Phylogenetic analysis of the genus Fagopyrum (Polygonaceae), includ ing the Nepali species E megacarpum, based on nucleotide sequence of the rbcL-accD region in chloroplast DNA

Fagopyrum 18: 9-14 (2001)

Phylogenetic analysis of the genus Fagopyrum (Polygonaceae), includ­ing the Nepali species E megacarpum, based on nucleotide sequenceof the rbcL-accD region in chloroplast DNA

Takanori OHSAKOI, Shuichi FUKUOKA2, Hari Prasad BIMBJ, Bimal Kumar BANIYAJ,Yasuo YASUII and Ohmi OHNISHII*

'Plant Germ-Plasm Institute, Graduate SchoolofAgriculture, Kyoto University, Nakajo 1, Mozume-cho, Mukou 617-0001. Japan2Laboratory ofPlant Genetic Resources, National Institute ofAgrobiologicalResources, Kannon-dai 2-1-2 Tsukuba 305-8502,Japan'Nepal AgriculturalResearchCouncil, Khumaltar, Lalitpur, Nepal

Received April 25,2001; accepted in revised form July9, 2001.

Key words: chloroplast rbcL-accD region, Fagopyrum megacarpum, phylogenetic relationships, taxonomy

ABSTRACTThe phylogenetic relationships among Fagopyrum species including F. megacarpum, a species in Nepal, and two

new species recently found in China were inferred based on nucleotide sequences of two chloroplast genes, rbeL andaceD, and their intergenic spacer. In the phylogenetic analysis of the rbeL data, three species from Rumex and Persi­earia were included. Fagopyrum megaearpum was closely related to Rumex aeetosella and did not make a mono­phyletic group with the other Fagopyrum species, indicating that F. megacarpum should not be treated as a member ofFagopyrum from the viewpoint of phylogenetic taxonomy. To obtain a better resolution of the phylogenetic relation­ships, combined analyses of the three data sets, rbeL, aceD, and their intergenic spacer, were performed. A new out­crossing species, sp. nov. A, is morphologically similar to the self-compatible species F. gracilipes but was closelyrelated to another self pollinater F. rubifolium, a relative of F. graeilipes in molecular phylogeny. The other newspecies, sp. nov. B, shared some morphological characters with F. gilesii; such as a fleshy leaf and a stem with scatteredtrichomes, but was found to be distantly related to F gilesii on the molecular phylogenetic tree.

INTRODUCTION

At present about 18 species are known to be membersof the genus Fagopyrum incuding the two cultivatedspecies, F. esculentum and F. tatarieum. The center ofdistribution of the genus has been identified as southwestChina and most of species are found in China. However,F. megaearpum and F. snowdenii are found only in Nepaland in Africa, respectively. These species, which arefound outside southwest China, have previously beendescribed as members of genera other than Fagopyrum.

Fagopyrum megacarpum was originally described byHara (1972), but almost simultaneously Malick andSengupta (1972) described it as Eskemukerjea nepalensis(see Fig. 1). Hara (1982) excluded it from the Fagopy­

rum by making a new combination Eskemukerjeamegacarpum. Fagopyrum snowdenii was originally de­scribed as Harpagocarpus snowdenii from Uganda byHutchinson and Dandy (1926). These species wereamalgamated with the genus Fagopyrum by Hong(1988) on the basis of pollen morphology. The phyloge­netic positions of these species have not been investigat­ed. Ohnishi and coworkers have collected Fagopyrumsamples in China, described new species (Ohnishi,

"Corresponding author

9

1998; Ohsako and Ohnishi, 1998), and surveyed thephylogenetic relationships among them (Yasui andOhnishi 1998a, b; Ohsako and Ohnishi 2000). Aftercompletion of the experiments by Yasui and Ohnishi(1998a, b), living samples of F. megaearpum, F. gilesiiand two new species were collected. In the presentstudy, we determined the nucleotide sequences of theregion of the two genes, rbeL and aceD, and their inter­genic spacer in chloroplast DNA. The sequences of thechlorplast rbeL-aceD region were newly surveyed for F.

megacarpum, F. pleioramosum, F. gilesii, F. rubifolium,

F. macrocarpum, and two unnamed new species A andB. Nucleotide sequences of the other species wereadopted from Yasui and Ohnishi (1996, 1998a).

MATERIALS AND METHODS

Plant samples

Accessions used in the present study were shown inTable 1. Samples of F. megaearpum were collected atDunai, Upper Dolpa in 1999 by the Nepal-Japan cooper­ative expedition team led by H. P. Bimb and B. K.Baniya. Other samples were collected by Ohnishi, Yasuiand Ohsako. Nucleotide sequences of the chloroplast

10 Ohsako et al.

Fig. I. Fagopyrum megacarpum grown in a green house atKyoto, Japan.

rbcL-accD region of the following species were newlysurveyed; F megacarpum, F pleioramosum, F gilesii,F rubifolium, F macrocarpum and two unnamed newspecies A and B. Nucleotide sequences of the otherspecies were adopted from Yasui and Ohnishi (1996 ,1998a)

Sequencing of the chloroplast rbcL-accD region

Procedures of total DNA extraction, PCR amplifica­tion and direct sequencing of PCR products were asdescribed by Ohsako and Ohnishi (2000). The chloro­plast rbcL-accD region was amplified using primersrbcL-m292F (5'-TCGfGfCAAGAGfGCTATTT-3 ') andrbcL-2644R (5'-TATCCGCATTCATCACAAA-3 ') ex­cept for F megacarpum, for which rbcL-I F (5'-ATGT­CACCACAAACAGAAGACTA-3') and rbcL-2644Rwere used because rbcL-m292F and rbcL-2644R werefound not to work successfully. This made it impossibleto determine the 24bp 5' end nucJeotides of F mega car­pum . This loss of data, however, was assumed not toaffect the result of the phylogenetic inference, as there isno variation in that region among all the other species.The sequences of oligonucleotides used for PCR ampli­fication of rbcL-accD region and sequencing are avail­able from Ohsako.

Table I. Species investigated in the present study

Species KU accessionEMBLlGenBank

/DDBJ #*

Genus Fagopyrum

F esculentum' C9136 ABOOO310

F homotropicum' C9139 ABOOO312

F. cymosum' C9051 ABOOO304

F tataricum' C9029 ABOOO318

F leptopodum ' C9253 ABOOO31J

F statice' C9470 ABOOO316

F gracilipes' C8938 ABOOOJII

F capillatum' C9149 ABOOOJ03

F. rubifolium C9589 AB056686

F callianthum' C9152 ABOOOJ02

F pleioramosum C9567 AB056687

F macrocarpum C9565 AB056688

F lineare ' C9256 ABOOO314

F urophyllum (Kunming) I C8844 ABOOO320

F. urophyllum (Dali) I C8842 ABOOO321

F gilesii C9836 AB056689

F megacarpum AB056690

Species nova A C97106 AB056691

Species nova B C97107 AB056692

Outgroup

Persicaria hydropiper' AB008781

Persicaria senticosum' D86289

Rumex acetosella' D86290

KU: Plant Germ-Plasm Institute, Graduate School of Agricul­ture, Kyoto University.*: Accession number of the EMBLIGenBank/DDBJ': Nucleotide sequences were determined by Yasui andOhnishi (1998a).2: Nucleotide sequences were determined by Yasui andOhnishi (1996).

Phylogenetic analyses

Nucleotide sequences were aligned using Se-AL 1.0(Rambaut, 1996). Aligned multiple sequence matriceswere output in NEXUS files. Parsimony analyses wereperformed using PAUP* 4,0 (Swofford, 1999). Twocriteria were used for parsimony analyses: unweightedparsimony (UP) and implied weights (lW) method(Goloboff, 1993). lndels were added to data matrix andanalyzed together with nucleotide substitutions. Branch­and-bound searches were performed on PAUP* 4.0 withUP and IW (constant of concavity k =0) optimalitycriteria . In each of the UP and IW analyses, the furthestaddition sequence and the MulTrees options were ineffect. At first, three subsets, including the rbcl: gene,the aceD gene, and the intergenic spacer, were analyzed

Fagopyrum phylogeny based on rbcL-accD sequence 11

separately. The three outgroup species were includedonly in the rbeL data set. After perform ing separateanalyses, heterogeneity among the subsets was assessedby incongruence length difference (ILO) tests (Farris etal., 1995). Initially ILO tests between two of the threesets and among three sets were performed, No signifi­cant heterogeneity was recognized between or amongsubsets. Then combined analyses of two of the threesubsets were performed. ILD tests were performed be­tween a subset and combined data of the remaining twosubsets. These tests did not show significance. No heter­ogeneity was found among all possible pairs of subsets,and so we then proceeded to combine all three subsetsfor further phylogenetic analyses. The Bremer supportindex for each clade (Bremer, 1988) was calculated byeither reconstructing trees up to seven steps longer thanthe most parsimonious trees or performing parsimonyanalyses with the topo logical constraint not to make aclade monophyletic. Support for each branch was alsoexamined by bootst rap analysis (Felsenstein, 1985) with2000 rep lications of character resampling and recon­structing parsimonious trees with heuristic search.

RESULTS

rbcL phylogeny including four outgroup species

Five UP trees and five IW trees were obtained byparsimony analyses. One of the five UP trees is shownin Fig. 2. UP trees have 143 steps. Consistency index(CI), retention index (RI), and rescaled consistency in­dex (RC) are 0.8741, 0.9053, and 0.7913, respectively.Five lW trees have identical topo logy to those of the UPtrees . All Fagopyrum species except for F. megaearpumformed a monophyletic group. They are divided into twogroups , the cymosum group and the urophyllum groupas previously shown by Yasui and Ohnishi ( I998a, b).Fagopyrum gilesii and the two new species are includedin the urophyllum group. Fagopyrum megacarpum showsa close relationship to Rumex aeetosella, which makesthe genus Fagopyrum a paraphyletic group.

Combined analyses of two genes and their spacer

lLO tests showed no significant heterogeneity amongthe three subsets of the region investigated, i. e., rbeL,aceD, and their intergenic spacer. The three subsets werecombined into a single data for phylogenetic analyses.Five UP trees and two lW trees were obtained by parsi­mony analyses of the combined data. Topology of IW

ming)

Persicaria hydropiper

esculentum

F. homotropicum

2 F. leptopodum

86/2 F. statice

Sp. nov . B

f--- F. gilesii

F. graciJipes

1 F capillatum........ - .24/0

1

6'/1""F1F. rubifoJium

6 /1 Sp. nov. AThe urophyllum group

10 ~F.lineare100/7 2

~cF. urophyll um (Kun

53/1 F. urophyllum (Dali)3

F. callianthum2

8 88/2 1 F. pleioramosum94/3

63/1 F. macrocarpum

6 IF.

The cymos um group ~, 99/5L!-

14 42/1 1F. tataricum

100/10 IF. cymosum9

F. megacarpum

~ 15Rumex acetosella

2720

100/12 6Persicaria senticosum

Fig. 2. One of the five unweighted parsimonious trees based on nucleotide sequence of rbcL gene of chloroplast DNA. Brancheswith a broken line indicates the clade that is not supported by the other parsimonious trees. Branch lengths and bootstrap percentage!Bremer support values are shown above and below the branches, respectively.

12

39

20/100

40

The urophyllum group

47

44/100

The cymosum group

6246/100

126

Ohsako et al.

1

0/40

5

3/86

11/72

10

7

6/100

6

F. gilesii

1..~3F. gracilipes0/4 4 .

6 F. cepitleium

4/93 3 F rubifolium2 .

2/88 2 Sp. nov. A

114

4/93

11

2119/100

15

11/100

F. esculentum

F. homotropicum

4 F. tataricum

F. cymosum

Fig. 3. One of the four unweighted parsimonious trees based on nucleotide sequenc e ofthe rbcL-accD region ofchloroplast DNA.Branches with a broken line indicate clades that are not supported by the other parsimonious tree(s) . Branch lengths and Bremersupport/bootstrap percentage values are shown above and below the branches, respectively.

trees is identical to that of UP trees. One of the five UPtrees is shown in Fig. 3. UP trees have 474 steps. CI, RI,and RC were 0.8945, 0.9196, and 0.8226, respectively.Relationships among clades within the urophyllumgroup are more resolved in the combined phylogenythan in the rbcL phylogeny. For example, the branchingpattern of the basal node of the urophyllum group iscongruent among all ofthe five combined trees whereasthey were not among the rbcL trees (broken line in Fig.2). The cymosum group and the urophyllum group arealso recognized with long branches (62 and 47 steps, re­spectively), strong Bremer support (46 and 44, respec­tively), and high bootstrap frequency (both 100%).Fagopyrum megacarpum was found to be distantly re­lated to other Fagopyrum species. Species nov. A, anew species morpholog ically similar to F. gracilipes,was found to be closely related to F rubifolium . Thesethree species plus F capillatum formed a clade withstrong Bremer (4) and bootstrap (93%) supports. Theother new species, sp. nov. B, was found to be distantlyrelated to F gilesii although they are morphologicallysimilar to each other.

DISCUSSION

Phylogenetic relationship of F. megacarpum to otherFagopyrum species

In the present study we refined the phylogenetic rela­tionships of Fagopyrum as reported by Yasui andOhnishi (1998a) by adding six additional species. Themost important result of the present study is the findingthat F megacarpum, a species found only in Nepal,showed a close relationship with Rumex rather than itscongener based on rbcL phylogeny. All other membersexcept for F megacarpum are monophyletic. Thesephylogentic relationships indicate that F megacarpumshould not be included in the genus Fagopyrum fromthe viewpoint of phylogenetic taxonomy (de Queiroz andGauthier, 1992) which state that taxonomy should reflectevolutionary relationships of organisms. The taxonomi­cal treatment of F megacarpum has fluctuated since itsoriginal description. At almost the same time of Hara's(1972) description of F megacarpum, Malick andSengupta (1972) reported the species as Eskemukerjeanepalensis, a monotype of a new genus. Hara (1982)excluded the species from the genus Fagopyrum by de­fining a new combination Eskemukerjea megacarpum.Hong (1988) reintroduced it into Fagopyrum as F

Fagopyrum phylogeny based on rbcL-accD sequence 13

megacarpum based on pollen morphology. The presentresult strongly supports the classification by Malick andSengupta (1972) and Hara (1982). The pollen morpho­logical similarity among F megacarpum and otherFagopyrum species shown by Hong (1988) might bedue to homoplasy that should not be recognized as akey of the genus.

Phylogenetic position of new species within the uro­phyllum group

The phylogenetic position of two new species and F.gilesii was surveyed based on the nucleotide sequenceof the rbcL-accD region. Two new species were includ­ed in the urophyllum group. This was previously shownin Ohsako and Ohnishi (1998) by phylogenetic analysesof other cpDNA regions. This result is consistent withtheir morphology; surface of achene is smooth and lus­trous, that is synapomorphy of the urophyllum group(Ohnishi and Matsuoka, 1996). Species nov. A (C97106)is more closely related to F rubifolium rather than to Fgracilipes. This result is consistent with that of Ohsakoand Ohnishi (2000). The speciation of F gracilipes, F.capillatum, F. rubifolium, and sp. nov A (C971 06) shouldbe studied more intensively using sensitive molecularmarker such as AFLP. Species nov. B (C97106) differ­entiates from other species near the base of the urophyl­lum group. This species retains morphological charac­ters similar to F gilesii such as fleshy leaves and slightlytrichomous stems. The geographical distributions of Fgilesii and sp. nov. B border each other in southwesternChina: being the Lancan River valley and the JinshaRiver valley, respectively (Ohnishi and Konishi, 2001).The molecular phylogeny, however, does not supporttheir close relationship . Species nov. Band F gilesiiare distantly related to each other. Fagopyrum gilesii isclosely related to F leptopodum and F statice, althoughthis relationship is not strongly supported by the Bremerindex (I) and the bootstrap frequency (60%). Speciesnov. B occupies a basal position in the urophyllumgroup and has no obvious sister group. One probablereason for the morphological similarity between sp. nov.Band F gilesii is that they share ancestral characterstates. This might be the case for the trichomes on thestem as all members of the urophyllum group have tri­chomes, except for three species showing monophyly, Fcallianthum, F pleioramosum, and F. macrocarpum. Incontrast, fleshy leaves are also seen in the distant speciesF callianthum and F. rubifolium. This character mighthave evolved independently in the four lineages. Speciesnov. B differs from F. gilesii in the morphology of theinflorescence and flower. Fagopyrum gilesii has aunique compact inflorescence and larger flowers thanthose of sp. nov. B. Molecular phylogeny supports thatthese differences are sufficient to discriminate sp. nov. B

from F. gilesii as a new species. A detailed descriptionof these two new species is now in preparation.

ACKNOWLEDGEMENT

The samples of Fagopyrum megacarpum were collected ina field survey of the collaborative research program on "In situconservation of Plant Genetic Resources in Nepal (78JP­C02A)" , funded by International Plant Genetic Resources In­stitute (TPGRI). The authors are grateful to Mr. M. L. Vaidyaand Mr. D. R. Sharma for their assistance in the field survey.

This is contribution No. 109 from Plant Germ Plasm Insti­tute, Fac. of Agriculture, Kyoto University.

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