independent domestications of cultivated tree peonies...

Independent domestications of cultivated tree peoniesfrom different wild peony species

JUN-HUI YUAN,*† AMANDINE CORNILLE,‡§¶ TATIANA GIRAUD,‡§¶ FANG-YUN CHENG† and

YONG-HONG HU*1

*Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, 3888

Chenhua Road, Shanghai 201602, China, †Landscape Architecture College, Beijing Forestry University, No. 35 Tsinghua East

Road, Haidian District, Beijing 100083, China, ‡Universit�e Paris-Sud, Ecologie, Syst�ematique et Evolution, UMR8079, 91405

Orsay, France, §CNRS, Ecologie, Syst�ematique et Evolution, UMR8079, 91405 Orsay, France, ¶AgroParisTech, Orsay, France2

Abstract

An understanding of plant domestication history provides insights into general mech-

anisms of plant adaptation and diversification and can guide breeding programmes

that aim to improve cultivated species. Cultivated tree peonies (genus Paeonia L.) are

among the most popular ornamental plants in the world; yet, the history of their

domestication is still unresolved. Here, we explored whether the domestication in

China of historically cultivated peonies, that is, the common and flare cultivated tree

peonies, was a single event or whether independent domestications occurred. We

used 14 nuclear microsatellite markers and a comprehensive set of 553 tree peonies

collected across China, including common tree peonies, flare tree peonies and the

wild species or subspecies that are potential contributors to the cultivated tree

peonies, that is, Paeonia rockii ssp. rockii, P. rockii ssp. atava, P. jishanensis and

P. decomposita. Assignment methods, a principal component analysis and approxi-

mate Bayesian computations provided clear evidence for independent domestications

of these common tree and flare tree peonies from two distinct and allopatric wild spe-

cies, P. jishanensis and P. rockii ssp. atava, respectively. This study provides the first

example of independent domestications of cultivated trees from distinct species and

locations. This work also yields crucial insight into the history of domestication of

one of the most popular woody ornamental plants. The cultivated peonies represent

an interesting case of parallel and convergent evolution. The information obtained in

this study will be valuable both for improving current tree peony breeding strategies

and for understanding the mechanisms of domestication, diversification and adapta-

tion in plants.

Keywords: annual grass, convergence, East Asia, hybridization, ornamental cultivated tree,

Paeonia suffruticosa

Received 30 April 2013; revision received 14 October 2013; accepted 16 October 2013

Introduction

An understanding of plant domestication can provide

insight into the history of human civilization (Diamond

2002) and into general mechanisms of biological adapt-

ation; it can also guide modern breeding programmes

aimed at the further improvement of crop species (Gross

& Olsen 2010; Miller & Gross 2011). The most fundamen-

tal and debated questions regarding the evolution of cul-

tivated plants concern their geographic origins, whether

domestication took place once or multiple times for a

given crop and whether genetic bottlenecks occurred dur-

ing their evolution (Diamond 2002; Gross & Olsen 2010).

A single origin of crops from one geographic area

and from a single progenitor was initially considered

Correspondence: Fang-Yun Cheng, Fax: XXXXX;

E-mail:[email protected]; Yong-Hong Hu, Fax: XXXXXXX;

E-mail: [email protected] and Jun-hui Yuan,

Fax: XXXXXXX; E-mail: [email protected] 3

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© 2013 John Wiley & Sons Ltd

Molecular Ecology (2013) doi: 10.1111/mec.12567

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the default domestication scenario for most crop species

(Gl�emin & Bataillon 2009). However, for several annual

crops, accumulating molecular evidence now suggests

that multiple domestications from distinct locations and

from wild contributors occurred. Confirmation of multi-

ple independent geographic origins has been made for

barley (Morrell & Clegg 2007), Asian rice (Londo et al.

2006) and the common bean (Phaseolus vulgaris; Bitocchi

et al. 2012, 2013). Independent domestication from dif-

ferent species was also reported for two species of

domesticated cotton (Gossypium hirsutum and G. barva-

dense L.; Westengen et al. 2005), two domesticated rice

species from Asia and Africa (Oryza sativa and O. glab-

errima Steud.; Londo et al. 2006) and beans (Phaseolus L.;

Chacon et al. 2005). The occurrence of strong genetic

bottlenecks during the process of domestication has

been documented for most annual propagated seed

crops (Tenaillon et al. 2004; Hyten et al. 2006; Liu &

Burke 2006; Haudry et al. 2007). In contrast, for peren-

nial tree crops, the investigation of these questions is

still in its infancy (e.g. Besnard & Berville 2000; Breton

et al. 2006; Myles et al. 2010; Cornille et al. 2012; Besnard

et al. 2013).

Trees are fascinating models for studying domestica-

tion because their peculiar life history traits permit

unique domestication processes (Gross & Olsen 20114 ;

Cornille et al. 2012). Whereas the domestication history

of some trees cultivated for food purposes has recently

been elucidated (Gross & Olsen 2011; Cornille et al.

2012; Besnard et al. 2013; Delplancke et al. 2013; Roullier

et al. 2013; Wincker 2013), the domestication of orna-

mental plants for aesthetic purposes, such as the rose

(Iwata et al. 2000)5 , has not received much attention. Cul-

tivated tree peonies (genus Paeonia L.) are among the

most popular ornamental plants worldwide and have

been used as ornamental and traditional medicinal

plants for over 1600 years in China, their centre of ori-

gin (Foster & Yue 1992; Li 2011). Although tree peonies

display a rich ethnobotanical history that is deeply

embedded in the Chinese culture, the history of the

domestication of cultivated tree peonies remains unre-

solved (Cheng 2007; Li 20066 ; Zhou 2007; Zhang et al.

2011)7 . The most important traits that have changed dur-

ing domestication are the number and size of the flow-

ers and the number and colours of the petals.

Historically cultivated tree peonies include (i) the com-

mon tree peonies (CTPs), Paeonia suffruticosa Andrews,

comprising 700–800 local varieties distributed through-

out temperate regions in the world (Li 2011) and (ii) the

flare tree peonies (FTPs), comprising 200–300 varieties

that are less extensively commercialized (Wang 1998;

Cheng et al. 2005; Li 2005, 2011; Cheng 2007). Flare tree

peonies are widely cultivated in the middle Gansu

Province and present scattered distributions elsewhere

in northwestern China (e.g. Ningxia, Qinghai and

Shaanxi) and in some provinces of northern China

(Wang 1998; Cheng et al. 2005; Cheng 2007; Li 2011).

These two cultivated tree peonies differ in ecology (e.g.

the flare tree peonies have greater ecological plasticity

and a higher degree of resistance to environmental

stress) and in morphology, with all flare tree peonies

presenting reddish-purple flares at the bases of the pet-

als that allow for easy identification (Cheng et al. 2005)

(Fig. 1). These marked differences led to the as yet

untested hypothesis that independent domestications of

these two groups of cultivated peonies occurred (Li

1998, 2005; Wang 1998; Cheng 2007).

China is pinpointed as the likely geographic origin of

cultivated tree peonies because it hosts the richest tree

peony resources in the world; these resources include

endemic wild species and greater than 1000 varieties.

Tree peonies were introduced to Japan during the Tang

dynasty (724–749), and more than 200 varieties of Japa-

nese tree peonies have been developed. Beginning in

the 1780s, some common tree peonies were introduced

into western countries, such as England and France

(Cheng 2007; Li 2011). Almost 110 varieties of tree peo-

nies are currently grown in Europe (Cheng 2007). In the

1880s, a wild tree peony species, P. delavayi, was intro-

duced into France, where nearly 13 cultivars designated

P. 9 lemoinei have been obtained by hybridizing P. dela-

vayi and P. suffruticosa (Wister 1995; Cheng 2007).

American tree peony cultivars were developed later by

further hybridizing P. 9 lemoinei with Japanese culti-

vars (Cheng 2007; Li 2011). The creation of these recent

cultivars has been well documented (Cheng 2007);

therefore, we focused in this work on the domestication

history of the historically cultivated peonies: the com-

mon and flare tree peonies in China.

Historical records and morphological comparisons

suggest that CTP and FTP cultivars originated indepen-

dently from different wild species in China. Cultivated

CTPs are likely to have originated from the wild species

Paeonia jishanensis T. Hong & W.Z. Zhao (Rehder 1920;

Li 2005), while one of the two subspecies of P. rockii

S.G. Haw & Lauener, T. Hong & J.J. Li ex D.Y. Hong

(P. rockii ssp. atava and P. rockii ssp. rockii) growing in

the central southern region of Gansu Province (Fig. 1) is

the likely progenitor of FTPs (Cheng et al. 2005; Li

2011). Molecular phylogenetic analysis suggests that

another wild species, P. decomposita Hand. Mazz., which

is distributed in Sichuan Province (Fig. 1), may also

have been involved to a lesser extent in the domestica-

tion of FTPs (Zhou 2007). This hypothesis remains con-

troversial, however, because P. decomposita displays a

restricted distribution in remote mountain areas in

northwestern Sichuan province and is therefore unlikely

to have been easily transplanted in the past (Li 2011). It


2 J . - H. YUAN ET AL.

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has also been proposed based on morphological fea-

tures and gene sequences that recent wild-to-crop and

crop-to-crop (i.e. FTPs to CTPs and conversely) hybrid-

izations and introgressions have been major forces in

the breeding of cultivated tree peonies (Cheng 2007;

Zhou 2007; Li 2011). The available phylogenies of culti-

vated and wild tree peony species are, however, not

resolved well enough to provide a basis for elucidation

of the domestication history of the CTP and FTP culti-

vars (Zhou 2007; Zhao et al. 2008; Zhang et al. 2011).

Here, we explored the domestication history of com-

mon and flare tree cultivated peonies in their centre of

origin using 14 nuclear microsatellite loci and a compre-

hensive set of 553 trees collected throughout China

(Fig. 1). This set included 103 flare tree peony varieties,

72 common tree peony varieties (e.g. P. suffruticosa) and

the main potential contributors to their evolution,

P. rockii ssp. rockii, P. rockii ssp. atava, P. jishanensis and

P. decomposita (Fig. 1), that are all diploid (Hong 2010)

and allogamous (Cheng et al. 2005; Cheng 2007) and

grow in allopatry (Fig. 1). The specific objectives of this

study were to test (i) whether the four wild species or

subspecies, the FTPs and the CTPs all form distinct

genetic clusters; (ii) whether recent wild-to-crop and

crop-to-crop hybridizations contributed to the genomes

of the FTP and CTP cultivars; (iii) whether the FTP and

CTP cultivars have been domesticated independently

from distinct wild species or subspecies and (iv)

whether footprints of genetic bottlenecks expected dur-

ing domestication can be detected.

Materials and methods

Sample collection and DNA extraction

Leaf material was carefully collected from domesti-

cated varieties and wild species to accurately represent

the genetic, morphologic and geographic diversity of

cultivated and wild tree peonies. The choice of varie-

ties and species was made based on interviews with

horticulturalists, botanists and local peony experts, on

field surveys and on an examination of the literature

Gansu province

Sichuan province

Hubei province

Henan provinceShaanxi province

Shanxi province

Lanzhou

Xian

Louyang

Yanan

Lingxia

Lingtao

Tianshui35° N

31° N

Kilometers

0 200

38° N

110° E105° E

P. rockii ssp. rockii

P. jishanensisP. decomposita

Common tree peony (CTP)

P. rockii ssp. atavaFlare tree peony (FTP)

100

N

e

Fig. 1 Geographic origins of the 553 tree peony samples used in this study. The samples included the two cultivated peony groups,28the common tree peony (CTP, red) and the flare tree peony (FTP, purple), and four allopatric wild species or subspecies, Paeonia

decomposita (blue), Paeonia jishanensis (yellow), Paeonia rockii ssp. rockii (green) and Paeonia rockii ssp. atava (pink), with pictures of

their typical flowers.

COLOR


INDEPENDENT DOMESTICATIONS OF TREE PEONIES 3

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(Li 1998, 2005, 2011; Cheng et al. 2005). Our sampling

covered all extant sites of Paeonia rockii ssp. rockii,

P. rockii ssp. atava and P. jishanensis according to the

records in the literature and specimens available in

most herbariums worldwide (Fig. 1; Table S1, Support-

ing information). We sampled a single population of

P. decomposita because this species was not likely to

have contributed to the cultivated tree peonies (Li

2011). A minimum distance of 20 m was kept between

sampled wild individuals.

Most wild tree peonies, including P. rockii (ssp. rockii

and ssp. atava) and P. decomposita, can only reproduce

sexually, but P. jishanensis can also propagate asexually

by stolons. Cultivated peony varieties are often propa-

gated clonally by grafting, division or layering, although

breeders sometimes use seeds. To avoid the inclusion of

clones in the data set, we sampled a single tree per vari-

ety. A total of 103 FTP varieties of P. rockii propagated

commercially, including 49 traditional and 54 modern

varieties, all of great ornamental value, were collected

(Fig. 1, Table S1, Supporting information). A total of 72

CTPs representing varieties of P. suffruticosa, including

28 traditional and 44 commercial modern varieties, were

sampled (Cheng et al. 2005; Li 2005).

DNA was extracted from the collected leaves using

an improved cetyltrimethylammonium bromide (CTAB)

method (Yuan et al. 2010) and examined by electropho-

resis on a 1% agarose gel in TAE buffer. The DNA was

then diluted to a concentration of 5–10 ng/lL for subse-

quent polymerase chain reaction (PCR) amplification.

Microsatellite loci and PCR amplification

We used 14 polymorphic microsatellite loci (Table S2,

Supporting information) that were previously shown to

be independent and to not belong to the chloroplast gen-

ome of Paeonia (Yuan et al. 2012). The PCR conditions

were as described by Yuan et al. (2012). Positive and nega-

tive controls were systematically run. The PCR products

were examined on an ABI PRISM X3730XL DNA analyser

with GeneScan 600LIZ size standards (Applied Biosys-

tems, Foster City, CA, USA). Each chromatogram was

read using GeneMapper 4.0 (Applied Biosystems). The

genotyping results are presented in Table S3 (Supporting

information). Samples with uncertain alleles were double

checked by repeating the PCR amplifications.

Analysis of genetic diversity within each wild peonyspecies or subspecies and within cultivated tree peonies

Although our sampling was designed to avoid clones

within cultivated peonies, footprints of clonality could

still be present if some varieties differing by only a few

mutations have been propagated by grafting. In wild

species, siblings can be collected unintentionally in the

field. Because these features could result in spurious

genetic structure due to the presence of closely related

individuals in the data set, we checked for the existence

of clonal genotypes using GENCLONE 2.0 (Arnaud-

Haond & Belkhir 2007) 8and estimated relatedness

between pairs of individuals within each species by

calculating the rxy of Lynch & Ritland (1999) using

RE-RAT (Schwacke & Rosel 2005).

We tested for the occurrence of null alleles with

MICRO-CHECKER 2.2.3 (Van Oosterhout et al. 2004).

Allelic richness (AR) and private allele (AP) frequencies

for a sample size of four were calculated with ADZE

(Szpiech et al. 2008). Observed (Ho) and expected (HE)

heterozygosities and Weir & Cockerham F-statistics

were estimated using GenALEx 6.5 (Peakall & Smouse

2012). Inbreeding coefficient (FIS), deviation from the

Hardy–Weinberg equilibrium, genotypic linkage dis-

equilibrium and the significance of differences between

FST values were assessed using exact tests conducted

with GENEPOP 4.0 (Rousset 2008).

We tested for the occurrence of a recent genetic bot-

tleneck in cultivated and wild tree peonies using the

method implemented in BOTTLENECK (Cornuet &

Luikart 1996; Piry et al. 1999), which is based on the

detection of significant heterozygosity excess relative to

equilibrium expectations. Heterozygosity excess is an

ephemeral pattern that can only be detected within the

first several generations after a decline of population

size; it initially occurs because allelic diversity is lost

faster than heterozygosity, resulting in the appearance

of greater heterozygosity than expected based on the

number of alleles in the population (Cornuet & Luikart

1996). We used all three available models for microsat-

ellite evolution, that is, infinite allele (IAM), stepwise-

mutation (SMM) or two-phase (TPM) models, which

yielded different results (Table S4, Supporting informa-

tion). We report only the results obtained under the

TPM model, with the setting values of 10% SMM, 90%

IAM and 10% variance, because the TPM model has

been reported to be the most conservative and powerful

model available (Cornuet & Luikart 1996; Luikart et al.

1998). Models were run for 10,000 replicates. The recent

occurrence of a genetic bottleneck was further investi-

gated using the Garza–Williamson index (M, an empiri-

cal value of the ratio of number of alleles to range in

allele size). This index, analysed here using Arlequin

3.5 (Excoffier & Lischer 2010), is expected to be lower

than the critical Mc value of 0.68, a value obtained by

simulations based on the empirical data, in bottlenecked

populations (Garza & Willamson 2001) 9. The method

implemented in BOTTLENECK has low power unless

the decline is greater than 90% (Cornuet & Luikart

1996); this approach is most powerful when genetic



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bottlenecks are severe and recent (Williamson-Natesan

2005). The Garza–Williamson index method is more

likely to correctly detect genetic bottlenecks if the bot-

tleneck lasted several generations or if the population

made a rapid demographic recovery (Williamson-Nate-

san 2005).

Genetic differentiation among wild species andcultivated tree peonies

STRUCTURE 2.3.2 (Pritchard et al. 2000; Falush et al.

2003) was used to infer species delimitation and the

contribution of wild species or subspecies to the culti-

vated peony genome. Analyses were run without prior

information. STRUCTURE performs model-based clus-

tering, in which Markov chain Monte Carlo (MCMC)

simulations are used to identify K distinct clusters. The

algorithm attempts to minimize deviations from

Hardy–Weinberg and linkage equilibrium within clus-

ters. Ten independent runs were conducted for each

K. Each run was pursued for 2,000,000 MCMC interac-

tions using the following conditions: an initial burn-in

of 200,000, an admixture model, correlated allelic fre-

quencies and the same a for all samples. STRUCTURE

was run with the full data set (N = 553) and with the

pruned data set (N = 323) that excluded clonal and

related individuals (i.e. rxy > 0.5). STRUCTURE was run

for K ranging from one to eight for species delimitation

analyses. The DK statistic, which is designed to identify

the number of clusters above which an additional

increase in likelihood is weaker, was calculated follow-

ing the method of Evanno et al. (2005). For analyses of

wild species contribution to the cultivated peony gen-

ome, STRUCTURE was run at K = 2 on data sets

including either CTPs or FTPs and each of the four wild

peony species by pairs. The P. rockii ssp. rockii and

P. rockii ssp. atava structure had been previously well

studied (Yuan et al. 2012); we focus here instead on the

species history.

Principal component analysis (PCA) was performed

with allele frequencies calculated from the full and

pruned data sets using the Multivariate Statistical Pack-

age (MVSP) version 3.13b (Kovach Computing Services,

Anglesey, Wales, UK).

Approximate Bayesian inference

Using approximate Bayesian computation (ABC), we

investigated whether domestication of the cultivated

common tree peonies and flare tree peonies was a single

event or whether there have been independent domesti-

cations. Population structure does not strongly affect

ABC results (St Onge et al. 2012); thus, we did not take

into account intraspecies structure to avoid useless

complex scenarios including higher numbers of parame-

ters (divergence times and effective population sizes).

Models were constructed based on an FST matrix, histor-

ical records and previous phylogenetic studies of the

domestication history of tree peonies. We excluded

P. decomposita from the ABC analyses because it

appeared to be the most genetically divergent species

based on our microsatellite data and because we had a

low sampling size (N = 11). Historical records and pre-

vious phylogenetic studies pointed to P. jishanensis as

the wild progenitor of CTP cultivars, whereas FTP culti-

vars were suggested to have originated either from

P. rockii ssp. rockii or atava or P. ishanensis. The three

models tested here were therefore designed to distin-

guish between (a) single domestication of CTPs and

FTPs from one wild peony species, P. jishanensis (model

a, Fig. 2); (b) independent domestications of FTPs from

P. rockii ssp. atava and of CTPs from P. jishanensis

(model b, Fig. 2) and (c) independent domestications

of FTPs from P. rockii ssp. rockii and of CTPs from P. ji-

shanensis (model c, Fig. 2). For all models, we assumed

five distinct groups, as indicated by STRUCTURE analy-

ses: the three wild species or subspecies, P. rockii ssp.

rockii (PRR), P. rockii ssp. atava (PRA) and P. jishanensis

(PJ), and two cultivated peony groups, the CTPs and

FTPs. We used ABCtoolbox (Wegmann et al. 2010) with

fastsimcoal (Excoffier & Foll 2011) to compare the three

scenarios (Fig. 2). Because the juvenile period of Paeonia

lasts 4–6 years, we assumed a generation time of

5 years. We estimated the effective size of each group

(NX) and the divergence times between groups X and Y

(TX-Y). We assumed a uniform prior distribution for NX

[600; 4.105] and TX-Y [1; 5.104]. Our priors were large

because no reliable historical data were available.

For all models, microsatellite data sets were simu-

lated for 14 loci (Pdel02-2, Pdel05, Pdel06, Pdel07,

Pdel20, Pdel22, Pdel9b, Pdel33, Pdel35, Jx02-2, Jx05-2,

Jx17 and Jx29) that carry perfect repeats (Wang et al.

2008; Yuan et al. 2010, 2012). We generated 106 genetic

data sets from coalescent simulations using model

parameters drawn from prior distributions under the

three previously specified scenarios. For each simula-

tion, we calculated 31 summary statistics. Within each

group, we computed H, the mean heterozygosity across

loci. We also calculated FST (Weir & Cockerham 1984)

and genetic distances (dl)2 (Goldstein et al. 1995)

between pairs of groups. Following Wegmann et al.

(2009, 2010), 10we used the R package ‘PLS’ (i.e. partial

least squares, Mevik & Wehrens 2007) to find the

appropriate number of PLS components to use (8, in

our case). The same set of 31 summary statistics was

also calculated on the observed data set, was PLS-tran-

sformed and then was used to calculate the Euclidean

distance to each simulation.



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We assumed a generalized stepwise model of micro-

satellite evolution (Estoup et al. 2002). The mutation rate

was allowed to vary across loci, with locus-specific

mutation rates drawn from a gamma distribution (a, a/

l), in which l is the mutation rate per generation and a

is a shape parameter. We assumed a uniform prior distri-

bution for l [0.001, 0.01] and a uniform distribution for a

[1, 30].

We compared the three models by calculating their

Bayes factors (Wegmann et al. 2010) by estimating their

relative posterior probabilities based on the 1% of simu-

lated data sets that most closely matched the observed

data, that is, 1000 simulated data sets. Once the best model

had been chosen, we estimated demographic parameters

under this scenario using a general linear model (ABC-

GLM) post-sampling regression adjustment for the 1000

retained simulations (Leuenberger & Wegmann 2010;

Wegmann et al. 2010). We report the mode and 95% confi-

dence interval for each model parameter estimate.

The performance of the method for discriminating

between competing historical models was assessed by

analysing test data sets (called pseudo-observed data

sets) that were simulated with the same number of loci

and individuals as the observed data sets. We simu-

lated 1000 such data sets for each competing model

using parameter values drawn from the same prior dis-

tributions as for the original analyses. We determined

the relative posterior probabilities of competing models

for each pseudo-observed data set using the model

choice procedure described above (Wegmann et al.

2010). Confidence in model choice was then estimated

based on the percentage of times that a given scenario

did not have the highest posterior probability of the

competing scenarios when it was actually the true sce-

nario (type I error) and on the percentage of times that

a given scenario had the highest posterior probability

when it was not the true scenario (type II error).

Results

Genetic diversity in cultivated common and flare treepeonies

Eight pairs of samples were assigned to clonal groups by

GENCLONE: one pair each in CTPs, FTPs and Paeonia

jishanensis and five pairs in P. rockii ssp. rockii. The per-

centage of pairs with pairwise relatedness (rxy) greater

than 0.5 (i.e. full-siblings) was 1.1% in CTPs (N = 27

pairs), 0.72% in FTPs (N = 38), 13.55% in P. jishanensis

(N = 63), 9.1% in P. decomposita (N = 5), 2.85% in P. rockii

ssp. rockii (N = 1075) and 11.86% in P. rockii ssp. atava

(N = 217). To avoid biases, STRUCTURE analyses were

then conducted both on the full (N = 553) and pruned

data set (N = 323), that is, keeping a single individual per

pair of related individuals and per pair of clone mates.

Tests for linkage disequilibrium with the Bonferroni

correction showed no significant linkage disequilibrium.

No locus pair deviated significantly from equilibrium in

more than six or seven of 30 total sampled sites, sug-

gesting that all loci were physically unlinked.

Moderate genetic diversity was found in the cultivated

common (HO = 0.61) and flare (HO = 0.58) tree peonies

(Table 1). The four wild species or subspecies showed

significantly lower genetic diversity compared to FTPs

and CTPs (Wilcoxon-sign-rank [WSR] tests on HO

between FTPs/CTPs and each wild species or subspecies,

TPJ-PRR

Present

(b)(a)

TPJ-CTP/

TPJ-FTP

PJ CTP FTP PRA PRR PJ CTP FTP PRA PRR PJ CTP PRA FTP PRR

TPJ-PRA

TPJ-PRR

Present

TPJ-CTP/

TPRA-FTP

TPJ-PRA

Time (c)

TPJ-PRR

Present

TPJ-CTP/

TPRR-FTP

TPJ-PRA

TimeTime

Fig. 2 Alternative domestication models of FTP and CTP. We considered three models describing the domestication of CTP and FTP.

Model a assumed a single domestication of CTP and FTP from Paeonia jishanensis. Model b assumed independent domestication of

CTP from Paeonia jishanensis and of FTP from Paeonia rockii ssp. atava. Model c was identical to model b, except that it assumed the

domestication of FTP from Paeonia rockii ssp. rockii. NX: effective population size of group X and TX-Y: the divergence time between

groups X and Y. PJ: Paeonia jishanensis, PRR: Paeonia rockii ssp. rockii, PRA: Paeonia rockii ssp. atava, FTP: flare tree peonies; CTP:

common tree peonies.

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all P < 0.05) (Table 1). Allelic richness (AR) was found to

be significantly higher in cultivated CTPs and FTPs than

in wild species or subspecies (Table 1; CTPs: AR = 2.54,

WSR, P ≤ 0.003; FTPs: AR = 2.53, WSR, P ≤ 0.002). FTPs,

CTPs and wild peonies all displayed significant FIS(Tables 1 and S4, Supporting information), indicating

heterozygote deficiency. Null alleles were identified in

some populations of wild and cultivated common and

flare tree peonies. The presence of these alleles and a

Whalund effect may explain the observed deviations

from Hardy–Weinberg equilibrium (Tables 1 and S4,

Supporting information).

The BOTTLENECK analysis showed no significant het-

erozygosity excess in the cultivated common and flare

tree peonies but revealed significant heterozygosity

excess in some populations of wild peonies, namely

P. jishanensis, P. rockii ssp. atava and P. rockii ssp. rockii

(Table S4, Supporting information). The Garza–William-

son index was lower than the threshold value (i.e.

Mc = 0.68) in cultivated peonies (CTPs: Mc = 0.28 �

0.12, FTPs: Mc = 0.26 � 0.26) and in all populations of

wild tree peonies (Table S4, Supporting information).

Together, these results suggest that the wild tree peonies

P. jishanensis, P. rockii ssp. atava and P. rockii ssp. rockii

have suffered from demographic bottlenecks, while culti-

vated common and flare tree peonies have not.

Common and flare tree peonies form well-separatedgenetic clusters

The STRUCTURE results obtained with the full data set

(N = 553) (Fig. 3 and S1, Supporting information) and

those obtained with the pruned data set (N = 323) (Fig.

S2, Supporting information) were highly similar; we

therefore used the full data set for further analyses. The

DK value was the greatest at K = 4 (Fig. S3, Supporting

information; DK = 918, Pr|ln L = �13923). However, at

K = 4, the CTPs remained clustered with P. jishanensis

(Fig. 3 and S1, Supporting information). At K = 5, most

taxonomic species or subspecies (e.g. P. jishanensis,

P. rockii ssp. rockii and P. rockii ssp. atava), except

P. decomposita, which mingled within P. rockii ssp. rockii,

were assigned to distinct clusters in the main mode (i.e.

the clustering solution found in 90% of the simulation rep-

licates). In addition, a genetic structure appeared within

P. rockii ssp. rockii. No additional well-delimited clusters

corresponding to subdivisions of previous clusters were

detected beyond K = 5, except for a structure within

P. rockii ssp. atava and P. rockii ssp. rockii (Fig. 3 and Fig.

S1, Supporting information). Albeit intraspecies structure

was found, K = 5 appeared to be the clustering solution

that was most relevant to our questions aimed at deter-

mining the genetic differentiation of tree peony species.

At K ≥ 3, the cultivated CTPs and FTPs were clearly

assigned to different clusters, while the cultivated FTPs

and wild P. rockii ssp. atava were clustered together

(Fig. 3 and Fig. S1, Supporting information). At K = 4,

the CTPs and P. jishanensis remained assigned to the

same cluster. This result is in agreement with the PCA

results (Fig. 4), which show that the CTPs were distinct

from the FTPs, with the CTPs lying very close to

P. jishanensis and the FTPs appearing mingled within

P. rockii ssp. atava (Fig. 3). These results support the

hypothesis of independent origins of the two groups of

cultivars from distinct wild peony species.

Genetic differentiation (FST) between each cultivated

and each wild peony species or subspecies was signifi-

cant (P < 0.001). Paeonia decomposita appeared to be the

most differentiated from the other species (Table 2).

The lowest differentiation was found between the culti-

vated FTPs and wild P. rockii ssp. atava (FST = 0.05), in

agreement with the STRUCTURE and PCA plots

(Fig. 3,4 and S1; Supporting information). The CTPs

appeared closer to the FTPs (FST = 0.07), P. rockii ssp.

rockii (FST = 0.09) and P. rockii ssp. atava (FST = 0.11)

than to P. jishanensis (FST = 0.12) (Table 2).

Rare wild-to-crop and crop-to-crop hybridizationsduring recent cultivated tree peony history

The recent contribution of each wild tree peony species

to common or flare tree peonies was further investi-

gated by running STRUCTURE at K = 2 by pairs of spe-

cies, including CTPs or FTPs and each wild species or

subspecies (Fig. S4 and Tables S4, S5 and S6, Support-

ing information).

Table 1 Genetic polymorphism within the cultivated common

and flare tree peonies and the four wild peony species or sub-

species

N HO HE FIS AR AP PNA

Paeonia

jishanensis

31 0.40 0.52 0.27*** 2.19 0.74 0.07

Paeonia

decomposita

11 0.36 0.33 �0.12*** 1.57 0.27 0.02

Paeonia rockii

ssp. rockii

275 0.50 0.72 0.31*** 2.51 1.03 0.13

Paeonia rockii

ssp. atava

61 0.45 0.58 0.25*** 2.31 0.46 0.08

FTP 103 0.58 0.66 0.14*** 2.53 0.44 0.05

CTP 72 0.61 0.66 0.08*** 2.54 0.60 0.03

N: sample size; HO and HE: observed and expected heterozyg-

osities, respectively; FIS: inbreeding coefficient; AR and AP: alle-

lic richness and private allelic richness, respectively, averaged

across loci estimated by rarefaction using a standardized sam-

ple size of four; PNA: proportion of null alleles; FTP: flare tree

peony, CTP: common tree peony.

***P-value < 0.0001.



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At K = 2, all pairs of species were separated into two

well-delimited clusters, except the pair that included

the cultivated FTPs and P. rockii ssp. atava (Fig. S4b,

Supporting information); this pair showed a substruc-

ture within P. rockii ssp. atava. Further increasing K

failed to separate the FTPs and P. rockii ssp. atava (Fig.

S4b, Supporting information).

Some rare footprints of introgression were detected

between cultivated and wild species (admixed individu-

als in Fig. S4a and S4b and Tables S5 and S6, Supporting

K = 2

K = 3

K = 4

K = 5

K = 6

K = 7

K = 8

P. jish

anensis

P.decomposit

a

P. rock

i ssp

. rock

iii

P. rock

ii ssp

. atava

Flare t

ree peo

ny

(FTP)

Common tree

peony

(CTP)

Fig. 3 Proportions of ancestry of cultivated common tree peonies, flare tree peonies and the four wild tree peony species or subspe-

cies genotypes (N = 553) obtained using K = 2 to K = 8 ancestral gene pools (‘clusters’) inferred with STRUCTURE. Each individual

is represented by a vertical bar partitioned into K segments representing the amount of ancestry of its genome in K clusters. Where

several clustering solutions (‘modes’) were represented within replicate runs, the proportion of simulations represented by each

mode is given in Fig. S1.

PC

2 (

7.3

1%

)

PC 1 ( 9.14%)

–2

–4

–6

–8

0

2

4

6

8

–2–4–6–8 1086420–10

P. rockii ssp. rockiiP. jishanensis Common tree peony (CTP)P. rockii ssp. atava Flare tree peony (FTP) P. decomposita

Fig. 4 The first two axes of a principal component analysis representing the microsatellite data obtained from two cultivated groups,

the common tree peony (CTP, red) and the flare tree peony (FTP, purple), and from the four allopatric wild species or subspecies,

Paeonia decomposita (blue), Paeonia jishanensis (yellow), Paeonia rockii ssp. rockii (green) and Paeonia rockii ssp. atava (pink).

COLOR

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information) and also between the two cultivated groups,

CTPs and FTPs (admixed individuals in Fig. S4c and

Tables S5 and S6, Supporting information).

Modelling the domestication history of common andflare tree peonies

We used ABC to statistically determine which of the

three models (Fig. 2) assuming alternative domestica-

tion histories of the cultivated CTPs and FTPs was the

most likely. The relative posterior probabilities (P) cal-

culated for each model provided the strongest statistical

support for model b, suggesting that FTPs and CTPs

were domesticated independently (Bayes factor for

model b = 18.4, Table 3). The model that assumed a sin-

gle domestication event of FTPs/CTPs from P. jishanen-

sis had the lowest relative posterior probability

(Table 3). For model b, we obtained estimates of effec-

tive population sizes of 16,737 [95% confidence interval:

608–242,674] for NPJ, 53,047 [4639–252,751] for NPRR,

44,977 [4638–254,751] for NPRA, 53,046 [8,654–363,703]

for NFTP and 32,874 [4639–367,723] for NCTP. Using a

generation time of 5 years, P. jishanensis and P. rockii

ssp. rockii diverged 220,297 years ago (TPJ-PRR, [96,538–

247,518]), P. jishanensis and P. rockii ssp. atava diverged

139,574 years ago (TPJ-PRA, [39,300–224,806]) and CTPs

and FTPs diverged independently from P. jishanensis

and P. rockii ssp. atava 74,931 and 35,168 years ago,

respectively (TPJ-CTP/TPRA-FTP, [14,978–177,337] and [2514

–118,057], respectively). The estimated mutation rate

per generation was l = 0.001 [1e�3–3e�3]. We used 1000

pseudo-observed data sets to check the coverage prop-

erty of the marginal posterior distributions estimated

with our approach. As seen from the histograms of the

posterior quantiles, the marginal posterior distributions

were slightly overestimated on average (Fig. S5, Sup-

porting information). These estimations should there-

fore be regarded with caution, particularly the

divergence time estimations that presented biased pos-

terior distributions. However, the observed values fell

within the simulated data (P = 0.35), suggesting that

the assumed model is capable of reproducing the

observed summary statistics (the 8 PLS components, in

our case).

We checked that the power of the analysis was suffi-

cient to discriminate between the competing models.

For model b against the other two models, the type I

error rate was 0.22 and the mean type II error rate was

0.20. Overall, ABC analyses provided strong support for

the independent domestication of CTPs and FTPs from

P. jishanensis and P. rockii ssp. atava, respectively.

Discussion

Independent domestications of the common and flaretree peonies: a model of parallel and convergentevolution

In combination, the STRUCTURE and ABC analyses

presented here provide strong support for the hypothe-

sis of independent domestications of cultivated com-

mon and flare tree peony varieties in China from

different wild peony species, Paeonia jishanensis and

P. rockii ssp. atava, respectively. These findings are con-

sistent with the morphological features of the cultivated

groups (CTPs and FTPs) (Rehder 1920; Li 2005; Cheng

2007). The CTPs are dwarf shrubs less than one metre

in height; they are typically characterized by the pres-

ence of nine (often less than 15) leaflets per compound

leaf and red carpels and filaments, characteristics that

are very similar to those of wild P. jishanensis (Li 2005,

2011). The cultivated FTPs are taller than one metre,

usually have more than 15 leaflets per compound leaf

and display the yellowish or white carpels and

Table 2 Pairwise genetic differentiation (FST) between cultivated tree peonies and four wild peony species or subspecies

Paeonia jishanensis Paeonia decomposita Paeonia rockii ssp. rockii Paeonia rockii ssp. atava FTP

Paeonia decomposita 0.35

Paeonia rockii ssp. rockii 0.14 0.23

Paeonia rockii ssp. atava 0.15 0.26 0.10

FTP 0.15 0.27 0.07 0.05

CTP 0.12 0.26 0.09 0.11 0.07

All FST values were significant (P < 0.001).

FTP: flare tree peony, CTP: common tree peony.

Table 3 Relative posterior probabilities (P) and Bayes factors

(BF) for the three historical models compared by approximate

Bayesian computations. The models are described in Fig. 2

Model P BF

a 4.88e�2 5.13e�2

b 9.49e�1 18.4

c 2.66e�3 2.67e�3



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filaments typically found in P. rockii ssp. atava (Cheng

et al. 2005; Li 2005; Cheng 2007). The allopatric distribu-

tions of the two wild contributors, P. jishanensis and

P. rockii ssp. atava, across China also point to indepen-

dent geographical domestications of cultivated tree peo-

nies. Together with the cultivated olive (Besnard &

Berville 2000; Breton et al. 2006; Besnard et al. 2013), this

study is one of the rare existing examples of docu-

mented independent geographical domestications in

perennial tree crops. Our results do not clearly define

the contribution of P. decomposita to flare tree peony

domestication because of the low numbers of samples

of this species in our survey. To answer this question,

further sampling that includes a subspecies studied by

Zhou (2007), that is, P. decomposita ssp. rotundiloba from

Maoxian County in Sichuan province, is needed.

The STRUCTURE results obtained in this study sup-

port a more recent domestication of FTPs than CTPs by

indicating little differentiation between FTPs and its

wild progenitor, P. rockii ssp. atava, in contrast to the

clear differentiation that was found between CTPs and

P. jishanensis. Although the ABC estimations of diver-

gence time sensus stricto of CTPs and FTPs from their

respective progenitors should be regarded with caution,

the estimated divergence time between P. rockii ssp.

atava and FTPs was half the time of CTPs from P. jishan-

ensis. Together, the genetic findings are in accordance

with historical clues suggesting that domestication of

FTPs may be more recent than domestication of CTPs.

Traditional CTP cultivars were referred to as ‘the king of

flowers’ in the Tang Dynasty (approximately 627–850)

(Li 2005, 2011), while the FTPs were developed later dur-

ing the Ming to the Qing Dynasties (around 1398–1797)

(Cheng et al. 2005; Li 2011).

Despite their independent domestications, FTP and

CTP cultivars share a common domestication syndrome

with respect to the number and size of flowers (multiple

vs. single in wild species) and the number and colours

of the petals (colourful, e.g., patchy white, pink, purple

and red vs. mostly white in wild species or subspecies).

Cultivated tree peonies thus represent an interesting

case of parallel and convergent evolution, with similar

phenotypes in the two groups despite their independent

domestication from different locations and different spe-

cies. The relatively recent domestication of tree peonies

and the fact that they display a strong syndrome with

respect to a few traits provides a good model for study-

ing the genes and processes involved in adaptation

(Hohenlohe et al. 2010). In addition, this study suggests

that future studies targeting the genes involved in the

marked domestication syndrome are warranted.

In contrast to previous suggestions (Zhou 2007), we

show here that hybridizations have not been a major

force in shaping the genomics of cultivated tree peonies.

The low level of gene flow between CTPs and FTPs and

between cultivated and wild tree peonies contrasts with

the pattern found in other cultivated fruit tree crops,

which experience frequent wild-to-crop gene flow and

vice versa (Duputie et al. 2007; Miller & Gross 2011;

Cornille et al. 2012, 2013; Delplancke et al. 2012).

Although tree peonies are outcrossing species, they can

be vegetatively propagated, more easily than fruit trees

that require grafting. Early propagation by clonality

may have prevented introgression between wild and

cultivated peonies. The lack of widespread clonal struc-

ture found is due to our sampling scheme, which was

designed to avoid the use of multiple samples from a

given variety.

Moderate but higher genetic diversity in cultivatedpeonies than in wild peony species: implications for theconservations of genetic resources and future breedingprogrammes

As a human-mediated evolutionary process, domestica-

tion impacts contemporary patterns of genetic variation

in cultivated populations. The moderate level of genetic

diversity estimated in cultivated and wild tree peony

species in this study is consistent with findings from

previous studies (Pei et al. 1995; Zou et al. 1999; Meng

& Zheng 2004; Yuan et al. 2010, 2011, 2012; Zhang et al.

2011). No clear footprints of recent genetic bottlenecks

were detected in cultivated peonies in this study. In

particular, the genetic diversity of the cultivated CTPs

and FTPs appeared higher than the diversity of their

wild progenitors, P. jishanensis and P. rockii ssp. atava.

This unexpected result is not due to introgression in

cultivated tree peonies as introgression events were

found to be rare, nor is it due to undersampling,

because we have well covered the present-day distribu-

tion of these wild peony species. Genetic bottlenecks

are expected to occur during domestication, and the

allelic diversity in domesticated species is expected to

be a subset of that found in their wild progenitors

(Gross & Olsen 2010). Footprints of bottlenecks have

been reported in several annual seed-propagated crops,

such as maize, wheat, soybean and sunflower (Tenaillon

et al. 2004; Hyten et al. 2006; Liu & Burke 2006; Haudry

et al. 2007). Long generation times and overlapping gen-

erations in trees can reduce the occurrence of domesti-

cation bottlenecks (Austerlitz et al. 2000). The particular

breeding system of cultivated trees may also limit the

loss of genetic diversity due to selection; as with apples

(Cornille et al. 2012), many cultivated tree peonies have

been selected from seedlings derived from open pollina-

tion in gardens.

In contrast to the lack of recent genetic bottlenecks in

cultivated tree peonies, a bottleneck footprint has


10 J . - H . YUAN ET AL.

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occurred in a few populations of wild tree peonies. The

lower diversity of wild tree peony species or subspe-

cies, such as P. rockii ssp. atava and P. jishanensis, com-

pared to cultivated peonies is likely due to their

distribution decreasing in extent during recent decades

because of habitat change caused by natural factors and

human overexploitation (Li 2005; Hong 2010; Yuan et al.

2011, 2012). Several wild peony species have been listed

as rare and endangered taxa in the Red Book of Chinese

Plant Species (Fu 1992). In contrast, the diversity in

domestic cultivars is maintained by vegetative propaga-

tion that occurs as part of agricultural activity. Continu-

ous habitat destruction and fragmentation in wild tree

peonies is expected to result in smaller and more iso-

lated populations, raising the inbreeding coefficient at

the species level and further increasing the risk of pop-

ulation extinction, as reported for P. rockii (Cheng 2007;

Yuan et al. 2011, 2012). Given the low genetic diversity

and fragmentation of wild populations, more efficient

and stringent conservation policies should be estab-

lished for wild tree peonies to maintain their genetic

variation as a resource for future breeding programmes.

Conclusion

This study provides the first example of the indepen-

dent domestications of perennial tree crops and illus-

trates the complexity of the process of domestication of

such crops, which can involve contributions to the

genetic makeup of cultivated tree genomes from multi-

ple species, populations and locations (Myles et al. 2010;

Cornille et al. 2012; Besnard et al. 2013). The results of

this study also provide crucial insights into the domes-

tication history of some of the most famous ornamental

trees, the peonies, that are valuable for improving cur-

rent tree peony breeding strategies. Future research

requires extending the investigation of the origin of the

worldwide peony gene pool far from its centre of origin

in China.

The parallel and convergent evolution of cultivated

tree peonies represents an interesting case study for

understanding the mechanisms of domestication, diver-

sification and adaptation in plants. Future studies focus-

ing on adaptive genetic variation in tree peonies will

likely yield important insights into the processes of evo-

lution. It would be interesting for instance to determine

whether the same genes, or even the same mutations,

are involved in the traits shared by the FTPs and CTPs.

Acknowledgements

We thank Myriam Heuertz and two referees for comments on

a previous version of this manuscript. We also deeply thank

Mr. Jiajue Li, Mr. Xingyun Cheng, Mrs. Yialing Tian, Dr. Jia

Wang, Mr. Jianhong Wang and others for their help with the

sample collections. We thank Dr. Shi-Liang Zhou for kindly

providing the experimental platform. This study was funded

by the National Natural Science Foundation of China (No.

31171984 and 31070617) and Technologies R&D Program of

Shanghai (10391901200).

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J.Y. and F.C. conceived and designed the experiments

and provided samples; J.Y., F.C. and Y.H. obtained

funding; J.Y. performed the experiments; J.Y. and A.C.

analysed the data and contributed analysis tools; J.Y.,

A.C., T.G. and Y.F. wrote the article.

Data accessibility

Sampling locations and microsatellite genotypes are depos-

ited in the DRYAD repository, doi:10.5061/dryad.vk43j.



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Supporting information

Additional supporting information may be found in the online ver-

sion of this article.

Fig. S1 The proportions of ancestry of genotypes of cultivated

common tree peonies, flare tree peonies and the four wild tree

peony species, Paeonia jishanensis, Paeonia rockii ssp. atava, Paeo-

nia rockii ssp. rockii and Paeonia decomposita (N = 553) from

K = 2 to K = 8 ancestral gene pools (‘clusters’) inferred with

the STRUCTURE program using the full data set. Each individ-

ual is represented by a vertical bar partitioned into K segments

representing the amount of ancestry of its genome in K clus-

ters. For cases in which several clustering solutions (‘modes’)

were represented within replicate runs, the proportion of simu-

lations represented by each mode is given.

Fig. S2 The proportions of ancestry of genotypes of cultivated

common tree peonies, flare tree peonies and the four wild tree

peony species, Paeonia jishanensis, Paeonia rockii ssp. atava, Paeo-

nia rockii ssp. rockii and Paeonia decomposita (N = 323) from

K = 2 to K = 8 ancestral gene pools (‘clusters’) inferred with

the STRUCTURE program using the pruned data set (i.e.

excluding clonal and related individuals). Each individual is

represented by a vertical bar partitioned into K segments rep-

resenting the amount of ancestry of its genome in K clusters.

Where several clustering solutions (‘modes’) were represented

within replicate runs, the proportion of simulations repre-

sented by each mode is given.

Fig. S3 Bayesian inference of the number of ancestral gene

pools (‘clusters’, DK) from cultivated common tree peonies,

flare tree peonies and the four wild tree peony species or sub-

species, Paeonia jishanensis, Paeonia rockii ssp. atava, Paeonia rock-

ii ssp. rockii and Paeonia decomposita (N = 553).

Fig. S4 The proportions of ancestry in two ancestral gene pools

inferred with the STRUCTURE program and based on the full

data sets for (a) the common tree peony (red, N = 72, left) and

(b) the flare tree peony (purple, N = 103, left) and each of four

wild tree peony species (right) and (c) for the common tree

peony (red, left) and the flare tree peony (purple, right). Details

are provided in Tables S6 and S7.

Fig. S5 Distribution of the posterior quantiles based on 1000

pseudo-observed data sets. P-values were computed with a

Kolmogorov–Smirnov test of uniformity of the posterior quan-

tile distribution.

Table S1 Description of the cultivated common tree peony,

flare tree peony and wild tree peony accessions analysed with

their geographic origins, providers, latitudes (N), longitudes

(E), altitudes and sampling sizes.

Table S2 Repeat motifs, annealing temperatures (Ta), primer

sequences and range of alleles detected for each of the 14 mi-

crosatellite loci.

Table S3 The genotypes of the 553 accessions at 14 microsatel-

lite loci.

Table S4 Genetic polymorphism within the cultivated common

and flare peonies and the four wild peony species or subspe-

cies at the population level.

Table S5 Mean membership coefficient and the a value at

K = 2 between (a) cultivated common tree peonies and (b) flare

tree peonies (Genepool 1) and each of the four wild tree peony

species or subspecies and (c) between the cultivated common

tree peonies and flare tree peonies determined using the

STRUCTURE program.

Table S6 The mean proportions of assignment to each of the

two ancestral gene pools in pairwise comparisons (K = 2)

including common tree peonies as Genepool 1 and each of the

four wild tree peony species or subspecies or flare tree peonies

as Genepool 2.

Table S7 The mean proportions of assignment to each of the

two ancestral gene pools in pairwise comparisons (K = 2)

including flare tree peonies as Genepool 1 and each of the four

wild tree peony species or subspecies or common tree peonies

as Genepool 2.



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