全 文 ：Journal of Systematics and Evolution 46 (3): 315–321 (2008) doi: 10.3724/SP.J.1002.2008.08007
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com

Phylogeny of Dioscorea sect. Stenophora based on chloroplast
matK, rbcL and trnL-F sequences
1Xing GAO 2Yu-Ping ZHU 1Bao-Cheng WU 1Ya-Mei ZHAO
2Jian-Qun CHEN* 1Yue-Yu HANG*
1(Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Jiangsu Province Key Laboratory for
Plant Ex Situ Conservation, Nanjing 210014, China)
2(Department of Biology, Nanjing University, Nanjing 210093, China)
Abstract Seventeen species, one subspecies and one variety of Dioscorea sect. Stenophora Uline were investi-
gated for their phylogenetic relationships based on a sequence analysis of chloroplast matK and rbcL genes and
trnL-F intergenic spacer by maximum parsimony and maximum likelihood methods. The results showed that: (a)
sect. Stenophora was a strongly supported monophyletic group; (b) D. rockii, D. membranacea, D. banzhuana,
and D. simulans formed a moderately supported monophyletic group, and D. prazeri was weakly supported to be
sister to this group; (c) D. althaeoides and D. nipponica ssp. nipponica formed a moderately supported clade, and
D. nipponica ssp. rosthornii was not a member of this clade; (d) D. zingiberensis and D. sinoparviflora showed a
moderate to strong sister relationship; and (e) D. collettii var. hypoglauca and D. collettii var. collettii were sister
to each other, but with only weak support.
Key words chloroplast DNA sequence, Dioscorea sect. Stenophora, maximum likelihood, maximum parsi-
mony, molecular phylogeny.
Dioscorea L. is the largest genus in the family of
Dioscoreaceae, including more than 95% species of
this family. This genus contains about 600 species
(Coursey, 1967), with about 70 sections. These spe-
cies are distributed mainly in Southeast Asia, Africa,
Central America, South America, and other tropical or
subtropical regions, while a few occurred in Europe
and North America. Dioscoreaceae is a pivotal taxon
in the evolution of monocotyledons, and occupies a
basal position among all extant monocotyledonous
plants (Dahlgren, 1989; Chase et al., 2006).
One of the sections of Dioscorea, sect. Steno-
phora Uline contains about 30 species, 2 varieties and
1 subspecies. More than 90% of these species are
distributed in Asia; one and two species are found in
North America and Europe, respectively. This section
has 13 endemic taxa in China, and likely originated in
Himalayas–Hengduan Mountains (Wan et al., 1994).
It has many ancestral characteristics of the genus, such
as rhizome, diploid chromosome number (Pei et al.,
1979; Chin et al., 1985), and single pollen aperture
(Shu, 1987; Schols et al., 2003). Furthermore, the
earliest fossil record of the genus Dioscorea was from
sect. Stenophora. Therefore, this section was consid-
ered the oldest section in Dioscorea (Burkill, 1960).
Phylogenetic relationships and circumscription of
some species in the sect. Stenophora have been con-
troversial. In a major monographic study by Pei et al.
(1979), the authors discarded sect. Illigerastrum, and
moved D. simulans Prain & Burkill to sect. Steno-
phora, since it possessed rhizome and some other
characteristics of sect. Stenophora. In their treatment,
D. sinoparviflora C. T. Ting, M. G. Gilbert & N. J.
Turland was recognized as a new species, separated
from D. zingiberensis C. H. Wright because the latter
exhibited a discontinuous distribution in China, and D.
nipponica ssp. rosthornii (Prain & Burkill) C. T. Ting
was elevated to a subspecies from a variety of D.
nipponica ssp. nipponica Makino.
In an earlier study by the same research group,
more species were recognized, such as D. banzhuana
C. Pei & C. T. Ting and D. biformifolia C. Pei & C. T.
Ting. The former was distinguished from D. chingii
Prain & Burkill by their anatomic difference in rhi-
zomes, and the latter was separated from D. panthaica
Prain & Burkill due to the difference in chromosome
numbers. Dioscorea collettii var. hypoglauca (Palibin)
C. Pei & C. T. Ting was also recognized as a variety
of D. collettii Hook. f. var. collettii (The Research
Group of Dioscorea, Kiangsu Institute of Botany,
1976).
In a recent study by Ting and Michael (2000), it


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Received: 21 January 2008 Accepted: 21 April 2008
* Authors for correspondence. E-mail: chenjq@nju.edu.cn (Chen J-Q)>.
Journal of Systematics and Evolution Vol. 46 No. 3 2008

316
was revealed that D. collettii var. hypoglauca and D.
collettii var. collettii had morphological characters
that exhibit continuous variation. Both D. simulans
and D. biformifolia were the only two species in the
section that possessed compound leaves, but they
could not be distinguished by their morphological
characters.
With the development of molecular systematics,
more and more researchers use DNA sequences to
solve complicated taxonomic problems and to infer
phylogenetic relationships among organisms, includ-
ing species in Dioscoreaceae. Kawabe et al. (1997)
studied the phylogenetic relationships of six species in
sect. Stenophora by analyzing the nucleotide sequence
variation of the phosphoglucose isomerase (Pgi)
locus. The result indicated that D. tokoro Makino and
D. tenuipes Franch. & Sav. belonged to a mono-
phyletic group, while D. gracillima Miq., D. nip-
ponica Makino ssp. nipponica, D. quinqueloba
Thunb. and D. septemloba Thunb. formed a separate
clade. Through combining the molecular data of
plastid genes (rbcL and matK) and the morphological
characters of roots and fruits, Wilkin et al. (2005)
reconstructed the interspecies phylogeny of 67 taxa
from Dioscorea, including 5 species of sect. Steno-
phora, and found that these 5 species fell into one
clade and was sister to all other species of Dioscorea.
In this study, we sequenced three chloroplast
markers, matK, rbcL and trnL-F, from most species in
the sect. Stenophora, and aimed to investigate inter-
specific relationships within the section.
1 Material and methods
1.1 Plant materials
A total of 19 taxa were investigated in this study
(Table 1), including 13 species, 1 subspecies and 1
variety of Dioscorea sect. Stenophora endemic to
China and 1 species from Europe, and three outgroup
species: D. esculenta Burkill, D. nitens Prain &
Burkill and D. bulbifera L. from sect. Combilium
Prain & Burkill, sect. Shannicorea Prain & Burkill
and sect. Opsophyton Uline, respectively. All of the
plant materials were collected from the field by the
authors, or kindly provided by Kunming Institute of
Botany. Voucher specimens have been deposited at
Herbarium of Institute of Botany, Jiangsu Province
and Chinese Academy of Sciences, China (NAS).
1.2 DNA extraction, PCR amplification and
sequencing
DNA was extracted from young fresh or silica
gel-dried leaves using a modified CTAB procedure of
Doyle and Doyle (1987). The primers for matK region
(MF and MR) were designed based on the matK
sequence of D. alata L. (AB040208) with the aid of
the software Oligo (Molecular Biology Insights, Inc.,
Cascade, Colorado, USA). The primers for the rbcL
gene (m3 and m4) were designed based on the rbcL
sequence of D. alata (AY667098), and the primers for
the trnL intron and the trnL-F spacer region was
referred to the sequences of the universal primers c
and f (Taberlet et al., 1991). Detailed sequences of all
the primers are listed in Table 2.
The polymerase chain reaction (PCR) was con-
ducted with the following program: a premelt of 3 min
at 94℃, followed by 35 cycles of 45 s denaturation at
94 ℃, 30 s annealing at 58 ℃, 1 min 30 s extension
at 72 ℃, plus a final extension of 5 min at 72 ℃.
Each 50 μL reaction contained 1× Mg-free DNA poly-
merase buffer, 2.5 mmol/L MgCl2, 1 U Taq
DNA-polymerase, 40 ng template DNA, 0.3 μmol/L
each primer and 0.12 mmol/L dNTPs. Amplified
products were checked on 0.8%–1.2% agarose gels.
The PCR products were purified by TIAN gel Midi
Purification Kit (QIAGEN) and directly sequenced on
an ABI 3730 automated sequencer. The sequencing
primers were the PCR primers listed in Table 2. All
sequences have been submitted to GenBank (Table 1).
1.3 Data analysis and phylogenetic assessment
For all three data sets, alignment of DNA se-
quences was initially performed with CLUSTAL X
(Thompson et al., 1997), with subsequent manual
adjustment. The lack of sequences for trnL-F, for D.
membranacea Pierre, D. rockii Prain & Burkill and D.
prazeri Prain & Burkill was treated as missing data.
We conducted preliminary separate phylogenetic
analyses using the parsimony criterion for each of the
three markers, and found no incongruent grouping
among any of the comparisons. Thus, the data were
combined to form a 3-gene matrix. Both maximum
parsimony (MP) and maximum likelihood (ML)
analyses were performed on this 3-gene matrix. For
MP analyses, a branch and bound search was done in
PAUP version 4.0b10 (Swofford, 2002). All charac-
ters were equally weighted, gaps were treated as
missing data, and character states were treated as
unordered. The search options were “on” for Multrees
and “furtherest” for addition sequence. Bootstrap
support (Felsenstein, 1985) was estimated with 1000
replicates of heuristic search, with the following
settings: simple addition sequence, one tree held at
each step during stepwise addition, TBR branch-
swapping algorithm, steepest descent option in effect,
MaxTrees setting of 5000, and “MulTrees” option
GAO et al.: Phylogeny of Dioscorea sect. Stenophora


317
Table 1 Taxa collected in this study, and their locality, voucher information and GenBank accession numbers
GenBank Accession No. Taxon Locality Voucher
matK trnL-F rbcL
Dioscorea nipponica Makino
ssp. nipponica (穿龙薯蓣)
Lin’an, Zhejiang,
China (浙江临安)
Y. Y. Hang & P. F. Zhang (杭悦宇,
张鹏飞) 0648570 (NAS)
AY957600* DQ841308 AF307455*
D. nipponica ssp. rosthornii
(Prain & Burkill) C. T. Ting
(柴黄姜)
Tianshui, Gansu,
China (甘肃天水)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648571 (NAS)
DQ974184 DQ841309 DQ408178
D. althaeoides R. Knuth (蜀葵
叶薯蓣)
Dêqên, Yunnan,
China (云南德钦)
Y. P. Zhu & Y. M. Zhao (朱昱苹, 赵
亚美) 0648572 (NAS)
EU407548 EU301741 EU407550
D. tokoro Makino (山萆薢) Anhua, Hunan, China
(湖南安化)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648573 (NAS)
DQ974186 DQ841312 DQ408180
D. zingiberensis C. H. Wright
(盾叶薯蓣)
Mt. Hengshan,
Hunan, China (湖南
衡山)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0646476 (NAS)
AY973831* DQ841318 AY939889*
D. sinoparviflora C. T. Ting, M.
G. Gilbert & N. J. Turland
(小花盾叶薯蓣)
Lijiang, Yunnan,
China (云南丽江)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648574 (NAS)
DQ974179 DQ841326 DQ408171
D. deltoidea Wall. ex Griseb.
(三角叶薯蓣)
Kunming, Yunnan,
China (云南昆明)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648575 (NAS)
EF614207 DQ841305 EF614218
D. biformifolia C. Pei & C. T.
Ting (异叶薯蓣)
Eshan, Yunnan, China
(云南峨山)
Y. P. Zhu & Y. M. Zhao (朱昱苹, 赵
亚美) 0648576 (NAS)
EU407549 EU301742 EU301740
D. gracillima Miq. (纤细薯蓣) Mt. Lushan, Jiangxi,
China (江西庐山)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648577 (NAS)
DQ974190 DQ841315 DQ408164
D. collettii Hook. f. var. collettii
(叉蕊薯蓣)
Jinghong, Yunnan,
China (云南景洪)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648578 (NAS)
DQ974178 DQ841300 DQ408173
D. collettii var. hypoglauca
(Palibin) C. Pei & C. T. Ting
(粉背薯蓣)
Mt. Hengshan,
Hunan, China (湖南
衡山)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648579 (NAS)
DQ974176 DQ841319 EF614220
D. futschauensis Uline ex R.
Knuth (福州薯蓣)
Yongtai, Fujian,
China (福建永泰)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648580 (NAS)
DQ974175 DQ841316 DQ408166
D. spongiosa J. Q. Xi, M.
Mizuno & W. L. Zhao (绵萆
薢)
Mt. Hengshan,
Hunan, China (湖南
衡山)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648581 (NAS)
DQ974191 DQ841317 DQ974194
D. banzhuana C. Pei & C. T.
Ting (板砖薯蓣)
Mengzi, Yunnan,
China (云南蒙自)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648582 (NAS)
DQ974182 DQ841301 DQ408174
D. simulans Prain & Burkill (马
肠薯蓣)
Guilin, Guangxi,
China (广西桂林)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648583 (NAS)
EF614206 DQ841320 EF614217
D. caucasica Lipsky Lyon, France (法国里
昂)
Y. F. Zhou & B. C. Wu (周义锋, 吴
宝成) 0648584 (NAS)
DQ974188 DQ841297 DQ408182
D. esculenta (Lour.) Burkill (甘
薯)
Lingshui, Hainan,
China (海南陵水)
Y. F. Zhou & B. C. Wu (周义锋, 吴
宝成) 0648585 (NAS)
AY956497* DQ841298 AY904794*
D. nitens Prain & Burkill (光亮
薯蓣)
Lijiang, Yunnan,
China (云南丽江)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648586 (NAS)
EF614205 EF614223 EF614215
D. bulbifera L. (黄独) Jinghong, Yunnan,
China (云南景洪)
C. H. Huang & K. Y. Guo (黄春洪,
郭可跃) 0648587 (NAS)
AY956488* EF619352 AY904791*
* Obtained from Genbank.


Table 2 Specific primers used to amplify and sequence DNA fragments in this study
Primer Direction Primer sequence (5 to 3)
matK MF forward ATT TGC GAT CTA TTC ATT CAA T
matK MR reverse TGA GAT TCC GCA GGT CAT T
rbcL m3 forward TAT CTT AGC GCC ATT CCG AGT A
rbcL m4 reverse CGC GGA TAA TTT CAT TAC CTT C
trnL-F c forward CGA AAT CGG TAG ACG CTA CG
trnL-F f reverse ATT TGA ACT GGT GAC ACG AG

Journal of Systematics and Evolution Vol. 46 No. 3 2008

318
in effect. For ML analyses, an optimal model of
nucleotide evolution (K81uf+I+Γ, with parameter
values for the proportion of invariant sites (I=0.667)
and the gamma distribution (Γ=0.856)) was selected
using the Akaike Information Criterion as imple-
mented in Modeltest v. 3.07 (Posada & Crandall,
1998). The ML bootstrap analysis (100 replicates) was
conducted with PHYML v. 2.4.4 (Guindon & Gas-
cuel, 2003) under the model with all parameters as
estimated by Modeltest.
2 Results
For matK and rbcL, we obtained 1036 and 1069
nucleotides (nt) of clean sequences, respectively, for
15 taxa in this study (Table 1). No insertion or dele-
tion (indel) was observed in any species for these two
genes. For trnL-F, we obtained approximately 750 nt
for 19 taxa (Table 1). Several indels were found that
separated the three outgroup species from the ingroup,
and a few other autapomorphic ones were also en-
countered in this fragment. For all 22 taxa analyzed,
matK and rbcL had data for virtually the entire length
of the genes, except that D. nipponica ssp. nipponica
and D. esculenta had about 200 and 260 nt missing in
matK, respectively. Three species, D. membranacea,
D. rockii, and D. prazeri lack data for trnL-F.
For parsimony analyses, 1728 equally most par-
simonious trees were found in the branch and bound
search, and one of them is presented in Fig. 1. As the
likelihood analysis generated a tree with similar
topology, the ML tree is not presented. Bootstrap
values above 50% from the parsimony and likelihood
analyses are also shown on this MP tree.
As can be evaluated from the branch length, the
evolutionary divergence between the outgroup and the
ingroup is significant. Within the ingroup, relation-
ships among major clades are not well resolved, either
due to lack of variation in the three molecular mark-
ers, or to a rapid radiation of the taxa in this study, or
both. However, several moderately to strongly sup-
ported groups are clearly identified in this study. First,
D. rockii, D. membranacea, D. banzhuana, and D.
simulans form a moderately supported monophyletic
group, and D. prazeri is weakly supported to be sister
to this group. Second, D. althaeoides and D. nip-
ponica ssp. nipponica form a moderately supported
clade. Surprisingly, D. nipponica ssp. rosthornii is not
a member in this clade. Third, D. zingiberensis and D.
sinoparviflora show a moderate to strong sister rela-
tionship. Finally, D. collettii var. hypoglauca and D.
collettii var. collettii, as expected, group together, but
with only weak support. Relationships of other species
sampled in the sect. Stenophora cannot be resolved in
this study.
3 Discussion
In a previous study that sampled 5 species of
sect. Stenophora and 62 other species representing
most major lineages of the genus Dioscorea, Wilkin et
al. (2005) showed that the section represents a
strongly supported monophyletic group. Our study,
sampling a total of 19 taxa in the sect. Stenophora,
strongly supports the monophyly of the section (Fig.
1). Within the section, despite poor resolution of
relationships among major lineages, several groups
had been identified which shed light on phylogenetic
relationships of some species that were previously not
clear. Our study also indicated that the sect. Steno-
phora represents a natural lineage in this special genus
that might have experienced a rapid radiation in its
evolutionary history.
3.1 Phylogenetic position of D. simulans
Dioscorea simulans was placed in the sect.
Stenophora by Pei et al. (1979) after the sect. Illigere-
strum was abolished. The result of our study strongly
supported their treatment because D. simulans was in
the same clade with some species in sect. Stenophora
(D. banzhuana, D. membranacea, and D. rockii) with
moderate bootstrap support. Previously, several
studies highlighted unique features of D. simulans.
First, it is the only species in sect. Stenophora that has
both simple and compound leaves. The venation of
middle leaflets of the palmate compound leaf is
arc-shaped (Hang, 2007). Second, while most species
in Dioscorea have dioecious flowers (Burkill, 1960),
D. simulans is hermaphroditic and androdioecious
(Hang, 2007). Hermaphroditic flowers also appear in
the genera of Trichopus Gaertn. and Avetra Perrier,
which are regarded as the most basal taxa in Dio-
scoreaceae. Finally, Pei et al. (1979) and Hang (2007)
reported that there was only a trace amount of dios-
genin in D. simulans, which is different from other
taxa in sect. Stenophora. The species in this section
generally have a high concentration of diosgenin. In
the new phylogenetic relationship revealed in this
study, it seems that these features all represent auta-
pomorphies in this species.
3.2 Phylogenetic relationship between D.
zingiberensis and D. sinoparviflora
Dioscorea zingiberensis and D. sinoparviflora
are endemic to China. They were recognized as two
closely related but independent species by Pei et al.
GAO et al.: Phylogeny of Dioscorea sect. Stenophora


319


Fig. 1. One of the 1728 equally most parsimonious trees found in the branch and bound search of the combined matK, rbcL and trnL-F matrix. The
nodes labelled with asterisks are collapsed in the strict consensus tree of all equally most parsimonious trees. Bootstrap values above 50% from the
parsimony and likelihood analyses are shown above and below branches, respectively.


(1979), since they have similar morphology, except
slight difference in the flower size and the
length/width ratio of the capsule. D. sinoparviflora is
distributed along the Jinsha River and the Red River
in Yunnan Province, which is located in the center of
origin of the sect. Stenophora, the Himala-
yas-Hengduan Mountains. On the other hand, D.
zingiberensis is mainly distributed the south of
Qinling Mountain in central China. Thus, D.
zingiberensis is considered to have evolved from D.
sinoparviflora via geographic isolation (Pei et al.,
1979; Wu et al., 2003). Our study of analyzing three
chloroplast markers has clearly confirmed this rela-
tionship (Fig. 1). In addition, unambiguous genetic
variations were observed in matK and trnL-F of the
two species, thus justifying independent species status
of both taxa.
3.3 Phylogenetic positions of D. nipponica ssp.
nipponica, D. althaeoides, and D. nipponica ssp.
rosthornii
Dioscorea nipponica ssp. nipponica and D. nip-
ponica ssp. rosthornii are distributed in the south and
north of Qinling Mountain respectively. The former
has 20 chromosomes and a rhizome with cork layer
readily peeling or flaking off, whereas the latter has 40
chromosomes and a persistent cork layer (The Re-
search Group of Dioscorea, Kiangsu Institute of
Botany, 1976). Other aspects of their morphology are
identical. In this study, D. nipponica ssp. nipponica
was shown to be sister to D. althaeoides with moder-
ate bootstrap support, and D. nipponica ssp. rosthornii
was not related to these two species (Fig. 1). It seems
that D. nipponica ssp. rosthornii needs to be elevated
to the species status and its position in the section
requires further investigation.
3.4 Phylogenetic relationship between D. collettii
var. collettii and D. collettii var. hypoglauca
Burkill (1960) defined D. collettii var. collettii
and D. collettii var. hypoglauca as two species. Based
on the specimens from China, several investigators
described some new species with similar characteris-
tics of D. collettii var. collettii (Prain & Burkill, 1908;
Journal of Systematics and Evolution Vol. 46 No. 3 2008

320
Knuth, 1924). As these new species had overlapping
distribution and little difference in morphology, they
were regrouped into two taxa (The Research Group of
Dioscorea, Kiangsu Institute of Botany, 1976), with
D. collettii var. collettii and D. collettii var. hypo-
glauca. These two taxa overlap in their distribution in
Sichuan Province, China, from where D. collettii var.
collettii extends towards southwest and D. collettii
var. hypoglauca goes towards northeast up to Taiwan
Island. These two taxa are unique among species of
the sect. Stenophora, as they have 3 fertile stamens.
The anther connective is often 1-2 times as wide as the
anther in D. collettii var. collettii, whereas in D.
collettii var. hypoglauca it is significantly shorter, ca.
1/2 the width of the anther (Ting & Michael, 2000).
The chromosomal number of the former was found to
be 20, in contrast to 40 in the latter. On the other hand,
Hang (2007) reported that D. collettii var. collettii and
D. collettii var. hypoglauca exhibit continuous mor-
phological variation from regions where the two taxa
showed a continuous distribution. In addition, 40
chromosomes were observed in D. collettii var. collet-
tii, and 20 chromosomes in D. collettii var. hypo-
glauca. In this study, we found that the two taxa are
closely related, but the bootstrap support is low. It
seems that more sampling of both taxa is needed
before a firm conclusion can be drawn regarding their
status.
3.5 Significance of combining data from different
genes in phylogenetic reconstruction
In recent years, one of the major difficulties in
the molecular phylogeny study has been the incon-
gruence between the phylogeny of genes and the
phylogeny of taxa (Wendel & Doyle, 1998; Sang,
2002). The phylogenetic tree generated from one
single gene reveals the evolution of the gene, not
necessarily the phylogeny of sampled taxa. Thus, the
phylogenetic tree based on the combined analysis of
multiple genes may enhance our ability to reconstruct
the true organismal phylogeny (Miyamoto & Fitch,
1995; Sang, 2002). In this study, the combined 3-gene
analysis clearly out-performed any of the three indi-
vidual genes. We hope that a future study sampling
more genes and more taxa will provide a better resolu-
tion for relationships among taxa within the sect.
Stenophora.
Acknowledgements We thank Dr. Si-Hai YANG
from Nanjing University for helping with data analy-
sis, and Bin Wang and Dr. Yin-Long Qiu from Uni-
versity of Michigan, Ann Arbor for helping with data
analysis and editing English. This work was supported
by Chinese Academy of Sciences Botanical Garden
and Taxonomic Research Projects, Grant. No.
KSCX2-YW-Z-028.
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