A single domestication for potato based on multilocus AFLP genotyping

Abstract

The cultivated potato, Solanum tuberosum, ultimately traces its origin to Andean and Chilean landraces developed by pre-Colombian cultivators. These Andean landraces exhibit tremendous morphological and genetic diversity, and are distributed throughout the Andes, from western Venezuela to northern Argentina, and in southern Chile. The wild species progenitors of these landraces have long been in dispute, but all hypotheses center on a group of ≈20 morphologically very similar tuber-bearing (Solanum section Petota) wild taxa referred to as the S. brevicaule complex, distributed from central Peru to northern Argentina. We present phylogenetic analyses based on the representative cladistic diversity of 362 individual wild (261) and landrace (98) members of potato (all tuber-bearing) and three outgroup non-tuber-bearing members of Solanum section Etuberosum, genotyped with 438 robust amplified fragment length polymorphisms. Our analyses are consistent with a hypothesis of a “northern” (Peru) and “southern” (Bolivia and Argentina) cladistic split for members of the S. brevicaule complex, and with the need for considerable reduction of species in the complex. In contrast to all prior hypotheses, our data support a monophyletic origin of the landrace cultivars from the northern component of this complex in Peru, rather than from multiple independent origins from various northern and southern members.

• evolution
• sect. Petota
• Solanum

Full text

A single domestication for potato based
on multilocus amplified fragment length
polymorphism genotyping
David M. Spooner*
†
, Karen McLean
‡
, Gavin Ramsay
‡
, Robbie Waugh
‡
, and Glenn J. Bryan
‡
*U.S. Department of Agriculture, Agricultural Research Service, Vegetable Crops Research Unit, Department of Horticulture, University
of Wisconsin, 1575 Linden Drive, Madison, WI 53706-1590; and ‡Genome Dynamics Programme, Scottish Crop Research Institute,
Invergowrie, Dundee DD2 5DA, United Kingdom
Communicated by S. J. Peloquin, University of Wisconsin, Madison, WI, August 29, 2005 (received for review November 15, 2004)
The cultivated potato, Solanum tuberosum, ultimately traces its
origin to Andean and Chilean landraces developed by pre-Colom-
bian cultivators. These Andean landraces exhibit tremendous mor-
phological and genetic diversity, and are distributed throughout
the Andes, from western Venezuela to northern Argentina, and in
southern Chile. The wild species progenitors of these landraces
have long been in dispute, but all hypotheses center on a group of
⬇20 morphologically very similar tuber-bearing (Solanum section
Petota) wild taxa referred to as the S. brevicaule complex, distrib-
uted from central Peru to northern Argentina. We present phylo-
genetic analyses based on the representative cladistic diversity of
362 individual wild (261) and landrace (98) members of potato (all
tuber-bearing) and three outgroup non-tuber-bearing members of
Solanum section Etuberosum, genotyped with 438 robust ampli-
fied fragment length polymorphisms. Our analyses are consistent
with a hypothesis of a ‘‘northern’’ (Peru) and ‘‘southern’’ (Bolivia
and Argentina) cladistic split for members of the S. brevicaule
complex, and with the need for considerable reduction of species
in the complex. In contrast to all prior hypotheses, our data
support a monophyletic origin of the landrace cultivars from the
northern component of this complex in Peru, rather than from
multiple independent origins from various northern and southern
members.
evolution 兩sect. Petota 兩Solanum
The origin of crop plants has long fascinated botanists,
archaeologists, and sociologists with the following funda-
mental questions: When, where, how, why, and how many times
did crop domestication occur? What are the wild progenitors of
these crops? How do crops differ from their progenitors, what
selective processes, and how many genetic changes produce these
changes? Did crops have single or multiple and separate origins
(1–5)? Single (diffusionist) vs. multiple origin (in situ) hypoth-
eses of crop origins has long been the subject of debate (6– 8).
We have used multilocus molecular data from amplified frag-
ment length polymorphisms (AFLPs) to reassess a single vs.
multiple origin of landrace cultivars of cultivated potato.
Primitive indigenous cultivated (landrace) potatoes are widely
distributed in the Andes from western Venezuela south to
northern Argentina, and in Chiloe´ Island and the adjacent
Chonos Archipelago of south-central Chile. The Chilean land-
races are secondarily derived from the Andean ones (9), likely
after hybridization with the Bolivian and Argentinean species
Solanum tarijense (10). Potato landraces have been classified into
21 species (11, 12), 7 species (9), 9 species (13, 14), or as a single
species, S. tuberosum, with eight cultivar groups (15). The
landraces are very diverse, with hundreds of clones differing in
tuber colors and shapes, and leaf, floral, and growth habit
variations. Ploidy levels in cultivated potato range from diploid
(2n⫽2x⫽24), to triploid (2n⫽3x⫽36), to tetraploid (2n⫽
4x⫽48), to pentaploid (2n⫽5x⫽60). 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
(16, 17); they possess all ploidy levels of the cultivars, as well as
hexaploids (2n⫽6x⫽72).
The wild species progenitors of these Andean landraces have
long been in dispute, but all hypotheses center on a group of ⬇20
morphologically similar wild species referred to as the S. brevi-
caule complex, distributed from central Peru to northern Ar-
gentina (18–22). Members of the complex are morphologically
similar to the landraces. Potato domestication from these wild
species involved selection for underground characters of shorter
stolons, larger tubers, (often) colored and variously shaped
tubers, and the reduction of bitter tuber glycoalkaloids; above-
ground characters of wild and cultivated species are similar but
with cultivated types exhibit high vigor and extensive segregation
for flower and foliage traits. The S. brevicaule complex includes
diploids, tetraploids, and hexaploids. Many members grow as
weeds in or adjacent to cultivated potato fields and form
crop–weed complexes (19). Morphological data (21) and single-
to low-copy nuclear restriction fragment length polymorphism
data (22) failed to clearly differentiate wild species in the S.
brevicaule complex from each other or from most landraces
(although the landraces often are taller and more vigorous as a
group than the wild species), and the most liberal taxonomic
interpretation from these studies was to recognize only three
wild taxa: (i) the Peruvian populations of the S. brevicaule
complex, (ii) the Bolivian and Argentinean populations of the
S. brevicaule complex, and (iii)S. oplocense (Bolivia and
Argentina).
Literally all hypotheses have suggested complex hybrid or
multiple origins of the cultivars from both northern and southern
members of the S. brevicaule complex (9, 13–15, 19–21, 23–25).
This study investigates these hypotheses through phylogenetic
analyses that incorporate the first comprehensive sampling of
landraces, the putative progenitors, and outgroups.
Materials and Methods
Plant Materials. We sampled 362 individual wild (261) and
landrace (98) members of tuber-bearing relatives of potato
(Solanum section Petota) and three sister group representatives
in Solanum section Etuberosum (S. etuberosum and S. palustre).
These accessions came from the United States Potato Genebank
(www.ars-grin.gov兾nr6) and the Commonwealth Potato Collec-
tion of the Scottish Crop Research Institute (www.scri.ac.uk兾
cpc). They were identified mostly from living accessions planted
at the genebank by visiting potato taxonomists (mainly Jack
Hawkes and Carlos Ochoa) over 30 years. The 264 wild species
Abbreviations: AFLP, amplified fragment length polymorphism; RAPD, randomly amplified
polymorphic DNA; RFLP, restriction fragment length polymorphism.
†To whom correspondence should be addressed. E-mail: dspooner@wisc.edu.
© 2005 by The National Academy of Sciences of the USA
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accessions are mostly members of the S. brevicaule complex and
are largely the same ones used in prior morphological (21) and
molecular (22) studies of the complex, and are labeled as those
species. We included tetraploid S. stoloniferum to have a data set
comparable to the prior morphological and molecular studies.
The members of the S. brevicaule complex are so similar that
their identities have frequently changed, and we mostly use the
identities from these prior studies for consistency. We add
additional cultivated accessions and wild species to represent the
entire four-clade diversity of section Petota from Spooner and
Castillo (26), and they included members of the Phureja Group
(diploid, Andean), Stenotomum Group (diploid, Andean), An-
digenum Group (tetraploid, Andean), and Chilotanum group
(tetraploid, lowland Chile). Our study included 230 diploids
(2n⫽2x⫽24), 120 tetraploids (2n ⫽4x ⫽48), and 12 hexaploids
(2n⫽6x⫽72). S. gourlayi included its diploid and tetraploid
cytotypes, and S. oplocense included its tetraploid and hexaploid
cytotypes. Designations of ploidy and classes of species (culti-
vated, S. brevicaule group north, S. brevicaule south, outgroups,
and ploidy) are presented in Fig. 1.
AFLP Genotyping. Plants were grown in the glasshouse, and DNA
was extracted from frozen plant leaf tissue taken from single
plants by using the DNeasy Plant DNA Extraction kit (Qiagen,
cat. no. 69181). AFLP assays were performed by using a mod-
ification of the protocol of Vos et al. (27), using the 6-bp cutting
enzymes PstI and EcoR I and the 4-bp cutting enzyme MseI. The
six AFLP primer combinations used were EAAC ⫹MCCA,
EACA ⫹MCAC, PAC ⫹MACT, PAG ⫹MACC, PAT ⫹
MAAC, and PCA ⫹MAGG. PCR reactions were set up on
384-well plates by using a Beckman Biomek 2000 liquid handling
device. Electrophoresis was carried out on the Bio-Rad Sequi-
Gen GT system on 5% acrylamide and 7 M urea in 1⫻TBE
buffer (100 mM Tris兾100 mM boric acid兾2 mM EDTA). A dual
buffer system of 1⫻TBE and 1⫻TBE supplemented with 0.5 M
NaOAc was used to create an ionic gradient, which resulted in
better separation of the larger fragments. A Promega fmol DNA
Cycle Sequencing system (Promega Q4100) marker (prepared
according to the protocol but using only d兾ddT Nucleotide Mix)
was run to estimate fragment sizes. Gels were dried onto paper
and visualized by exposure to x-ray film (Kodak BIOMAX MR).
Gels were scanned by using a standard flat-bed scanner at 300
dpi and 8-bit grayscale format. The TIFF images were then
imported into AFLP-QUANTAR software supplied by Keygene
(Wageningen, The Netherlands). To enable automatic position-
ing of marker bands, a standard genotype was run on each gel.
The AFLP data matrices are published as supporting informa-
tion on the PNAS web site.
Phylogenetic Analysis. Phylogenetic reconstructions were per-
formed by using PAUP* 4.0b8 (28), using Wagner parsimony (29).
The non-tuber-bearing species S. etuberosum and S. palustre
(section Etuberosum) were designated as the outgroup, but
members of these species, and clades 1, 2, and 3 of Spooner and
Castillo (26), fall at the base of the tree and are listed as
outgroups in Fig. 1; all remaining wild and cultivated species are
members of clade 4. To find multiple tree islands, we used a
four-step search strategy, modified from Olmstead and Palmer
(30). (i) One million replicates initially were run by using random
order entry starting trees with nearest-neighbor interchange. (ii)
The shortest trees from this analysis were used individually as
starting trees with the tree-bisection-reconnection (TBR)
method. (iii) The resulting trees were searched with nearest-
neighbor interchange, retaining all most parsimonious trees
(MULPARS). (iv) The resulting trees were searched with TBR
and MULPARS. The last two analyses were terminated at 10,000
trees. The resulting trees were used to compute a strict consensus
tree. A bootstrap analysis was conducted on 500 replicates with
TBR and MULPARS. The above analyses were done twice, once
with the entire data set and again only with the diploids.
AFLP data were also analyzed by neighbor-joining using
NTSYS-PCR2.02k (31). The program SIMQUAL was used to compute
similarity matrices using the “Jaccard” option, which ignores
shared absent bands, and which is an appropriate algorithm for
AFLPs scored as dominant markers.
Concordance Tests Among the Present AFLP Data to the Prior Restric-
tion Fragment Length Polymorphism (RFLP), Randomly Amplified
Polymorphic DNA (RAPD), and Morphological Studies. We tested
concordance among our new AFLP results to the morphological
(21) and single- to low-copy RFLP and RAPD data (22) of the
S. brevicaule complex. For each data set, we constructed parallel
matrices containing accessions in common between studies. We
then made pairwise distance matrices for all four data sets. For
the AFLP and RAPD data, we used the Jaccard matrix, for the
RFLP data we used a simple-matching coefficient, and for the
morphological data we used the distance algorithm, all present
in NTSYS-PCR2.02k (31). We then performed pairwise compari-
sons of these matrices with the Mantel test (32) as performed in
NTSYS-PCR. This statistic varies from 0 (no correspondence of
matrices) to 1 (perfect correspondence).
Results and Discussion
Cladistic Results. The six AFLP primer combinations produced
438 characters of which 3.6% of the data matrix had missing
values, caused by occasional failed reactions or faint bands.
Wagner parsimony analysis of all 362 accessions (Fig. 1) pro-
duced 10,000 (our designated upper tree limit) most parsimo-
nious 13,672-step trees with a consistency index of 0.033 and a
retention index of 0.571, and a rescaled consistency index of
0.019. The topology of the entire data set is ver y similar to the
four clade cladistic structure of Spooner and Castillo (26).
S. etuberosum and S. palustre form a monophyletic basal out-
group, sister to S. bulbocastanum,S. polyadenium,S. stenophyl-
lidium, and S. tarnii (clades 1 and 2), sister to S. acroscopicum,
S. andreanum,S. chilliasense,S. pascoense, and S. paucissectum
(clade 3); this clade also includes two accessions of S. acroscopi-
cum and two accessions of S. multidissectum, members of the
S. brevicaule group. The bootstrap analysis (Fig. 1) showed
⬎50% support for the basal clades of sect. Etuberosum and
clades 1–3, and in some internal branches of clade 4, but poor
support (⬍50%) in the external branches of clade 4. Wagner
parsimony analysis of the 230 diploid accessions (data not
shown) produced 177 most parsimonious 8,393-step trees with a
consistency index of 0.0534, and a retention index of 0.5617 and
a rescaled consistency index of 0.0300; bootstrap support was
similar to the total taxon tree. The topology of the entire data
set and the reduced data set of the diploids differs very little.
The topology of the entire data set is in concordance with
the morphological (21), RAPD, and RFLP results (22) in
defining a northern (species from Peru, together with S.
achacachense from northern Bolivia) and southern (species
from Bolivia and northern Argentina) clade of the S. brevicaule
complex. This geographic split does not exactly follow country
borders, but very closely so. For example, the northern clade
contains S. achacachense PI 558032 from the department of La
Paz, Bolivia bordering Peru, and the southern clade contains
S. leptophyes PI 458378 from the department of Puno border-
ing Bolivia.
Also in concordance with prior results, the AFLP data fail to
resolve many species in the complex. Species that fail to form
clades in the northern S. brevicaule group are S. abancayense,
S. bukasovii,S. canasense,S. leptophyes,S. marinasense,S.
multidissectum, and S. multiinterruptum, whereas S. candollea-
num and S. pampasense form clades. Species that fail to form
clades in the southern complex are S. ambosinum,S. brevicaule,
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Fig. 1. Strict consensus parsimony cladogram of 10,000 equally parsimonious 13,176-step Wagner trees based on the entire AFLP data set of 362 accessions,
with six AFLP primer combinations producing 438 characters. Outgroups consist of three accessions in Solanum section Etuberosum and clades 1–3 tuber-bearing
wild potatoes (section Petota) (26). The remaining ingroup consists of members of clade 4, labeled as the northern and southern S. brevicaule groups and
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cultivated species . The southern S. brevicaule group includes species from North and Central America and species from South America that have not traditionally
been considered part of this group. For space considerations, the taxa are staggered on the tree. Diploid accessions are colored black, tetraploid accessions are
blue, and hexaploid accessions are red.
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S. canasense,S. leptophyes,S. oplocense,S. sparsipilum, and S.
sucrense, whereas S. avilesii,S. hoopesii,S. incamayoense,
S. spegazzinii,S. ugentii,S. vernei, and S. vidaurrei [diploid] (to the
exclusion of one accession of S. gourlayi [tetraploid]) form clades.
The AFLP data support a monophyletic origin of all of the
cultivars. S. bukasovii 568954 and S. acroscopicum 230495,
however, fall within the cultivated clade. Accession 230495 was
labeled in the genebank as S. acroscopicum (a diploid species),
but chromosome counts show it to be tetraploid and likely to be
a misidentified cultivated species. S. bukasovii 568954 could be
a progenitor or an unrecognized cultivated diploid species.
Hosaka (10) showed that S. tarijense was a likely maternal
contributor in the origin of landraces of the Chilotanum group,
because they share a 241-bp chloroplast deletion. Our examina-
tion of three accessions of S. tarijense, and the related species
S. arnesii,S. berthaultii, and S. chacoense, show them to form a
clade, separate from the cultivars.
Phenetic Results. Because AFLPs are dominant and anonymous
marker data, a case has been made that they should be
analyzed with phenetic methods (33). The neighbor-joining
tree (data not shown) outlines nearly the same set of species
groups including the northern and southern S. brevicaule
groups, places the cultivated species as a single group, and
places the outgroup distant to the tuber-bearing species. A
phenogram could represent the phylogeny when similarities
are mainly due to shared derived characteristics (34). In our
case, conclusions are the same with cladistic or neighbor-
joining procedures, and controversies of proper methods to
use are therefore moot.
Concordance of the Present AFLP Data to Prior Morphological and
Molecular Data in the
S. brevicaule
Complex. The Mantel tests
showed high correlations (r) of the present AFLP results to the
prior RAPD and single- to low-copy nuclear RFLP results of the
S. brevicaule complex (22) (r⫽0.740–0.845) (Table 1) and much
lower correlations to the morphological data (r⫽0.204). Despite
much lower correspondence of AFLP, RFLP, and RA PD results
to the morphological results (Table 1), the morphological data
still showed a north–south partitioning by a canonical variates
analysis (21).
The utility of AFLPs to examine relationships of closely
related species has been documented elsewhere. For example,
Powell et al. (35) showed AFLPs and nuclear RFLPs to be
significantly correlated in diversity studies of cultivated soybean
(Glycine max [L.] Merrill) and its progenitor species G. soja
Hort.; Milbourne et al. (36) showed good correlations between
AFLPs and RAPDs in S. tuberosum; and Russell et al. (37)
showed good correlations of AFLPs and nuclear RFLPs in
Hordeum. Such correlations, and the concordance of AFLP to
nuclear RFLP, RAPD, and morphological results reported here,
support the utility of AFLPs for examining relationships of
S. tuberosum and the S. brevicaule complex.
Single Domestication for Potato. All cladistic and phenetic analy-
ses, of both the diploids and tetraploids, show all landrace
populations to form a monophyletic clade, derived from the
northern members of the S. brevicaule complex. These S. brevi-
caule northern group member ‘‘species’’ are poorly defined, and
ongoing studies may reduce them to a single species, with the
earliest valid name of the group being S. bukasovii. The conclu-
sions of a single origin of cultivated potato from the northern
species of the S. brevicaule group differs from all conventional
domestication hypotheses (9, 13, 15, 19–21, 23–25) in two
fundamental respects: (i) a single origin is here supported, rather
than a series of multiple independent origins; and (ii) the origin
is confined to the northern component of the S. brevicaule
complex, rather than to other southern complex species that are
commonly mentioned as progenitors, such as S. sparsipilum or
S. vernei (e.g., refs. 9 and 23). A ‘‘single’’ origin is here supported
to mean an origin from a single species, or its progenitor
(S. bukasovii), in the broad area of southern Peru. Because
landrace potatoes are currently spread throughout the Andes
and Chile, they clearly were diffused from Peru both north
and south, assuming present-day distributions of the original
cultivars.
The single origin of potato parallels results suggesting single
origins of other crops including barley (38), cassava (39), maize
(40), einkorn wheat (41), and emmer wheat (42). This differs
from multiple origins of common beans (43), cotton (44), millet
(45), rice (46), and squash (47). Allaby and Brown (48) criticize
the use of ‘‘anonymous’’ marker data of any type (including
AFLPs) to infer single crop origins. Results of computer simu-
lations have led these authors to postulate that monophyletic
origins can be erroneously inferred when using dominant marker
data analyzed by neighbor-joining methods. They assume that
pairs of markers, on average, are unlinked and simulate different
scenarios for cereal crops. Authors of some of the original studies
have replied, suggesting that the ‘‘intrinsic quality’’ of their data
outweighs any doubts through use of simulated data (49). It is
clear that further, more sophisticated simulations are required,
using information on known marker linkage relationships. More-
over, Allaby and Brown (48) do not comment on the applicability
of their studies to predominantly outbreeding, clonally propa-
gated crops, such as potato.
Diamond (50) discusses single vs. multiple crop origins in a
geographic perspective. He suggests that crops that spread east
and west (as einkorn wheat and emmer wheat in the Fertile
Crescent), rather than north and south (as squash and cotton in
the Americas), have a competitive advantage in rapid diffusion
because they take less time to adapt to new habitats. He further
contends that such rapid diffusion preempts adoption of com-
peting crops and favors single crop origins, in contrast to multiple
origins of crops spread north and south. His paper was written
when maize (51, 52) and potato (present results) were thought
to have multiple origins. The widespread north and south
diffusion of maize and potatoes, and their monophyletic origin,
prompts a reconsideration of his geographic interpretation of
single and multiple origins.
Reconsideration of the Taxonomy of the
S. brevicaule
Complex. The
data strongly suggest that (i) considerable reduction of species is
needed in the S. brevicaule complex and (ii) the complex is
polyphyletic. AFLP data suggest that some of the species in the
complex, however (mentioned above), may be valid. AFLP data
also suggest, as do the RAPD and nuclear RFLP data, that some
accessions of S. multiinterruptum are part of the distinctive clade
3 (series Piurana) (22). Taxonomic changes in species circum-
scriptions are underway as part of a broader-scale taxonomic
Table 1. Pairwise comparisons of the present AFLP results to the
morphological results (21) and RAPD and single- to low-copy
nuclear RFLP results (22) using the same accessions, as
performed with the Mantel test (32)
AFLP RFLP RAPD Morphology
AFLP — 0.760 0.761 0.182
RFLP 0.740 (166) — 0.590 0.123
RAPD 0.845 (76) 0.609 (82) — 0.069
Morphology 0.204 (211) 0.048 (128) 0.121 (69) —
0⫽no correspondence; 1 ⫽perfect correspondence. The results above the
diagonal compare the similarity matrices, and those below the diagonal
compare the tree topologies transformed to matrix values through cophen-
etic values (31). The numbers in parentheses below the diagonal indicate the
number of accessions common to each comparison.
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revision of the entire genus Solanum (53). Poor support for the
traditionally circumscribed S. brevicaule complex species sug-
gests that designations of species-specific progenitors of the
landrace cultivars with this outdated taxonomy (9, 25) are futile.
Rather, our results suggest that the Andean landrace cultivars
arose from the northern component of the S. brevicaule complex.
S. bukasovii is the earliest taxonomic name possessing priority as
this progenitor.
We thank the U.S. Department of Agriculture Foreign Agricultural
Service and the Scottish Executive Environment and Rural Affairs
Department for financial support.
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