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Extensive simple sequence repeat genotyping of
potato landraces supports a major reevaluation of
their gene pool structure and classification
David M. Spooner*
†
, Jorge Nu
´n
˜ez
‡
, Guillermo Trujillo
‡
, Marı´a del Rosario Herrera
‡
, Frank Guzma
´n
‡
, and Marc Ghislain
‡
*Vegetable Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Department of Horticulture, University of Wisconsin, 1575
Linden Drive, Madison, WI 53706-1590; and ‡Applied Biotechnology Laboratory, International Potato Center, Apartado 1558, Lima 12, Peru
Communicated by S. J. Peloquin, University of Wisconsin, Madison, WI, October 16, 2007 (received for review August 15, 2007)
Contrasting taxonomic treatments of potato landraces have con-
tinued over the last century, with the recognition of anywhere
from 1 to 21 distinct Linnean species, or of Cultivar Groups within
the single species Solanum tuberosum. We provide one of the
largest molecular marker studies of any crop landraces to date, to
include an extensive study of 742 landraces of all cultivated species
(or Cultivar Groups) and 8 closely related wild species progenitors,
with 50 nuclear simple sequence repeat (SSR) (also known as
microsatellite) primer pairs and a plastid DNA deletion marker that
distinguishes most lowland Chilean from upland Andean land-
races. Neighbor-joining results highlight a tendency to separate
three groups: (i) putative diploids, (ii) putative tetraploids, and (iii)
the hybrid cultivated species S. ajanhuiri (diploid), S. juzepczukii
(triploid), and S. curtilobum (pentaploid). However, there are many
exceptions to grouping by ploidy. Strong statistical support occurs
only for S. ajanhuiri,S. juzepczukii, and S. curtilobum. In combina-
tion with recent morphological analyses and an examination of the
identification history of these collections, we support the reclas-
sification of the cultivated potatoes into four species: (i)S. tubero-
sum, with two Cultivar Groups (Andigenum Group of upland
Andean genotypes containing diploids, triploids, and tetraploids,
and the Chilotanum Group of lowland tetraploid Chilean land-
races); (ii)S. ajanhuiri (diploid); (iii)S. juzepczukii (triploid); and (iv)
S. curtilobum (pentaploid). For other classifications, consistent and
stable identifications are impossible, and their classification as
species is artificial and only maintains the confusion of users of the
gene banks and literature.
cultivated 兩microsatellites 兩sect. Petota 兩Solanum tuberosum 兩taxonomy
The cultivated potato represents one of the most important
food plants worldwide, yet interpretation of its gene pool
structure remains controversial. Contrasting taxonomic treat-
ments of the landraces have continued over last century, with the
recognition of anywhere from 1 to 21 distinct Linnean species,
or of various Cultivar Groups within the single species S.
tuberosum (1). For consistency in usage in our article and to
maintain the names most familiar to scientists, we use the seven
species terminology of Hawkes (2). Indigenous cultivated (land-
race) potatoes are widely distributed in the Andes from western
Venezuela, south to northern Argentina, and with another set of
landraces in south-central Chile in Chiloe´ Island and the adja-
cent Chonos Archipelago. The Chilean landraces, although once
proposed to have arisen independently from central Chile (3),
are secondarily derived from the Andean ones (2), likely after
hybridization with the Bolivian and Argentinean species Sola-
num berthaultii (4), a species recently combined with the for-
merly recognized wild species S. tarijense (5). Three of the
Andean-cultivated species are hypothesized to be of hybrid
origins with cultivated potatoes and wild species: S. ajanhuiri [S.
stenotomum cultivated ⫻S. megistacrolobum wild (6)], S. juz-
epczukii [S. stenotomum ⫻S. acaule wild (7, 8)], and S. curti-
lobum [S. andigenum cultivated ⫻S. juzepczukii (7, 8)]. The latter
three ‘‘bitter potatoes’’ are grown in upland habitats and are not
grown nearly as extensively as S. tuberosum, as outlined by
Huama´n and Spooner (1).
The relationships and extent of genetic differentiation be-
tween the Andean and Chilean landraces has long been contro-
versial. Based on cytoplasmic sterility factors, geographical
isolation, and ecological differences, Grun (9) suggested that
Chilean landraces were distinct from Andean landraces. Hawkes
(2) distinguished the tetraploid Chilean from Andean landraces
by characters of the leaf and flower pedicel. Plastid restriction
site data documented five genotypes (A, C, S, T, and W types)
in the diploid and tetraploid Andean landraces, and the Chilean
landraces had three types, A, T, and W (10, 11). The most
frequently observed type in the Chilean landraces (21 of 24 or
87.5% of the accessions examined) is type T, which is charac-
terized by a 241-bp deletion (12). Conversely, 5 of the 113 (4.4%)
accessions of S. tuberosum subsp. andigenum had the T type
(10–12).
Potato landraces have been classified into 21 species (13, 14),
7 species with seven subspecies (2) and 9 species with two
subspecies (15, 16), or as the single species S. tuberosum with 8
user-defined Cultivar Groups (1). Cultivar Groups are taxo-
nomic categories used by the International Code of Nomencla-
ture of Cultivated Plants to associate cultivated plants with traits
that are of use to agriculturists and are not meant to represent
natural groups or species in any classification philosophy. Ploidy
levels in cultivated potatoes range from diploid (2n⫽2x⫽24),
to triploid (2n⫽3x⫽36), to tetraploid (2n⫽4x⫽48), to
pentaploid (2n⫽5x⫽60). Huama´n and Spooner (1) examined
the morphological support for the various classifications of
potato landraces using representatives of all seven species from
the classification of Hawkes (2). The results showed some
morphological support for S. ajanhuiri,S. chaucha,S. curtilobum,
and S. juzepczukii, lesser support for S. tuberosum subsp. tubero-
sum, and no support for S. phureja and S. stenotomum. Whatever
morphological support for these entities was present was only by
using a suite of characters, all of which are shared with other taxa
(polythetic support). These results, combined with their likely
hybrid origins, multiple origins, and evolutionary dynamics of
continuing hybridization, led Huama´n and Spooner (1) to rec-
ognize all landrace populations of cultivated potatoes as a single
species, S. tuberosum, with the eight Cultivar Groups: Ajanhuiri
Group, Andigenum Group, Chaucha Group, Chilotanum
Group, Curtilobum Group, Juzepczukii Group, Phureja Group,
and Stenotomum Group (the latter containing all landraces of
the Goniocalyx Group).
Author contributions: J.N., G.T., M.d.R.H., F.G., and M.G. performed research; and D.M.S.
wrote the paper.
The authors declare no conflict of interest.
†To whom correspondence should be addressed. E-mail: david.spooner@ars.usda.gov.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0709796104/DC1.
© 2007 by The National Academy of Sciences of the USA
19398–19403
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The wild relatives of these landraces (Solanum section Petota) are
all tuber-bearing and include ⬇190 wild species that are widely
distributed in the Americas from the southwestern United States to
southern Chile (17); they possess all ploidy levels of the cultivars, as
well as hexaploids (2n⫽6x⫽72). Spooner et al. (18) studied the
origin of the landraces S. tuberosum subsp. andigenum,S. tuberosum
subsp. tuberosum,S. phureja, and S. stenotomum with amplified
fragment length polymorphisms (AFLPs). They discovered that (i)
in contrast to all prior hypotheses, these species were shown to have
a monophyletic origin; (ii) the wild species progenitors were from
a group of very similar wild potato species classified in the Solanum
brevicaule complex; and (iii) the landraces had their origin in the
highlands of southern Peru.
Although ploidy has been a major feature to define the
cultivated species, many cultivated potato germplasm collections
lack chromosome numbers, and many assumptions of ploidy are
likely in error. For example, Ghislain et al. (19) used simple
sequence repeats (SSRs) (also known as microsatellites) to
48
63
52 75
89
100
48
48
48
36
36
48
48
48
48
48
48
48
X
T
T
T
T
TT
T
T
T
T
T
T
T
X
X
X
X
X
All T except
2 as X
phu
cur
S. juzepczukii (juz)
S. ahanhuiri (ajh)
S. curtilobum (cur)
S. brevicaule group
S. acaule (4x) wild species
00.2
ajh
ajh
ajh
stn
cha
juz
A
Fig. 1. (Figure continues on the opposite page.)
Spooner et al. PNAS
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EVOLUTION
36
48
48
48
36
36
36 36 36
36
48
48 48
36
36 48
24
24
48
48
48
48
48
48
T
T
X
Pink - Solanum ajanhuiri (ajh, 2x), S. juzepczukii (juz, 3x), S. curtilobum (cur, 5x)
Dark blue - S. tuberosum subsp. andigenum (adg, 4x)
Green - S. chaucha (cha, 3x)
Gray - S. tuberosum subsp. tuberosum (tub, 4x)
Red - S. phureja (phu, 2x)
Light blue - S. stenotomum (stn, 2x)
B
Fig. 1. Jaccard’s tree based on a dissimilarity matrix of 742 potato landraces and 8 wild species examined with 50 microsatellite primer pairs. (Inset) Overall
view of the entire tree. (A) Detail of the left-hand side of this tree, which contains mostly putative polyploid accessions (the ‘‘polyploid cluster’’) and accessions
of S. ajanhuiri,S. curtilobum,S. juzepczukii, and wild species. (B) Mostly putative diploid accessions and some triploids (the ‘‘diploid cluster’’). Chromosome
numbers (36 and 48) are labeled for accessions not 24 within the main red S. phureja cluster (in red) on the left right side of the tree or previously identified as
S. phureja but falling outside of the red cluster. The circled ‘‘T’’ (possessing a 241-bp deletion) and ‘‘X’’ (no deletion) designate accessions unexpected to occur
in these clusters because they either lack the T cytoplasm thought be largely confined to lowland Chile (area in the wedge) or lack this deletion (area outside
of the wedge containing mostly accessions from Venezuela to Argentina).
19400
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assess diversity in the S. phureja collection at the International
Potato Center. Solanum phureja is widely grown in the Andes
from western Venezuela to central Bolivia and has been defined
by short-day adaptation, diploid ploidy (2n⫽2x⫽24), and a lack
of tuber dormancy. SSR results, in combination with chromo-
some counts, uncovered fully 31% (32 of 102 accessions exam-
ined) triploid and tetraploid accessions from the International
Potato Center collection of S. phureja that were long assumed to
be exclusively diploid.
The purpose of our study is to reexamine the support for
classification categories for landrace potatoes, using nuclear SSR
markers developed for optimal utility in S. tuberosum regarding
polymorphism, quality scores, and genomic coverage (20), sup-
plemented with a plastid DNA deletion marker as discussed
below. Nuclear SSRs have been shown to be ideal markers for
detecting phylogenetically significant diversity within cultivated
potatoes (19, 21, 22). In addition, their codominant nature allows
them to identify polyploids when three or four bands (alleles) are
found, as was shown by Ghislain et al. (19). This is particularly
important for our study, where few cultivated accessions have
been characterized for chromosome number yet ploidy has been
so important conceptually in defining the cultivated species.
Our study also used the 241-bp plastid deletion marker distin-
guishing most populations of Chilean from Andean potato
landraces (4, 12, 23).
Results and Discussion
SSR Neighbor-Joining (NJ) Tree. NJ results highlight a tendency to
separate three broad groups: (i) putative diploids and triploids
(all accessions in the diploid cluster Fig. 1), (ii) putative tet-
raploids and triploids (most accessions in the polyploid cluster of
Fig. 1), and (iii) the hybrid cultivated species S. ajanhuiri
(diploid), S. juzepczukii (triploid), and S. curtilobum (penta-
ploid), grouped with the wild species. Bootstrap support above
50% is common in many small groups of species in the terminal
branches of the NJ tree (not shown because they greatly com-
plicate the graphic). However, bootstrap support above 50% is
present only in the lower nodes supporting S. ajanhuiri,S.
juzepczukii, and S. curtilobum and the wild species.
However, there are many exceptions of clustering by ploidy.
Landraces of S. goniocalyx, as in the morphological study of
Huama´n and Spooner (1), were invariably intermixed with
those of S. stenotomum and are so labeled as this species on
Fig. 1. There are 28 putative triploid landraces of S. chaucha
present on the main polyploid cluster of the tree and 123 on
the main diploid cluster. There are 28 putative tetraploids (S.
tuberosum subsp. andigenum and subsp. tuberosum)onthe
diploid cluster and 28 putative diploids (S. phureja and S.
stenotomum) on the tetraploid cluster. In addition, S. phureja
(the only cultivated species with extensive chromosome
counts) shows 18 of the 89 accessions to be triploid or
tetraploid. Regarding the S. phureja, Fig. 1 shows the position
of all accessions formerly identified as this species in the
International Potato Center collection (24) but shown to be
polyploid in the study of Ghislain et al. (19). Most are present
in the main ‘‘S. phureja cluster’’ (red dots in the diploid cluster
of Fig. 1), but this cluster also contains two accessions of S.
tuberosum subsp. andigenum, two of S. stenotomum, and one of
S. chaucha, and with 10 accessions elsewhere on the diploid
cluster and 16 on the polyploid cluster. As expected, most (22
of 27) of the S. tuberosum subsp. tuberosum accessions clus-
tered together (the area in the wedge in the polyploid cluster
in Fig. 1). However, this cluster also contained three accessions
of S. tuberosum subsp. andigenum.
241-bp Plastid Deletion. We determined the presence or absence
of the 241-bp plastid deletion for all 742 cultivated accessions
examined. As expected, most (22 of 23) of the S. tuberosum
subsp. tuberosum accessions in the main cluster of this subspecies
(designated by the wedge in the polyploid cluster of Fig. 1)
possessed this deletion. Also in the area of the wedge are three
tetraploid accessions from Peru (dark blue); two of these possess
the deletion, and one lacks it (the two accessions lacking the
deletion are marked with ‘‘X’’).
All four remaining accessions from Chile falling outside of this
cluster (gray dots marked with ‘‘X’’) lack the 241-bp deletion
characteristic of this subspecies, suggesting misidentifications of
possible recent introductions of the S. tuberosum subsp. andige-
num into Chile. Thirteen of the 251 S. tuberosum subsp. andi-
genum accessions (5.2%; marked with ‘‘T’’ outside of the gray S.
tuberosum subsp. tuberosum cluster) possessed the deletion,
similar to the 4.4% reported in prior studies (above). These 13
accessions are widely distributed throughout the Andes in
Venezuela (2 accessions), Colombia (1 accession), Ecuador (13
accessions), Peru (4 accessions), Bolivia (2 accessions), and
Argentina (1 accession). In addition, one of the S. stenotomum
accessions (putatively diploid) and two S. phureja accessions
(known as diploid) possessed this deletion, the first report of
diploid potatoes possessing this deletion, because none of the
accessions of S. stenotomum (215) and S. chaucha (150) previ-
ously screened was found with this marker (19, 25). Unfortu-
nately, these three accessions do not have reliable collection
information.
Reconsideration of the Classification of Cultivated Potato. In com-
bination with a recent morphological study (1), the SSR data
support the reclassification of the cultivated potatoes into four
species: (i)S. tuberosum,(ii)S. ajanhuiri (diploid), (iii)S. juzepczukii
(triploid), and (iv)S. curtilobum (pentaploid). We support dividing
S. tuberosum into two Cultivar Groups (Andigenum Group of
upland Andean genotypes containing diploids, triploids, and tet-
raploids, and the Chilotanum Group of lowland tetraploid Chilean
landraces). Because Cultivar Groups are taxonomic categories used
to associate cultivated plants with traits that are of use to agricul-
turists, this classification is convenient to separate these populations
that grow in different areas, are adapted to different day-length
regimes, and have some degree of unilateral sexual incompatibility
to the Andean populations. For the remaining ‘‘species’’ or Cultivar
Groups, consistent and stable identifications are impossible, their
classification as Linnean species is artificial, and their maintenance
as either species or Cultivar Groups only serves to perpetuate
confusion by breeders and gene bank managers, and the instability
of names in the literature. For example, Ghislain et al. (19) showed
S. phureja to be indefinable as traditionally recognized because
prior authors incorrectly assumed that their assumption of diploidy
was incorrect for 31% of the accessions, and our results showed
many accessions of S. phureja to cluster with the polyploids. The
recognition of S. phureja as either a species or Cultivar Group
(Phureja Group), therefore, is no longer tenable because it is no
longer diploid, does not exclusively possess low-dormancy tubers, is
not short-day adapted, and is not morphologically coherent (1). The
other species (or Cultivar Groups) have ploidy as a major identi-
fying criterion. The results from S. phureja and this study indicate
that chromosome counts from other accessions of cultivated pota-
toes will uncover a high proportion of counts not matching expec-
tations based on their identifications.
Ploidy has been of great importance in the classification of
cultivated potatoes, but our results show so many exceptions that it
is a poor character to define gene pools. Cultivated potato fields
contain mixtures of different ploidy levels (6, 26 –32). Bukasov (33)
was the first to count chromosomes of the cultivated potatoes and
used ploidy variation to speculate on hybrid origins. The strong
reliance on ploidy levels was clearly stated by Hawkes and Hjerting
(34): ‘‘The chromosome number of 2n⫽36 largely helps to identify
S. chaucha, but morphological characters can also be used.’’
Morphology is a poor character to define most species or Cultivar
Spooner et al. PNAS
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EVOLUTION
Groups except for the bitter potato species S. ajanhuiri,S. curti-
lobum, and S. juzepczukii. As shown by Huama´n and Spooner (1),
most traditionally recognized cultivated potato species have little
morphological support, and then only by using a suite of characters,
all of which are shared with other taxa (polythetic support).
The International Potato Center has collected cultivated pota-
toes for 30 years and has invested tremendous effort in their
identification. An examination of identification records at the
International Potato Center shows many changes over the years,
further showing the lack of stability of any character set to reliably
define most cultivated species.
Potato gene banks are in great need of an integrated and
comprehensive program of ploidy determinations; controlled and
replicated studies of tuber dormancy (which we suspect will high-
light grades of dormancy, not the present/absent determinations
that exist today); photographically documented determinations of
tuber and flesh colors and tuber shapes; and determinations of
tuber pigments, glycoalkaloid contents, carbohydrates, proteins,
amino acids, minerals, and secondary metabolites, using functional
genomics approaches, with all data publicly integrated into a readily
searchable web-based bioinformatics database. Such a multicom-
ponent system will serve the breeding community much better than
the outdated, unstable, and phylogenetically indefensible tradi-
tional classifications that exist today.
Materials and Methods
Plant Materials. A total of 742 potato landraces of all cultivated
potato species were examined: S. tuberosum subsp. andigenum,
putatively tetraploid (251 accessions); S. ajanhuiri, diploid
(22); S. chaucha, triploid (151 accessions); S. tuberosum subsp.
tuberosum, tetraploid (27 accessions); S. curtilobum, penta-
ploid (21 accessions); S. juzepczukii, triploid (35 accessions); S.
phureja, diploid (104 accessions); S. stenotomum, diploid (131
accessions); 7 diploid wild species accessions in the northern
S. brevicaule complex S. ambosinum Ochoa (1 accession), S.
bukasovii Juz. (4 accessions), and S. multiinterruptum Bitter (2
accessions); and the wild tetraploid species S. acaule Bitter (1
accession) (750 accessions in total with the 8 wild species).
Selection of these wild species is based on recent amplified
fragment length polymorphism (AFLP) studies that docu-
mented the northern S. brevicaule complex wild species to be
the progenitors of the cultivated potatoes and S. acaule
believed to be a wild species parent in the hybrid species S.
juzepczukii and S. curtilobum. We qualify landrace collection
ploidy as ‘‘putative’’ because only S. phureja has been counted
in detail (19), that showed extensive examples of incorrect
assumptions of ploidy as discussed above. Data of these
accessions that includes International Potato Center accession
number, taxonomic identification, ploidy when known, locality
of collection, and average number of SSR alleles per accession
are available as a supporting information (SI) Dataset.
DNA Extraction, SSR Primers, PCR Conditions, and Electrophoresis.
Genomic DNA was obtained by using standard protocols at the
International Potato Center (35). DNA concentration was calcu-
lated by using PicoGreen dsDNA quantitation reagent (Molecular
Probes) and a TBS-380 Fluorometer (Turner BioSystems). DNA
dilutions were performed to achieve a final concentration of 3
ng/
l, using 96-well plates. We used 50 nuclear SSRs (see SI
Dataset) screened from 88 that included the 22 from the Potato
Genetic Identity (PGI) kit (20), 13 from ESTs developed at the
Scottish Crop Research Institute (36), 30 identified by using the
potato EST database at TIGR, and 23 from the University of Idaho
(37). PCR reactions were performed in a 10-
l volume containing
100 mM Tris䡠HCl (Sigma), 20 mM (NH
4
)
2
SO
4
(Merck), 2.5 mM
MgCl
2
(Merck), 0.2 mM each dNTP (Amersham Biosciences), 0.3
M labeled M13 forward primer (LI-COR IRDye 700 or 800), 0.3
M M13-tailed SSR forward primer (Invitrogen), 0.2
M SSR
reverse primer (Invitrogen), 1 unit of Taq polymerase (GIBCO/
BRL), and 15 ng of genomic DNA. PCR was carried out in a
PTC-200 thermocycler (MJ Research). The program used was the
following: 4 min at 94°C, followed by 33 cycles of 50 sec at 94°C, 50
sec at annealing temperature (T°a), and 1 min at 72°C, then 4 min
at 72°C as a final extension step. PCR products were separated by
electrophoresis on a LI-COR 4300 DNA analyzer system. The
molecular weight ladder was the LI-COR IRDye 50–350 bp size
standard and was loaded into gel each eight samples.
SSR Allele Scoring. SSR alleles were detected and scored by using
SAGA Generation 2 software (LI-COR). Size calibration and an
SSR ‘‘smiling line’’ were performed by using the molecular weight
ladder (LI-COR IRDye 50–350). The SSR alleles were determined
for size in bp of the upper band of the allele and scored as present
(1) or absent (0). Missing data were scored as ‘‘9.’’
Data Analysis. Genetic analysis was performed by using the prog ram
DARwin (38). A dissimilarity matrix was calculated by using
Jaccard’s coefficient, 60% of minimal proportion of valid data
required for each unit pair, and 500 replicate bootstrapping. The
dendrogram was built by using the NJ method, using the seven wild
species accessions in the northern S. brevicaule complex as out-
group. The NJ method developed by Saitou and Nei (39) estimates
phylogenetic trees. Although based on the idea of parsimony (it
does yield relatively short estimated evolutionary trees), the NJ
method does not attempt to obtain the shortest possible tree for a
set of data. Rather, it attempts to find a tree that is usually close to
the true phylogenetic tree (40). This method allows the rooting of
trees on outgroups (in this case, the seven accessions of the S.
brevicaule complex). The polymorphic information content (PIC)
was calculated as PIC ⫽1⫺⌺(p
i
2
), where p
i
is the frequency of the
ith allele detected in all accessions (41). Data of somatic chromo-
some counts for accessions of S. phureja were obtained from
Ghislain et al. (19).
Plastid DNA Polymorphism Detection. The 241-bp deletion was
analyzed for all 742 cultivated accessions by using the primers from
ref. 23. PCR amplification was performed in a volume of 10
l
consisting of 18 ng of genomic DNA, 0.4
M each of primers
(Invitrogen), 1⫻PCR buffer (PerkinElmer), 2.5 mM MgCl
2
(PerkinElmer), 200
M each dNTP (Amersham Biosciences), and
0.25 unit of Taq DNA polymerase (GIBCO/BR L). Thermal cycling
was carried out in a PTC-200 thermocycler (MJ Research) (one
cycle of 4 min at 94°C, followed by 40 cycles of 45 sec at 94°C, 45
sec at 59°C, and 45 sec at 72°C, then terminated with one cycle of
4 min at 72°C). PCR products were separated by electrophoresis in
a 1% agarose gel, and lambda phage digested by PstI was used as
a molecular weight marker. The 241-bp plastid polymorphism was
determined for size in bp and scored as ‘‘T’’ (⬇200 bp for deleted
type) and ‘‘X’’ (⬇440 bp for undeleted type).
We thank David Douches and Lynn Bohs for comments on an earlier draft
of the manuscript. This work was supported by the International Potato
Center, U.S. Department of Agriculture, Generation Challenge Program
Grant SP1C2-2004-5, and National Science Foundation Grant DEB
0316614 entitled ‘‘A world-wide treatment of Solanum’’ (to D.M.S.).
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