Discovery and characterization of sweetpotato’s closest tetraploid relative

Abstract

The origin of sweetpotato, a hexaploid species, is poorly understood, partly because the identity of its tetraploid progenitor remains unknown. In this study, we identify, describe and characterize a new species of Ipomoea that is sweetpotato’s closest tetraploid relative known to date and probably a direct descendant of its tetraploid progenitor.
We integrate morphological, phylogenetic, and genomic analyses of herbarium and germplasm accessions of the hexaploid sweetpotato, its closest known diploid relative Ipomoea trifida, and various tetraploid plants closely related to them from across the American continent.
We identify wild autotetraploid plants from Ecuador that are morphologically distinct from Ipomoea batatas and I. trifida, but monophyletic and sister to I. batatas in phylogenetic analysis of nuclear data.
We describe this new species as Ipomoea aequatoriensis T. Wells & P. Muñoz sp. nov., distinguish it from hybrid tetraploid material collected in Mexico; and show that it likely played a direct role in the origin of sweetpotato’s hexaploid genome. This discovery transforms our understanding of sweetpotato’s origin.

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Rapid report
Discovery and characterization of sweetpotato’s closest tetraploid
relative
Author for correspondence:
Robert W. Scotland
Email: robert.scotland@plants.ox.ac.uk
Received: 23 November 2021
Accepted: 16 January 2022
Pablo Mu~noz-Rodrıguez
1
*, Tom Wells
1
*, John R. I. Wood
1,2
,
Tom Carruthers
2
, Noelle L. Anglin
3,4
, Robert L. Jarret
4
and
Robert W. Scotland
1
1
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK;
2
Royal Botanic Gardens, Kew,
Richmond, Surrey, TW9 3AB, UK;
3
International Potato Center, Avenida La Molina 1895, Distrito de La Molina, Lima 15023,
Peru;
4
United States Department of Agriculture, 1109 Experiment Street, Griffin, GA 30223, USA
New Phytologist (2022) 234: 1185–1194
doi: 10.1111/nph.17991
Key words: crop wild relatives, Ecuador,
genomics, herbarium specimens, Ipomoea
aequatoriensis, new species, tetraploid.
Summary
The origin of sweetpotato, a hexaploid species, is poorly understood, partly because the
identity of its tetraploid progenitor remains unknown. In this study, we identify, describe and
characterize a new species of Ipomoea that is sweetpotato’s closest tetraploid relative known to
date and probably a direct descendant of its tetraploid progenitor.
We integrate morphological, phylogenetic, and genomic analyses of herbarium and
germplasm accessions of the hexaploid sweetpotato, its closest known diploid relative Ipomoea
trifida, and various tetraploid plants closely related to them from across the American continent.
We identify wild autotetraploid plants from Ecuador that are morphologically distinct from
Ipomoea batatas and I. trifida, but monophyletic and sister to I. batatas in phylogenetic analysis
of nuclear data.
We describe this new species as Ipomoea aequatoriensis T. Wells & P. Mu~
noz sp. nov.,
distinguish it from hybrid tetraploid material collected in Mexico; and show that it likely played a
direct role in the origin of sweetpotato’s hexaploid genome. This discovery transforms our
understanding of sweetpotato’s origin.
Introduction
Sweetpotato, Ipomoea batatas (L.) Lam., is a hexaploid species
thought to have originated via allopolyploidy from a diploid and a
tetraploid ancestor (Yang et al., 2017). Ipomoea trifida (Kunth)
G. Don, a Circum-Caribbean species, was recently confirmed as
sweetpotato’s closest diploid relative and most likely the direct
descendant of its diploid progenitor (Mu~noz-Rodrıguez et al.,
2018). In contrast, the identity of the sweetpotato’s closest
tetraploid relative remains unknown. Identifying this entity is
key to untangling the evolutionary history of sweetpotato,
understanding its contemporary diversity and assembling its large
allohexaploid genome.
Whilst preparing a monograph of all American species of
Ipomoea L. (Wood et al., 2020), our attention was drawn to
herbarium specimens from Ecuador identified as I. batatas but
differing in their shorter and blunter sepals (Fig. 1a,b), sepal
morphology being an important taxonomic character in Ipomoea
(Austin, 1978; Wood et al., 2020). These specimens were restricted
to coastal Ecuador (Fig. 1c) and were of wild provenance, in
contrast to most populations of I. batatas that are only known from
cultivation or as escapes.
As part of our research, in parallel to studying herbarium
specimens, we also grew tetraploid Ipomoea specimens from seeds
available in germplasm collections (Supporting Information
Table S1). Tetraploid collections (2n=4x=60) are of particular
interest because their ploidy is intermediate between hexaploid
sweetpotato (2n=6x=90) and its closest diploid relative, I. trifida
(2n=2x=30), meaning that they may represent intermediate
stages in sweetpotato evolution. The germplasm material studied
by us included other tetraploid specimens from the same areas of
Ecuador as the distinctive herbarium material we had identified
during our studies (Figs S1–S5), as well as material of the Mexican
sweetpotato variety I. batatas var. apiculata J.A. McDonald & D.F.
*These authors contributed equally to this work.
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Research

Austin and the Mexican hybrid species Ipomoea tabascana
J.A. McDonald & D.F. Austin, both of them also tetraploids
(Notes S1). Ipomoea tabascana is a modern hybrid between
I. batatas and I. trifida known from a single collection (McDonald
& Austin, 1990; Srisuwan et al., 2006). Modern tetraploid
hybrids such as this may confound data interpretation, hence the
importance of including in our study examples of known hybrid
origin: it is essential to be able to distinguish between truly
autotetraploid entities and other tetraploids of modern hybrid
origin.
To place the Ecuadorian specimens in a phylogenetic context, we
conducted a preliminary phylogenetic analysis using rpl32-trnL,a
small, noncoding, rapidly-evolving chloroplast DNA region.
Ipomoea batatas contains two different chloroplast lineages (Roul-
lier et al., 2013), the ancestral lineage (chloroplast lineage 1) and a
second, more recent lineage that is most likely the result of
introgression with chloroplast capture from I. trifida (lineage 2)
(Mu~noz-Rodrıguez et al., 2018). The preliminary analysis of this
small chloroplast DNA region showed that the herbarium
specimens and the germplasm material from Ecuador were the
same entity, and that they were more closely related to sweetpotato
chloroplast lineage 1 than to any other lineage (Methods S1;
Fig. S6). A subsequent literature review showed that we were not
the first to recognize these tetraploids from Ecuador (Martin &
Jones, 1972; Martin et al., 1974; Austin et al., 1993), but previous
studies lacked the taxonomic and phylogenetic framework required
to accurately infer their relationship with sweetpotato.
Here, we provide the first comprehensive study of these
Ecuadorian tetraploids and show that they represent a distinct
species that is sweetpotato’s closest wild relative. We describe this
new species as Ipomoea aequatoriensis T. Wells & P. Mu~noz and
show it is most likely the direct descendant of the sweetpotato’s
tetraploid progenitor.
Materials and Methods
Herbarium collections and germplasm material
We studied American material from germplasm collections (CIP
and USDA) and herbaria (AAU, BM, E, FL, FTG, GUAY,
(a)
(c)
(b) (d)
(e)
Fig. 1 Ipomoeaaequatoriensis is morphologically distinct from Ipomoea batatas and Ipomoea trifida. Sepals are (a) oblong/ovate in cultivated I. batatas (Balls
5483) and (b) obovate in I. aequatoriensis (Jativa and Epling. 1191). (c) Map of the Americas showing the distribution of specimens included in the
morphological analysis. Closed symbols indicate specimens also included in the genomic analyses. All hexaploid I. batatas specimens in this study are of
cultivated origin and are not included in the map. (d) Principal component analysis and (e) linear discriminant analysis of 12 quantitative morphological traits
widely used in sweetpotato morphological studies. Ellipses indicate 95% confidence level. In (c–e), I. batatas (green dots), I. aequatoriensis (blue triangles),
I. trifida (red squares), hybrids Ipomoea tabascana (black triangle) and I. batatas var. apiculata (orange triangles). The Colombian specimens affinis to
I. aequatoriensis are indicated by light blue triangles.
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HUEFS, K, LPB, OXF, QAC, QAP, QCA, QCNE, RB, ST, US,
USZ, XAL, acronyms according to Thiers (2018)). We included
specimens of cultivated hexaploid I. batatas (L.) Lam. and diploid
I. trifida (Kunth) G. Don from across their geographical distribu-
tion; representatives of the 14 other close wild relatives of
sweetpotato (Wood et al., 2020) including the Mexican hybrid
tetraploid I. tabascana J.A. McDonald & D.F. Austin , known from
a single collection (Notes S1) (McDonald & Austin, 1990; Austin
et al., 1991); two specimens of the tetraploid sweetpotato variety
I. batatas var. apiculata J.A. McDonald & D.F. Austin, also from
Mexico and apparently restricted to the vicinity of the city of
Veracruz (Notes S1); the tetraploid material from Ecuador; a
tetraploid accession from Colombia; and multiple herbarium
collections of wild plants resembling the tetraploid Ecuadorian
material and collected in the same geographical area (Dodson &
Gentry, 1978; Austin, 1982; Dodson et al., 1985; McDonald &
Austin, 1990; Wood et al., 2020). See Tables S2 and S3 for
passport data of all specimens and indication of analyses they were
included in.
Quantitative morphological analyses
Character selection and measurement We identified and anal-
ysed herbarium specimens and germplasm material of I. trifida (57
specimens), I. batatas (55 specimens), the Ecuadorian tetraploids
(44 specimens), I. tabascana (one specimen) and I. batatas var.
apiculata (five specimens) (Table S2). We measured 12 morpho-
logical characters found to be informative in taxonomic treatments
of Ipomoea or commonly used to study sweetpotato germplasm
collections (Table S2) (Austin, 1978; Huaman, 1991; Wood et al.,
2020). Measurements were taken using digital callipers or, in the
case of digitized herbarium specimens, using the biological-image
analysis software FIJI (Schindelin et al., 2012).
Clustering analyses We first ran a principal component analysis
(PCA) to investigate phenotypic clustering between I. batatas,
I. trifida and the various tetraploid entities. We used FACTOMINER
package v.2.4 (L^eet al., 2008) in R and divided the tetraploid
material into three groups based on geographical distribution and
past determinations: (1) Ecuadorian, (2) I. tabascana, and (3)
I. batatas var. apiculata. We then used R package MASS v.7.3.54
(Venables et al., 2002) to assess how well individual specimens
could be classified into their assigned groups through a linear
discriminant analysis (LDA). We plotted the results of both
analyses using the GGPLOT2 package v.3.3.5 (Wickham, 2016), with
ellipses depicting 95% confidence level added using the stat_ellipse
function.
Analysis of genomic data
We sequenced 13 new specimens using Illumina whole genome
sequencing and incorporated them in our previously-existing
dataset of sweetpotato crop wild relatives (CWRs) (Mu~noz-
Rodrıguez et al., 2018) (Table S2). This material included six
Ecuadorian tetraploids (PI 561246, PI 561248, PI 561255, PI
561258, K300/CH71.3, CH81.2), one Colombian tetraploid
(K500/CH80.3), diploid I. trifida specimen s from Colombia (F. de
la Puente 1054) and Mexico (F. de la Puente 2961), three I. batatas
var. apiculata (D.F. Austin 7480, PI 518474 and K233) and one
I. tabascana (PI 518479).
DNA processing and sequencing We extracted DNA using the
Plant Tissue Mini protocol for Qiagen DNEasy Plant Mini Kit. We
created genomic libraries using the NEBNext Ultra DNA Library
Prep Kit for Illumina v.3.0 (New England BioLabs, Ipswich, MA,
USA). Sequencing was done at Novogene facilities in Cambridge,
UK, using Illumina NovoSeq6000. We obtained 150 bp paired
end whole genome data, on average 11 Gb per sample. We filtered
the sequence files using default parameters in TRIMMOMATIC
(Bolger et al., 2014) and checked the quality of the reads using
FASTQC. We used default settings in BBTOOLS’ tadpole (https://
sourceforge.net/projects/bbmap/) to correct the reads.
Assembly of single copy nuclear regions for phylogenetic
analysis We assembled 386 putative single copy nuclear DNA
regions of all samples using a reference-guided assembly. A detailed
description of how these nuclear regions were identified is provided
in Methods S2. We mapped the reads to the reference 386 nuclear
probes using BBMAP (paired only =t,local =t). We used
SAMTOOLS (Danecek et al., 2021) to extract all reads mapped to
the reference probes and to remove duplicate reads, and Picard
Tools (http://broadinstitute.github.io/picard) to realign the reads
mapped around indels. We used BCFTOOLS (Danecek et al., 2021)
for variant calling, indel normalization and variant filtering, and
VCFTOOLS’vcf-sort (Danecek et al., 2011) to sort the VCF files.
Phylogenetic analysis of nuclear DNA regions We used phylo-
genetic analysis of nuclear data to confirm the close relationship
between the Ecuadorian tetraploids and sweetpotato. We used
consensus sequences and included the tetraploids from Ecuador
and Colombia, 10 I. batatas specimens, 10 I. trifida specimens, one
I. tabascana specimen, two I. batatas var. apiculata specimens, one
specimen of each of the other 14 species closely related to
sweetpotato and one I. cryptica J.R.I. Wood & Scotland as
outgroup (Mu~noz-Rodrıguez et al., 2018, 2019). We obtained
consensus sequences from VCF variant files using BCFTOOLS
consensus (Danecek et al., 2021) and masked all positions in the
consensus sequences with read coverage lower than 59. The use of
consensus sequences in phylogenetic analysis can obscure potential
subgenome differentiation in polyploids. However, the lack of a
reference genome makes it impossible to assign the alleles in the
nuclear regions to specific subgenomes. To minimize the potential
effects of divergent subgenomes, we only included likely homozy-
gous variant positions in this analysis. Heterozygous sites were
therefore masked and not considered in the main phylogenetic
analysis but were included in additional phylogenetic analyses
(Methods S3).
We used the BIOPYTHON script sequence_cleaner to remove sequences
shorter than 500 bp or with more than 10% ambiguous sites. We
excluded three further regions of the analysis (solyc06g073230.2.1_1,
solyc08g043170.2.1_1 and solyc11g012820.1.1_1)asnoneofthe
sequences in those regions passed the filters, as well as one I. batatas
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var. apiculata herbarium specimen (D.F. Austin 7480) with almost
80% missing data.
We aligned each of the regions independently using MAFFT
v.7.310 (Katoh & Standley, 2013) and removed poorly
aligned regions using GBLOCKS (half gaps) (Castresana, 2000;
Talavera & Castresana, 2007). We generated summary files of
all edited alignments using AMAS (Borowiec, 2016) (Table S4).
A further 12 alignments that had no variable sites were
excluded, so this analysis was done using 371 putative single-
copy nuclear DNA regions. We also used AMAS to concatenate
the alignments.
We inferred three different phylogenies: (1) partitioned maxi-
mum likelihood (ML) analysis of concatenated alignments with
automated model selection +merge in IQ-TREE v.1.6.12 (Nguyen
et al., 2015; Kalyaanamoorthy et al., 2017); (2) Approximate ML
analysis of unpartitioned concatenated alignments in multi-
threaded double-precision FASTTREE v.2.1.10
54,71
(GTR +gamma
model); and (3) independent gene tree inference using IQ-TREE
v.1.6.12 with automated model selection followed by species tree
inference using the coalescent in ASTRAL III (Zhang et al., 2018).
We used the GNU parallel tool (Tange, 2011) to parallelize and
speed up several steps in the pipeline.
Principal component analysis We conducted a PCA of I. batatas,
I. trifida, the Ecuadorian tetraploids, the Colombian tetraploid
specimen K500/CH81.3 and the hybrids I. tabascana and I. batatas
var. apiculata. We used a subset of 20 I. batatas and 20 I. trifida
samples to try to minimize bias due to uneven population sizes
compared to the other entities (Priveet al., 2020). We also
conducted additional analyses including all I. batatas and I. trifida
samples instead of a subset (Methods S4).
We mapped the nuclear reads to a sweetpotato sample (accession
CIP 400435) and called variants using the same procedure as
earlier. We filtered out all variants with coverage lower than 59and
ran a linkage disequilibrium pruning step using PLINK (--indep-
pairwise 50 10 0.1). We then used PLINK (--double-id --allow-extra-
chr --set-missing-var-ids @:# --make-bed --pca --geno 0.20 --snps-
only --max-alleles 2) (Chang et al., 2015; Purcell, 2021) for the
PCA and plotted the results using TIDYVERSE (Wickham et al.,
2019) and GGPLOT2 (Wickham, 2016) in RSTUDIO (RStudio Team,
2021). This analysis used 419 single nucleotide polymorphisms
(SNPs) from across the 386 Ipomoea nuclear regions, both
homozygous and heterozygous.
K-mer analyses We used GENOMESCOPE2.0 (Ranallo-Benavidez
et al., 2020) to assess heterozygosity from k-mer frequencies of raw,
unaligned sequencing reads, in a representative Ecuadorian sample
(PI 561248) sequenced at high-coverage. Relative frequency
patterns can then be used to infer whether a tetraploid sample is
autopolyploid or allopolyploid. We carried out initial k-mer
counting and histogram construction on the filtered but unaligned
sequencing reads using JELLYFISH (Marc
ßais & Kingsford, 2011). We
ran both JELLYFISH and GENOMESCOPE2.0 with a maximum
coverage of 100 000 and the default k-mer value of 21. We also
ran the same analysis in three Mexican hybrid tetraploids sequenced
at lower coverage (Methods S5).
Assembly of whole chloroplast genomes We used GETORGANELLE
(-F embplant_pt; SPAdes options: "--threads 20 --only-assembler -k
21,33,55,77,93")(Jinet al., 2018) to de novo assemble the chloro-
plast genomes of the new samples. When GETORGANELLE failed to
produce a circular genome assembly in the first attempt, we ran a
second attempt using --reduce-reads-for-coverage INF and --max-
reads INF options. GETORGANELLE successfully assembled all
samples except one I. trifida sample (F. de la Puente 2961). To
assemble the genome of this one sample, we used a reference-guided
assembly using I. trifida (F. de la Puente 1054) as reference.
Phylogenetic network using chloroplast genomes This analysis
includes all I. batatas,I. trifida and I. tabascana specimens from our
previous study, together with the 15 newly sequenced samples. We
aligned the whole chloroplast genome sequences using MAFFT
v.7.310 (FFT-NS-2) and removed poorly aligned regions using
GBLOCKS (no gaps). We used POPART (http://popart.otago.ac.nz) to
infer a Median Joining Network (reticulation tolerance 0.50
(Bandelt et al., 1999)) with 602 segregating sites, 182 of them
parsimony-informative.
Results
Morphological differentiation
The tetraploid Ecuadorian and Colombian material form a
cluster distinct from I. batatas and I. trifida in PCA and LDA of
12 morphological characters (Fig. 1e,f). The PCA shows three
clusters corresponding to I. batatas,I. trifida and the Ecuado-
rian/Colombian material, with some overlap at the margins,
predominantly between I. trifida and I. batatas (Fig. 1e). Hybrid
specimens from Mexico, i.e. I. tabascana (PI 518479) and
I. batatas var. apiculata (PI 518474), fall close to or within the
clusters of I. trifida and I. batatas. The three distinct clusters
were more pronounced in the LDA trained on 80% of the data
(Fig. 1e), which yielded a 90% success rate in accurately
identifying the test data and recovered the Mexican hybrids
within I. trifida.
Genomic differentiation
The phylogenetic analysis of nuclear regions recovers the six
tetraploid specimens from Ecuador and one from Colombia in a
clade sister to hexaploid I. batatas (Fig. 2a). This relationship is
recovered in all methods of phylogenetic inference with strong
support (Figs S7, S8). In addition, the tetraploid Ecuadorian and
Colombian specimens also form a distinct group from I. batatas and
I. trifida in the different PCA using nuclear SNPs (Figs 2b, S9). The
analysis using a subset of I. batatas and I. trifida samples, shown in
Fig. 2(b), aimed at preventing bias due to uneven population sizes
(Priveet al., 2020). In this analysis, the Ecuadorian and Colombian
tetraploids partially overlap with I. trifida in principal component
one (PC1) but clearly separate from all entities, including I. trifida,
in principal component two (PC2). The single specimen of the
Mexican hybrid I. tabascana and the three I. batatas var. apiculata
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specimens are intermediate between I. trifida and I. batatas in PC1
but cluster with both species in PC2 (Fig. 2b).
The analysis of nucleotide heterozygosity patterns suggests that
the Ecuadorian tetraploids have a genomic structure consistent
with an autopolyploid origin, with proportions of aaab consistently
higher than aabb (Table S5; Notes S1). This pattern is indicative of
two identical or highly similar subgenomes originating from a
whole genome duplication (Ranallo-Benavidez et al., 2020). The
same analysis for the hybrid I. tabascana and two specimens of
I. batatas var. apiculata, albeit using lower coverage data (Methods
S2), shows instead a higher proportion of aabb than aaab
(Table S5; Notes S1), which suggests that two distinct subgenomes
have been derived from a recent hybridization event.
Analysis of whole chloroplast genomes
The Median Joining phylogenetic network inferred using 602
segregating sites from the alignment of whole chloroplast genomes
shows the Ecuadorian plants are associated with the ancestral
sweetpotato lineage 1, whereas the single Colombian specimen we
sequenced (K500/80.3) is associated with the sweetpotato lineage
2. The hybrid I. tabascana and I. batatas var. apiculata are also
associated with sweetpotato lineage 2.
Discussion
Ipomoea aequatoriensis is a distinct species
We have identified a group of plants from Ecuador that are distinct
from cultivated sweetpotato and from all sweetpotato CWRs
known to date. These tetraploid plants are of wild provenance,
morphologically and geographically coherent, most likely autote-
traploid, isolated in the genetic space, and form a monophyletic
group most closely related to sweetpotato in phylogenetic analysis
of nuclear data. Their distinctiveness justifies recognition as a new
species I. aequatoriensis T. Wells & P. Mu~noz. A formal diagnosis is
presented here. Specimen citation, full description and ecological
notes are provided in the Notes S2. Specimens from Colombia,
although possibly also part of this species, require further study and
are not formally included in I. aequatoriensis (see Notes S2).
Ipomoea aequatoriensis T. Wells & P. Mu~noz, sp. nov. (Illustra-
tion in Fig. S10) TYPE: ECUADOR. Esmeraldas Province,
Quininde. Austin, D.F. 7803 (holotype FTG, Isotype CIP).
Diagnosis This species is most closely related to I. batatas (L.)
Lam. (Figs 2a, 3) which it resembles in corolla size, dense sub-
umbellate inflorescence and pubescent ovary, but differs in
possessing sepals that are consistently shorter (outer: <7vs
>7 mm; inner: <10 mm vs >12 mm) and stems that are thinner
(1–3mmvs2–6 mm diameter) with longer internodes (6–16 cm vs
2–10 cm), consistent with a twining (rather than trailing) habit. It
also closely resembles I. trifida (Kunth) G. Don, particularly in the
twining habit and chartaceous sepals, but differs in having obtuse
sepals (80°–160°vs 20°–70°) and laxer, more obviously umbellate
inflorescences with a greater number of flowers (5–24 vs 2–12) and
mostly entire, larger leaves (4–14 cm vs 2–10 cm long).
Identifying the tetraploid progenitor of sweetpotato
A major barrier to understanding the origin and evolution of
sweetpotato remains the difficulty of assembling its large
(b)(a)
Fig. 2 Molecular analyses identify Ipomoea aequatoriensis as a distinct entity, phylogenetically distinct and isolated in the genetic space. (a) Approximate
maximum likelihood analysis of 371 single-copy nuclear DNA regions. Numbers on the branches indicate Shimodaira–Hasegawa-like support values; black dots
indicate branches with 100% support. (b) Principal component analysis of Ipomoea batatas (green), Ipomoea trifida (red), I. aequatoriensis (blue) and the
hybrids Ipomoea tabascana and I. batatas var. apiculata (black and orange respectively). Principal component analysis inferred using 419 single nucleotide
polymorphisms (SNPs) from across the 386 nuclear probes. Ellipses indicate multivariate t-distribution. The Colombian specimen K500/CH81.3 discussed
throughout the text is indicated in light blue.
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allohexaploid genome (Isobe et al., 2019), which comprises three
subgenomes: two identical (BBBB) and one slightly different (AA)
in an AABBBB structure (Ting & Kehr, 1953; Ting et al., 1957;
Jones, 1965; Magoon et al., 1970; Nishiyama et al., 1975; Shiotani
& Kawase, 1987; Srisuwan et al., 2006; Gao et al., 2011; Yang
et al., 2017). These subgenomes are most likely derived from a
hybridization event between a diploid progenitor that contributed
the AA subgenome and a tetraploid progenitor that contributed the
BBBB subgenomes (Fig. 4). The AA subgenome is most likely
derived from a diploid ancestor shared with I. trifida (Yang et al.,
2017; Mu~noz-Rodrıguez et al., 2018), but the tetraploid progen-
itor that contributed the BBBB subgenomes remains unidentified
(Yang et al., 2017).
The new autotetraploid species I. aequatoriensis is the closest
wild relative of sweetpotato identified to date, and our results
strongly suggest it is the direct descendant of sweetpotato’s
tetraploid progenitor. A possible scenario for this is presented in
Fig. 4, and there are four lines of evidence for this conclusion. First,
the wild provenance of the samples we studied, which were not
cultivated, feral or derived from breeding programmes (Notes S3).
Second, I.aequatoriensis is consistently recovered as monophyletic
and sister to I. batatas in nuclear phylogenies, regardless of the
method of phylogenetic inference, both in our study (Figs 2a, S7,
S8) and in a recent pre-print (Yan et al., 2021). Third, its genetic
structure is indicative of an autopolyploid origin (Table S5; Notes
S1), a requirement for the tetraploid progenitor of the sweetpotato
Ipomoea aequatoriensis
(Ecuador)
Ipomoea 4x
(Colombia)
Ipomoea batatas
(Lineage 1)
Ipomoea batatas
(Lineage 2)
Ipomoea trifida
Ipomoea batatas var. apiculata
Ipomoea tabascana
Fig. 3 The analysis of chloroplast genomes shows Ipomoea aequatoriensis is associated with the sweetpotato ancestral lineage. Median Joining phylogenetic
network inferred using 602 segregating sites (182 parsimony-informative) and showing the relationships between Ipomoea batatas,Ipomoea trifida,
I. aequatoriensis and the hybrid entities, Ipomoea tabascana and I. batatas var. apiculata. The one Colombian specimen sequenced (K500/CH80.3), indicated
with an arrow, seems to carry a chloroplast related to sweetpotato lineage 2 chloroplast; we excluded it from our diagnosis of I. aequatoriensis pending further
investigation. The size of the circles indicates the number of samples, with samples grouping in larger circles being identical for the sites studied.
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because of the AABBBB structure of the sweetpotato genome.
Fourth, I. aequatoriensis is most closely related to sweetpotato
lineage 1 –the ancestral sweetpotato lineage –in the analyses of
chloroplast genomes in our study (Fig. 3) and that by Roullier et al.
(2013).
Poor taxonomy and modern hybrids complicate sweetpotato
studies
Previous attempts to identify sweetpotato’s tetraploid progenitor
have been hampered by taxonomic confusion, the lack of a well-
resolved phylogenetic framework for sweetpotato and its closest
relatives or the inclusion of probably feral specimens (Jones, 1967;
Nishiyama, 1971; Martin & Jones, 1972; Austin, 1988; Roullier
et al., 2013) (Table S1).
In addition, the existence of modern hybrids between I. batatas
and its closest diploid relative, I. trifida, further complicates data
interpretation. This is because hybridization between I. batatas (3n
gametes) and I. trifida (1ngametes) will most likely produce a
tetraploid (Orjeda et al., 1991), as in the case of I. tabascana
(Austin, 1977; Jarret et al., 1992; Bohac et al., 1993; Srisuwan et al.,
2006). Because of their parentage, such tetraploids are closely
related to hexaploid I. batatas in nuclear phylogenies (Figs 2a, S7).
Therefore, studies that rely purely on phylogenetic analysis of
nuclear DNA sequence data are likely to confuse these putative
hybrid tetraploids with the autotetraploid progenitor of hexaploid
I. batatas (Yan et al., 2021) (Notes S3). However, the incorpor ation
of other lines of evidence confirms the hybrid origin of these
tetraploid entities and shows they cannot be the tetraploid
progenitor of sweetpotato. First, I. batatas var. apiculata is
recovered with the known hybrid I. tabascana in all phylogenies
(Figs 2a, S7, S8) and both entities are in an intermediate position
between I. trifida and I. batatas in the PCAs using nuclear genomic
variants (Fig. 2b), implying a highly similar genetic structure.
Second, k-mer analysis of these samples suggests that they possess
two distinct subgenomes (Table S5; Notes S1). The k-mer analyses
require confirmation using higher-coverage sequence data (Meth-
ods S2), but our results are consistent with their apparent hybrid
origin (Srisuwan et al., 2006; Mu~noz-Rodrıguez et al., 2018).
Third, the hybrid entities are most closely related in the chloroplast
analysis to the derived sweetpotato chloroplast lineage 2 (Fig. 3),
which is the result of introgression with I. trifida and therefore
I. batatas (6X, lineage 1)
I. batatas (6X)
I. aequatoriensis (4X)
modern hybrids (4X)
I. trifida (2X)
6X
4X
I. batatas
I. aequatoriensi
s
Common
ancestor
2X
6X
6X
Modern hybrids
4X
Nuclear phylogeny Chloroplast phylogeny
I. batatas (6X, lineage 2)
modern hybrids (4x)
I. trifida (2X)
Chloroplast capture
I. trifida
Present diversity
2X 2X 2X
4X
(a)
(b) (c)
I. aequatoriensis (4X)
Fig. 4 One of several possible scenarios of sweetpotato evolution and origin of current diversity. Tetraploid plants closely related to sweetpotato have two
different origins: plants from Ecuador represent direct descendants from the autotetraploid progenitor of hexaploid Ipomoea batatas, whereas plants from
Mexico and Central America are the result of a more recent hybridization between hexaploid I. batatas and diploid Ipomoea trifida. (a) One possible scenario,
congruent with the data currently available, is presented here. An autotetraploid would have arisen from a whole genome duplication of a diploid common
ancestor with I. trifida. This autotetraploid would have hybridized with the diploid ancestorto produce an allohexaploid. Subsequent introgression between the
diploid ancestor lineage and the allohexaploid would result in chloroplast capture from I. trifida, explaining the two distinct I. batatas lineages in the chloroplast
phylogenies. This separate lineage would keep a hexaploid nuclear genome but a chloroplast most similar to the diploid progenitor, and therefore to modern
I. trifida, than to the ancestral sweetpotato lineage. Red and blue colours indicate the proportion of diploid (AA, red) and tetraploid (BBBB, blue) ancestral
genomes in the different entities. Small, coloured circles represent the chloroplast. Dashed lines indicate hybridization and dotted line indicates introgression
with chloroplast capture. (b) Summary nuclear phylogeny depicting the relationship between modern taxa, with Ipomoea aequatoriensis most closely related to
I. batatas. (c) Summary chloroplast phylogeny depicting the relationship between modern taxa, with I. aequatoriensis most closely related to I. batatas lineage
1, the ancestral sweetpotato lineage.
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postdates the origin of I. batatas (Mu~noz-Rodrıguez et al., 2018).
Finally, the PCA and LDA using morphology (Fig. 1d,e) consis-
tently show these specimens cluster with either I. trifida or
I. batatas, instead of forming a distinct group as is the case of
I. aequatoriensis.
In summary, a broader consideration of collection history,
nuclear and chloroplast sequence data, and genomic structure,
enables the identification of modern tetraploid hybrids, such as
I. tabascana and I. batatas var. apiculata, and rules them out as
sweetpotato’s tetraploid progenitor (Fig. 4). Our results also
suggest I. batatas var. apiculata should be treated as a distinct
entity of hybrid origin akin to I. tabascana rather than a subspecies
of I. batatas (Notes S1). Although we have not been able to study all
the material listed in earlier studies of tetraploid plants (Table S1;
Notes S4), future studies that consider the different criteria
presented here should allow the classification of those specimens as
either ancient autotetraploids or modern hybrids, and further
clarify their relationship to sweetpotato.
Ipomoea aequatoriensis, a key finding for sweetpotato
studies
The identification of the closest living relative of the tetraploid
progenitor of sweetpotato is key to untangling its genomic history
and contemporary diversity. Ipomoea aequatoriensis has all the
hallmarks of being that species, and therefore represents an
extraordinary discovery and a key finding for subsequent sweet-
potato studies.
Acknowledgements
This project was funded by a BBSRC research grant to RWS
(T001445/1). TW was funded by an Interdisciplinary DTP
BBSRC scholarship. PM-R was also funded by an Interdisciplinary
DTP BBSRC scholarship at the early stages of this project. The
authors thank botanical artist Rosemary Wise for the illustration of
Ipomoea aequatoriensis. The authors thank all herbarium curators
and germplasm managers for providing access to their material,
especially Masaru Tanaka at NARO –Japan, as well as the people
who did fieldwork and collected the specimens in this study.
Author contributions
RWS, JRIW, PM-R, TW and TC conceived the project; PM- R and
TW conducted the analyses; NLA and RLJ contributed material
and information about its provenance; PM-R, TW, RWS, JRIW
and TC wrote the manuscript. PM-R and TW contributed equally
to this work.
ORCID
Noelle L. Anglin https://orcid.org/0000-0002-3454-1142
Tom Carruthers https://orcid.org/0000-0003-1586-3557
Robert L. Jarret https://orcid.org/0000-0002-0426-6186
Pablo Mu~noz-Rodrıguez https://orcid.org/0000-0002-3580-
8136
Robert W. Scotland https://orcid.org/0000-0002-6371-2238
Tom Wells https://orcid.org/0000-0002-4664-7868
John R. I. Wood https://orcid.org/0000-0001-5102-3729
Data availability
Raw reads from the 2018 study and newly generated data are
available in the Sequence Repository Archive, BioProjects
PRJNA453382 and PRJNA796763 respectively. Original and
edited files with morphological and molecular analyses and scripts
are available via the Oxford Research Archive (https://ora.ox.ac.uk/
objects/uuid:055e2f01-bbb1-4a69-a3ae-dac009db31d1). Any
other information required to re-analyse the data is available from
the lead contact upon request.
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 Ipomoea aequatoriensis specimen PI 355830/K300/
CH71.3.
Fig. S2 Ipomoea aequatoriensis specimen K500/CH80.3.
Fig. S3 Ipomoea aequatoriensis specimen PI 561248/CIP 403553.
Fig. S4 Ipomoea aequatoriensis specimen PI 561258.
Fig. S5 Ipomoea tabascana specimen PI 518479/CIP 460824 and
Ipomoea batatas var. apiculata specimen PI 518474/CIP 403953.
Fig. S6 trnL-rpl32 chloroplast DNA barcode phylogeny.
Fig. S7 Nuclear phylogenies of Ipomoea Clade A3 indicating the
position of the Ecuadorian tetraploids and the modern hybrids.
Fig. S8 Nuclear phylogenies of Ipomoea Clade A3 indicating the
position of the Ecuadorian tetraploids and the modern hybrids.
Phylogenies inferred including IUPAC characters for heterozygous
sites.
Fig. S9 Additional principal component analyses.
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Fig. S10 Scientific illustration of Ipomoea aequatoriensis T. Wells &
P. Mu~noz.
Methods S1 Preliminary analysis of the trnL-rpl32 chloroplast
DNA region.
Methods S2 K-mer analysis of putative hybrid tetraploids.
Methods S3 Additional phylogenetic analysis of nuclear probes.
Methods S4 Additional principal component analyses.
Methods S5 K-mer analysis of putative hybrid tetraploids.
Notes S1 Modern hybrids closely related to Ipomoea batatas.
Notes S2 K-mer analyses diagrams.
Notes S3 Description and additional information for Ipomoea
aequatoriensis.
Notes S4 Hybrid specimens in other studies.
Table S1 Tetraploid accessions in previous studies, indicating past
and present identifications.
Table S2 Passport data of all samples included in morphological
analyses.
Table S3 Passport data of all samples included in phylogenetic
analyses.
Table S4 Statistics of the putative single copy nuclear regions used
in phylogenetic analysis.
Table S5 Patterns of nucleotide heterozygosity in k-mer spectra of
sequencing reads (k=21).
Please note: Wiley Blackwell are not responsible for the content or
functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
See also the Commentary on this article by S€arkinen et al.,234: 1107–1108.
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