Annual, outcrossing Ohnishi (2000). The nucleotide sequence of F. gilesii with heterostyly. Plant 20 to 50 cm high, lower than F. was deposited in the GenBank/EMBL/DDBJ database capillatum (60 to 150 cm high), bearing many small white under accession number AB086257. The sequence data flowers. Leaf blade sagittate to ovate. Stem, ochrea, in Ohsako and Ohnishi (2000) were used for the other and leaf heavily pubescent as in F. gracilipes. Achene ~3 species. The nucleotide sequences were manually mm long. aligned using Se-AL 1.0 (Rambaut, 1996). Phylogenetic Chromosome number 2n = 16. analyses were performed by the maximum parsimonious Distribution: Limited to Lijiang district of Yunnan prov- (MP), neighbor-joining (NJ) (Saitou and Nei, 1987), and ince, China. maximum likelihood (ML) methods using PAUP* 4.0 b10 Holotype: This species was first found in Baoshan village, (Swofford, 2002). MP analysis was performed with Lijiang district of Yunnan province by T. Ohsako on Octo- branch and bound search option. In MP analysis, infor- ber 30 in1997. Ohsako #97-55 in the Herbarium of Plant mative insertion/deletions (indels) were added to the data Germ-Plasm Institute, Kyoto University. matrix and analyzed together with the nucleotide substitutions. ML analysis was performed with the heu- Fagopyrum jinshaense Ohsako et Ohnishi, spec. nova ristic search option with 10 replications of random Fagopyro gilesii Hermsl. vel F. leptopodo Diels simile, sed stepwise additions of sequences. The HKY85 model differt a priore inflorescentia spicata, a secundo foliis car- (Hasegawa, Kishino, and Yano, 1985) was adopted as the nosis opacis (non nitidis). Planta annua, 10–30 cm. alta, nucleotide substitution model in the NJ and ML analyses. floribus numerosis sparsim dispositis; perigonium album, The HKY85 model allows unequal base frequencies in heterostyliciter multiplicabilis. Lamina foliorum sagit- sequences and different rates of transitions and transver- tata, carnosa, opaca, glabra. Caules basi leviter pilosi, in sions. In the ML analysis, under the HKY85 model, tran- parte superiore, praecipue in ramis floriferis, glabri et sition/transversion ratio and base composition were cerei. Achenia circa 1.5 mm. longa. estimated from the empirical data. Bootstrap analysis Numerus chromosomatum: 2n = 16. (Felsenstein, 1985) was performed for the support of each Distribution: China: districtus Deqin provinciae Yunnan, New Fagopyrum species from Yunnan, China 403 districtus Derong et Batang provinciae Sichuan. inflorescence of F. jinshaense is spike-like with moder- Holotypus: in herbario Institutum Plant Generativum ately elongating internodes as seen in the other species in Plasma, Universitas Kyotoensis conservatur, Ohnishi the urophyllum group, whereas F. gilesii has compact #97-60. head-like inflorescences due to reduced internodal elonga- tion (Fig. 1b, 1c). Fagopyrum jinshaense Ohsako et Ohnishi This new species is morphologically similar to F. gilesii Hemsl. and Chromosome number. Mitosis in root tip cells was F. leptopodum Diels., but can be critically distinguished observed in the present study for ten individuals for each from F. gilesii by spike-like inflorescence and from F. lep- species. All the individuals examined of a species topodum by fleshy lusterless leaf. Annual, outcrossing showed the same chromosome number. Both new spe- with heterostyly. Plant 5 to 30 cm high, bearing many cies, F. gracilipedoides and F. jinshaense, have the chro- white flowers sparsely. Leaf blade sagittate, fleshy, lus- mosome number of 2n = 16, i. e., showing that these terless and nonpubescent. Stem slightly trichomous at species are diploid. The chromosome number was deter- the base of the plant, though smooth and waxy at upper mined for the first time for the following species; F. rub- part, especially flower-bearing branches. Achene ~1.5 ifolium (2n = 32), F. macrocarpum (2n = 16), and F. gilesii mm long. Chromosome number 2n = 16. (2n = 16). Distribution: Deqin district of Yunnan province, Derong and Batang districts of Sichuan province, Mangkang dis- Interspecific crosses. The combinations of interspe- trict of eastern Tibet, China. cific crosses that were executed and the results are shown Holotype: This species was first found at Benzilan village, in Table 2. Note that although we did not check the ger- Deqin district of Yunnan province by O. Ohnishi on Octo- mination rate of seeds obtained in the crosses within a ber 4 in 1997. Ohnishi #97-60 in the Herbarium of Plant species (as a control) in this study, the germination rate Germ-Plasm Institute, Kyoto University. of seeds in selfing of self-compatible species or in freely outcrossed seeds in self-incompatible species is usually in Morphological characters. Whole plant of F. gracil- the range of 70–90% for the species in the urophyllum ipedoides is shown in Fig. 1A. Fagopyrum gracilipe- group. Interspecific crosses between F. gracilipedoides doides is similar to F. gracilipes and F. capillatum in and each of its relatives, F. gracilipes, F. capillatum, and gross morphology. The key to a clade of F. gracilipes and F. rubifolium, resulted in some seeds being set. When F. F. capillatum defined by Ohnishi and Matsuoka (1996), gracilipedoides was used as the female parent, crosses heavily pubescent ochrea and stem, is also seen in this between F. gracilipedoides and these species resulted in new species. On the other hand, F. gracilipedoides and a percentage seed set of 63.6%, 20.0%, and 61.5%, F. gracilipes show sharp contrast in flower morphology. respectively. In the reciprocal cases, the seed set was Fagopyrum gracilipedoides shows heterostyly that is typ- lower at 14.3% (F. gracilipes x F. gracilipedoides) and ically seen in self-incompatible species of Fagopyrum. 37.9% (F. capillatum x F. gracilipedoides). We, however, Individuals with pin flowers and those with thrum failed to make the cross with F. rubifolium as the female flowers are segregated in the population of F. parent because of the difficulty in emasculation of F. gracilipedoides. Most individuals of F. gracilipes bear rubifolium. The combinations of F. gracilipedoides and small homostylous flowers and heterostyly is seen only most of the other species resulted in the production of no exceptionally. This difference in flower morphology is or few seeds. Some of the hybrid seeds germinated and linked to differences in the mating system; F. gracilipe- grew to mature plants bearing flowers. However, no doides is self-incompatible while F. gracilipes is self-com- seed was obtained after self- and cross-pollination among patible and probably highly self-fertilizing. Fagopyrum the hybrids, and backcrosses to their parent species, gracilipedoides can not be distinguished from F. capilla- showing that hybrid plants were completely self- and tum by the mating system and related morphology. cross-sterile. Interspecific crosses between F. jinshaense They, however, apparently differ quantitatively in plant and F. gilesii resulted in seed set in all of the four pairs height and in sizes of plant organs such as leaf, flower, of individuals. The seed set percentage was 19.2% for and achene. Fagopyrum capillatum grows taller and has the pair for which the number of pollinated flowers was larger vegetative and reproductive organs than F. gracil- counted. Out of a total of 62 seeds obtained, five germi- ipoides. nated on wet filter paper at 25°C. Three of them ceased Fig. 1B shows a plant of F. jinshaense. Fagopyrum jin- growth after transfer to soil, and one of the two remaining shaense has similar morphological characters to F. gilesii plants obtained was dwarf and did not survive to except for the inflorescence (cf. Fig. 1b and 1c). The flowering. The only hybrid plant that grew to a mature major morphological characters shared by them are a stage showed complete sterility after backcrossing to both fleshy lusterless leaf, slightly trichomous basal stems, parent species. Attempts were made to cross F. gilesii to and heterostylous flowers with self-incompatibility. The six species. All of the ten reciprocal combinations of the 404 T. OHSAKO et al.

Fig. 1. A, a: A plant and inflorescence of F. gracilipepoides grown in green house, respectively. B, b: A plant and inflorescence of F. jinshaense grown in greenhouse, respectively. C, c: A plant and inflorescence of F. gilesii grown in green house, respectively. New Fagopyrum species from Yunnan, China 405

Fig. 2. One of the five most parsimonious trees of the urophyllum group of Fagopyrum inferred from nucleotide sequence of trnK intron region in cpDNA. A branch that is not supported by other parsimonious trees is shown by broken line. Number of steps of each branch is shown above the branch. Bootsrap percentage and Bremer support value of each clade are shown below the branch. crosses resulted in no seed set. present study the nucleotide sequence of the intron of the chloroplast trnK (UUU) gene was newly surveyed for F. Phylogenetic relationships based on the nucleotide gilesii and analyzed with the data of other Fagopyrum sequence of the chloroplast trnK gene intron. In the species investigated by Ohsako and Ohnishi (2000). The 406 T. OHSAKO et al. nucleotide sequences of the two new species have already incompatibility, whereas F. gracilipes is homostylous self- been determined for two accessions C97106 and C97107 compatible. Fagopyrum gracilipedoides is also similar to of then two nameless species (Ohsako and Ohnishi, another related species F. capillatum, which is closely 2000). As F. leptopodum and F. urophyllum show exten- related to F. gracilipes but retains heterostylous self- sive intraspecific nucleotide variation, two accessions of incompatibility as does F. gracilipedoides. A clear qual- each were included in the analyses to represent major itative morphological difference, including flower lineages. Fagopyrum statice differentiates into two morphology, could not be found between F. gracilipe- major lineages, but the accession C9469, representing one doides and F. capillatum. However, F. gracilipedoides is of the two lineages, was not included in the analyses as different from F. capillatum in quantitative traits such as the trnK intron sequence of C9469 was identical to that plant height (F. capillatum taller) and sizes of vegetative of F. leptopodum C9077 (Ohsako and Ohnishi, 2000). For and reproductive organs (F. capillatum larger). the other species with no or little variation, a single acces- Although a numerical survey for quantitative traits was sion was chosen. Fifteen accessions out of 13 species in not performed in the present study, difference in these the urophyllum group were analyzed. 110 nucleotide sub- traits is apparently discrete and no intermediate form stitutions and 13 indels (insertions or deletions) were was observed in nature. found in aligned sequences that were 1030 bp long. Fagopyrum jinshaense is similar to F. gilesii in Among these, 15 substitutions and 2 indels were poten- morphology. They share many characters, such as a lus- tially phylogenetically informative. A single inversion terless fleshy leaf and slightly trichomous stem. The was also observed, but this character was found to be only character that discriminates F. jinshaense from F. highly homoplasious (Ohsako and Ohnishi, 2000) and gilesii is the form of branching in the inflorescence, spike- was not used in the phylogenetic information for the like branching vs. head-like branching (Fig. 1b, 1c). analysis. Based on these nucleotide variation data, phy- Spike-like branching in the inflorescence is common in logenetic analyses were performed utilizing MP, NJ, and Fagopyrum and thus not autoapomorphy of F. ML methods. MP analysis resulted in five MP trees with jinshaense. For example, F. jinshaense is similar to F. tree length of 117 steps, consistency index (Kluge and leptopodum except for leaf morphology (F. leptopodum Farris, 1969) of 0.974, and retention index (Farris, 1989) has a lustrous leaf). A key for F. jinshaense could not be of 0.908. One of the MP trees is shown in Fig. 2. Topol- defined based on any single morphological character. It ogy of other trees, inferred by NJ and ML analyses, with is a combination of the following characters that distin- the HKY85 model was identical to that in Fig. 2. All of guish F. jinshaense from all other species; heterostylous five MP trees support four lineages at the base of the uro- self-incompatibility, spike-like inflorescence with moder- phyllum group. Five trees, however, are different in ate elongation of the internodes, and a fleshy leaf. relationships among four lineages and/or in branch length of a particular branch. In all trees F. gracilipedoides Reproductive isolation among new species and formed a clade with F. gracilipes, F. capillatum, and F. other members of Fagopyrum. In addition to mor- rubifolium. Fagopyrum jinshaense was found to be dis- phological differences, reproductive isolation is useful as tantly related to F. gilesii, and became a sister to a clade a criterion for discrimination of species. Cross experi- of three self-compatible species, i. e., F. pleioramosum, F. ments among the new species and other Fagopyrum spe- callianthum, and F. macrocarpum. The phylogenetic cies were performed to confirm the validity of the position of F. gilesii is somewhat ambiguous as the sister taxonomic treatment. Crosses between F. gracilipe- group of F. gilesii varied among the five MP trees. doides and its relatives, F. gracilipes and F. capillatum Fagopyrum gilesii became basal in the urophyllum group resulted in the production of self- and cross- sterile in the strict consensus tree. progenies. Only two hybrid plants were obtained from the cross between F. jinshaense and F. gilesii. A single plant that grew to maturity was sterile even after recip- DISCUSSION rocal backcrosses to both of the parental species. These Morphological characteristics of new species. Two results, although a critical result on F. rubifolium x F. new species, F. gracilipedoides and F. jinshaense have the gracilipedoides is still missing, indicate that the new spe- key character for the urophyllum group of Fagopyrum, i. cies are reproductively isolated from other Fagopyrum e., a lustrous small achene (Ohnishi and Matsuoka, 1996). species. This in turn confirms that the discrimination of Each of the new species, F. gracilipedoides and F. these species is reasonable on the basis of the biological jinshaense, has a species that shows morphological species concept. similarity, i.e. F. gracilipes and F. gilesii, respectively. In the present study crossability between F. gilesii and Fagopyrum gracilipedoides and F. gracilipes differ in some other species was also surveyed for a more complete flower morphology that is linked to a mating system. understanding of the reproductive isolation among Fagopyrum gracilipedoides retains heterostylous self- Fagopyrum species. No seed was obtained after ten New Fagopyrum species from Yunnan, China 407 pairs of crosses between F. gilesii and six other species in relationship between these morphologically similar spe- the urophyllum group (Table 2). Out of the 29 combina- cies could further be clarified by surveying intraspecific tions of species examined by Ohsako and Ohnishi (1998) variation in organellar and nuclear genomes as well as in and in the present study, only two combinations of inter- interspecific diversity. specific crosses, F. statice x F. leptopodum and F. macro- carpum x F. pleioramosum, resulted in the production of We are grateful to Dr. Clayton Campbell, Kade Research Ltd., fertile hybrids. These results indicate that the interspe- Morden, Manitoba, Canada for reading the manuscript, correct- cific reproductive barrier among these species is quite ing English and making numerous useful suggestions. We are also grateful to Prof. Tone Wraber, University of Ljubljana, strict with only a few exceptions among the closely Lubljana, Slovenia for reading the manuscript and translating related species. The reproductive isolation, as shown by the description of new species into Latin. This is contribution the experiments, agrees well with the absence of hybrid No. 117 from the Plant Germ Plasm Institute, Graduate School species within the genus Fagopyrum. Extensive inter- of Agriculture, Kyoto University. specific crossing experiments for the remaining combina- tions would help serve for further clarification of the REFERENCES patterns and processes of speciation within the genus. Bremer, K. (1988) The limits of amino acid sequence data in Molecular phylogenetic relationships among new angiosperm phylogenetic reconstruction. Evolution 42, 795–803. species and other Fagopyrum species. In the MP Farris, J. S. 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