Fagopyrum tataricum

Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004
Speciation ofFagopyrum tataricum Inferred from Molecular Data
Kyoko Yamane, Koji Tsuji, and Ohmi Ohnishi*
Graduate School ofAgriculture, Kyoto University, Nakajoh 1, Mozume-cho, Mukoh 617-0001, Japan
(*corresponding author)
ABSTRACT
A phylogenetic tree, reconstructed from cpDNA sequences, demonstrated that F. tatar~cum (including
both wild and cultivated forms), a close relative of F cymosum, was completely mcluded 1D the
Tibet-Himalayan clade of F. cymosum. This finding strongly indicated that the inbreeding species F.
tataricum arose by differentiation from the outcrossing species F. cymosum in the Tibet-Himalayan area.
The matKsequences gave an estimation that these two species diverged approximately 1.9 million years
ago. It was found that natural populations ofF. cymosum maintained a high amount ofgenetic variations
within the species, whereas F. tataricum possesses a fairly low level. Since F. cymosum and F tataricum
are crossable with each other, this suggested that F cymosum may be a good genetic resource for the
improvement of cultivated Tartary buckwheat.
Keywords: Fagopyrum, Fagopurum cymosum, Fagopyrum tataricum, matK, sequence, speciation
INTRODUCTION
Tartary buckwheat is a self-fertilizing diploid (2n=16) species and has both a cultivated, F
tataricum ssp. tataricum Gaertn. and a wild fonn, F tataricum ssp. potanini Batalin. Tartary
buckwheat is mainly cultivated in southern China and in the Himalayan hills. Wild Tartary is
found growing in the Yunnan and Sichuan provinces of China, the Tibetan plateau, and in the
high Himalayan hills of Nepal, India, and Pakistan (OHNISHI, 1994, 1995, 1998a, 2000), F
tataricum was always found to be distributed north of the northern most boundaries for the
distribution ofF cymosum (OHNISHI AND YASUI, 1998).
Tartary buckwheat is a nutritionally important food in many areas. It contains a'balanced
amino acid composition and a relatively high content of crude fiber as well as the vitamins B1
and B2. Moreover, Tartary buckwheat seeds contain a larger amount of rutin (>80 folds) than
do common buckwheat seeds (FABJAN ET AL., 2003). Rutin has antioxidative, antihypertensive
and anti-inflammatory activities with buckwheat being the only grain to contain rutin in its
seeds.
Fagopyrum cymosum Meisn., commonly known as perennial buckwheat, is a herbaceous,
perennial insect-pollinated heterostylous species. In contrast to the large differences between F.
cymosum and F tataricum in their morphological characteristics and breeding habits, KJSHlMA
et al. (1995) first reported that F cymosum was related to F. tataricum more closely than to F
esculentum based on a chloroplast DNA restriction site survey. YASUI AND OHNISHI (1998a and
b) confinned this by observing a synapomorphic deletion in the F cymosum-F. tataricum clade
of their phylogenetic tree. More recently, YAMANE ET AI. (2003) gained an insight into the
origin ofT tataricum as differentiating from F cymosum in the Tibet-Himalayan area based on
the nucleotide sequences from three chloroplast regions.
In the present paper, we attempted to estimate the speciation time ofF tataricum from F
cymosum based on matK sequences. Moreover, we show that F cymosum may be a good
genetic resource for the improvement of Tartary buckwheat, by demonstrating that there is a
large amount of genetic variation in F cymosum but a limited amount in F tataricum.
MATERIALS AND METHODS
The analysis ofthe trnKlmatK sequences
Data from the matK sequences ofsix, four and one accessions ofdiploid F cymosum, F.
tataricum, and F. esculentum (as an outside group), respectively, were obtained from GenBank.;
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Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004
the accessions numbers (GenBank number) being C9438 (AB093075), C9435 (AB093072),
C8924 (AB09371), C8926 (AB093074), C8927 (AB093081), YC9806 (AB093082) of F.
cymosum, C9532 (AB093083), C9542 (AB093084) of F. tataricum ssp. potanini, CT9835
(AB093085), C9044 (AB093086) ofF. tataricum ssp. tataricum, and C9106 (AB093087) ofF.
esculentum (YAMANEETAL. 2003). The locations ofthese accessions (village or town, province,
and country) are given in Figure 1. The F. tataricum samples were carefully selected from those
used by TSUJI AND OHNISHI (2000, 2001a, b) so that a small number of samples would
encompass the entire variation. The nucleotide sequences were aligned manually using
DNASIS version 3.0 (Hitachi Software Engineering Co. Ltd., Tokyo, Japan), with manual
modifications to minimize the number of gaps. We used DnaSP version 3.5 to estimate the
nucleotide diversity (1£) and the number of net nucleotide substitutions between the two
populations (DA) (NEI, 1987). The phylogenetic relationships were inferred by the
neighbor-joining (NJ) method (SAITOU AND NEI, 1987) which was performed using PAUP
4.0b10 (SWOFFORD, 1999). The number of nucleotide substitutions per site was estimated by
Kimura's (1980) two-parameter method and used to estimate the genetic distance. All
informative substitutions were used in the analyses, and indels were treated as missing data in
both methods. PAUP was also utilized to perform bootstrap analyses (1000 replicates) to
estimate the relative support for the clades (FELSENSTEIN, 1985).
Figure 1.
A NJ tree reconstructed using nucleotide sequences of the trnKl matK region.
Bootstrap values for 1000 replicates are shown above the branch, but values of less than 50%
are not shown.
Genetic variations of diploid R cymosum and R tataricum
In order to evaluate the possibility ofF cymosum as a genetic resource for F tataricum,
we reanalyzed the published data on allozyme variation in F. cymosum and F. tataricum
(OHNISHI 1998b; YAMANE AND OHNISHI, 2001; Table 1), chloroplast DNA sequences (YAMANE
ET AL., 2003; Table 2), and theAdh sequences (YAMANE, 2003; Table 2), and compared genetic
variations between F tataricum and diploid F. cymosum.
, Tab. 1 Levels ofallozyme variation at the species level in F. cymosum and F. tataricum.
F cymosum (2X)
F tataricum
YAMANE AND OHNISHJ (2001)* 1 OHNISHI (1998)* 1
Number ofinvestigated populations 10 148
Number ofinvestigated loci 9 17
Number ofpolymorphic loci 8 4
Percentage (%) ofpolymorphic locus 0.89 0.24
Other plants *2 0.50 (Outcrossing species) 0.42 (Selfinf,! species)
Average number ofallele per loci 3.20 1.40
*\calcu)ated based on the data in these papers.
*2; After HAMRICK AND GODT (1989).
Other plants ~ 1.99 (Outcrossing species) 1.69 (Selfing species)
318

Proceedings ofthe 9th /nlematiQIWI Symposium 01'1 Buckwheat, Prague 2004
Tab. 2 Nucleotide diversities (n) ofthe marK and the Adh sequences in F tataricum and diploid
F. cvmosum.
matK
Adh
Species
Yamane et a1. (2003~ Yamane (2003)
Diploid F cymosum (n=6) 0.0036 0.0074
P. tataricum (0=4) 0.0003 0.0022
krabidopsis thaiiana (selfmg species)'j - 0.0080
IArabis zemmidera (outcrossinll soecies) OJ
~
0.0053
*1; MIYASHITA ET AL. 1996 and 2001. n' number ofaccessions analyzed.
RESULTS
Phylogenetic position of R tataricum
A total of 2413 characters were analyzed for the trnK/matK sequences. As shown in
Figure 1, F. cymosum consisted of two major clades. The reconstructed tree showed that the
phylogenetic relationships within F. cymosum coincided with the geographic locations of the
accessions; one consisting of the accessions from Tibet, which includes the accessions of F
tataricum (Tibet-Himalayan clade), and the other consisting ofthe F. cymosum accessions from
the Yunnan and Sichuan provinces of China.
Fig. I A NJ tree reconstructed using nucleotide sequences ofthe trnKJ rnatK region. Bootstrap
values for 1000 replicates are shown above the branch, but values of less than 50% are
not shown.
61
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YaJeOe ('Tong!reI,,...Qlln.)
Polymorphisms in F. cymosum and F. tataricum
Table 1 shows the comparison for allozyme variability's between the two species.
Although the kind and number of enzymes analyzed were not the same in the two studies, the
level of allozyme variation was obviously different between the two species; i. e., natural
populations of F cymosum maintained a greater amount of allozyme variations within the
species, wherea

Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004
self-fertilizing plant species (HAMRICK AND GODT, 1989). Likewise, Table 2 shows that the
level of chloroplast and nuclear nucleotide variation was higher in F. cymosum than in F.
tataricum.
DISCUSSION
Speciation ofE tataricum from R cymosum
All the accessions ofF tataricum were included in one clade of the phylogenetic tree,
and the phylogenetically closest accession to F. tataricum was found to be diploid F cymosum
from Tongmai in Tibet (YAMANE ET AL., 2003; Figure 1). We therefore concluded that F.
tataricum speciated from F cymosum in the Tibet-Himalayan region. Interspecific hybrids
between F. cymosum and F tataricum are extremely difficult to obtain by conventional
breeding methods; i. e. the reproductive isolation between these two species is strict (WOO ET
AL, 1999). The breakdown ofthe self-incompatibility system in F. tataricum might have led to
rapid reproductive isolation of F tataricum from its progenitor. In traditional theories of
speciation, reproductive isolation may originate from an incidental by-product ofadaptation to
distinct environments (DOBZHANSKY, 1951).
We estimated the approximate time of divergence between F tataricum and the
accession of F cymosum from Tongmai to be approximately 1.9 MYA by utilizing the same
method as YAMANE ET AL. (2003). Interestingly, the speciation time of F. tataricum, 1.9 MY;
corresponds approximately to the end of the Pliocene era and the beginning of Quaternary
(Pleistocene) (YAMANE ETAL., 2003). During this period, the global climate around Tibet turned
cold and dry, according to pollen data and the age of Mt. Everest and other localities (ZHOU,
1985). F tataricum has always been found to be distributed north of the northern most
boundaries for the distribution ofF cymosum (OHNISHI AND YASUl, 1998). It therefore appears
that F tataricum has adapted to cooler environments. This may have played an important role in
the speciation ofF tataricum from F. cymosum.
Evaluation ofR cymosum as genetic resources
The present data indicated that F. cymosum contains a large amount of intraspecific
genetic variation. It also suggested that populations from different habitats evolved
independently and may possess newly arisen genotypes as a result of adaptation to differing
local environmental conditions. In contrast, F. tataricum demonstrated low intraspecific genetic
diversity in allozyme variations (Table 1), cpDNA variations and in the nucleotide sequence of
the Adh gene (Table 2). These findings lead to the assertion that F cymosum may be utilized as
an important genetic resource or gene reservoir which could be utilized in the improvement of
cultivated Tartary buckwheat. When F. cymosum was used as the male parent, hybrids between
F. cymosum and F tataricum can be successfully developed into mature plants through embryo
rescue methods (WOOETAL., 1999). Therefore, introgression ofuseful genes from F. cymosum
into cultivated Tartary buckwheat may help solve important agricultural problems such as water
stress injury, disease resistance, and increased yield in the future. In addition, a diploid
population ofF cymosum from the Tibet-Himalayan area was recently discovered (TSUJI ETAL.,
1999). Ithas unique achene morphology and it was revealed that this population had genetically
diverged from the other diploid populations of F cymosum. Therefore, conservation and
utilization of these types ofF cymosum may be necessary at the population level.
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