Nonadaptive molecular evolution of plastome during the speciation of Actaea purpurea and its relatives

Abstract

We have seen an explosive increase of plant plastid genome (plastome) sequences in the last decade, and the view that sequence variation in plastomes is maintained by the mutation‐drift balance has been challenged by new evidence. Although comparative genomic and population‐level studies provided us with evidence for positive evolution of plastid genes at both the macro‐ and micro‐evolution levels, less studies have systematically investigated how plastomes have evolved during the speciation process. We here sequenced 13 plastomes of Actaea purpurea (P.K. Hsiao) J. Compton, and its closest relatives, and conducted a systematic survey of positive selection in their plastid genes using the McDonald‐Kreitman test and codon‐based methods using maximum likelihood to estimate the ratio of nonsynonymous to synonymous substitutions (ω) across a phylogeny. We found that during the speciation of A. purpurea and its relatives, all plastid genes evolved neutrally or were under purifying selection. Genome size, gene order, and number were highly conserved. Comparing to A. purpurea, plastomes of Actaea japonica and Actaea biternata had low genetic diversity, consistent with previous studies. Our work not only sheds important light on the evolutionary history of A. purpurea and its kin, but also on the evolution of plastomes during plant speciation. Thirteen plastomes of 1representative populations of Actaea purpurea and its relatives were conserved with respect to size, structure, gene content, and order, with few variations. Positive selection analysis suggests that all plastid genes evolve neutrally or are subjected to purifying selection. Actaea japonica's low genetic diversity probably results from a recent expansion, not a recent selective sweep on plastid genes.

Full text

Ecology and Evolution. 2022;12:e9321.  
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https://doi.org/10.1002/ece3.9321
www.ecolevol.org
1 | INTRODUCTION
The plant genomes are compartmentalized as other eukaryotes,
composed of the nucleus and organelles. As the result of endo-
symbiosis, plastids and mitochondria have retained features of
their ancestral genomes but also transferred most of their genes
to the nuclear genome (Kleine et al., 2009). Genomes of plastids
(plastomes) in photosynthetic angiosperms are relatively conserved
in structure, gene number, and arrangement (Palmer & Stein, 1986).
A typical angiosperm plastome is circular and could be split into four
regions: a large single- copy region (LSC) and a small single- copy re-
gion (SSC), which are separated by two identical inverted repeats
(IRs) (Davis et al., 2014; Du et al., 2015). It normally includes ca.
80 protein coding genes (PCGs), 30 transfer RNA genes and four
Received:21February2022
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Revised:2May2022
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Accepted:30August2022
DOI: 10.1002 /ece3.9321
RESEARCH ARTICLE
Nonadaptive molecular evolution of plastome during the
speciation of Actaea purpurea and its relatives
Dan- Qing Zhang1,2 | Yi Ren1,2 | Jian- Qiang Zhang1,2
This is an op en access ar ticle under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provide d the original work is properly cited.
© 2022 The Authors. Eco logy and Evolut ion published by John Wiley & Sons Ltd.
1National Engineering Laboratory for
Resource Development of Endangered
Crude Drugs in Nor thwest China,
College of L ife Science s, Shaanxi Normal
University, Xi'an, China
2KeyLaboratoryofMedicinalPlant
Resource and Natural Pharmaceutical
ChemistryofMinis tryofEduc ation,
Shaanxi Normal University, Xi'an, China
Correspondence
Jian- Qiang Zhang, National Engineering
Laboratory for Resource Development of
Endange red Crude Drugs in Nor thwest
China, College of Life Sciences, Shaanxi
Normal U niversit y, Xi'an 710119, C hina.
Email: jqzhang@snnu.edu.cn
Funding information
Nationa l Natural Science Foundation of
China, G rant/Award Number: 31870194
and 32070236; Fundamental Research
Funds for the Central Universitie s, Grant/
Award Number: GK202103077
Abstract
We have seen an explosive increase of plant plastid genome (plastome) sequences
in the last decade, and the view that sequence variation in plastomes is maintained
by the mutation- drift balance has been challenged by new evidence. Although com-
parative genomic and population- level studies provided us with evidence for positive
evolution of plastid genes at both the macro- and micro- evolution levels, less studies
have systematically investigated how plastomes have evolved during the speciation
process. We here sequenced 13 plastomes of Actaea purpurea (P.K. Hsiao) J. Compton,
and its closest relatives, and conducted a systematic survey of positive selection in
their plastid genes using the McDonald-Kreitman test and codon-based methods
using maximum likelihood to estimate the ratio of nonsynonymous to synonymous
substitutions (ω) across a phylogeny. We found that during the speciation of A. pur-
purea and its relatives, all plastid genes evolved neutrally or were under purifying
selection. Genome size, gene order, and number were highly conserved. Comparing to
A. purpurea, plastomes of Actaea japonica and Actaea biternata had low genetic diver-
sity, consistent with previous studies. Our work not only sheds important light on the
evolutionary history of A. purpurea and its kin, but also on the evolution of plastomes
during plant speciation.
KEYWORDS
Actaea,adaptiveevolution,dN/dS,McDonald-Kreitmantest,plastome,speciation
TAXONOMY CLASSIFICATION
Genomics

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ribosomal RNA genes (Daniell et al., 2016). PCGs encode proteins
essential in photosynthesis, transcription, and translation (Kleine
et al., 2009). Plastomes normally have two notable features: (1) there
are many identical copies in plant cells, resulting in a high polyploidy;
(2) virtually lack of recombination (Greiner et al., 2011), but there are
still several genera present high recombination rates, which altered
the plastome structure, e.g., Onobrychis Mill. and Trifolium L. (Cai
et al., 2008;Moghaddametal.,2022).
It is recognized that the sequence variation in plastomes is main-
tained by the mutation- drift balance, and no adaptive evolution
would have occurred (reviewed in Bock et al., 2014). However, re-
cent evidence has shown that positive selec tion may have played an
important role in the evolution of plastomes (Bock et al., 2014; De
Santana Lopes et al., 2021; Wu et al., 2020).Forexample,Muirand
Filatov (20 07) inferred a selective sweep on the Silene L. plastome,
occurringbetween0.16and 1.06Mya(Million yearsago).Sambatti
et al. (2008) used reciprocal transplant experiments to show that
plastid genes were involved in drought adaptation in Helianthus pet-
iolaris Nutt. and H. annuus L. Several recent comparative genomic
studies in a plethora of plant groups also detected signatures of
positive selection from the patterns of sequence diversity (e.g., Ye
et al., 2018; Zhao et al., 2020). Although these studies provided us
with evidence for positive evolution of plastid genes at both the
micro- and macro- evolution levels, less studies have systematically
investigated how plastomes have evolved during the speciation pro-
cess. Plastomes could contribute to the speciation process through
establishment of reproductive barriers by genetic incompatibility
between the nucleus. For example, a recent study has shown that
adaptation to specific environmental factors could cause the evo-
lution of the hybridization barriers via the cytoplasm and nucleus
incompatibility (Zupok et al., 2021).
Actaea purpurea (P.K. Hsiao) J. Compton is a perennial herb
growing in the understory or the forest margins (Hsiao, 1979; Li &
Brach, 20 01). Its flowers are distinct from other species in the genus
by having purple sepals and less stamens with purple filaments and
yellow anthers, while flowers of other congeners are white and have
numerous white stamens (Chang et al., 2020). Phylogenetic studies
have shown that A. purpurea is sister to A. japonica Thunb. and A. bit-
ernata (Siebold & Zucc.) Prantl, and the three species formed a well-
supported clade (Compton, Culham, Gibbings et al., 1998, Compton,
Culham, & Jury, 1998). Chang et al. (2020) used three plastid markers
(trnL- trnF, rpl20- rps12, trnS- trnG) to study genetic divergence of the
group, and they found a striking pattern: all individuals of A. japonica
and A . biternata shared one haplotype. Compared with A. purpurea,
which had multiple haplotypes, the lack of genetic variation in A.
japonica and A. biternata might be caused by a historical selective
sweep, or a recent demographic expansion of A. japonica and A. bit-
ernata populations. If the selective sweep hypothesis is true, plas-
tid genes may have played an impor tant role in the divergence and
speciation of A. purpurea and its relatives. To discriminate the two
scenarios, we sequenced 13 plastomes of A. purpurea and its closest
relatives, and conducted a systematic survey of positive selection
in plastid genes of A. japonica + A. biternata, using both population
genetic-based test (the McDonald-Kreitman test; McDonald &
Kreitman, 1991) and codon- based methods using maximum likeli-
hood to estimate the ratio of nonsynonymous to synonymous sub-
stitutions (ω) across a phylogeny. We also investigated the structure
and gene content variation in this group. Our work sheds light on the
evolution of plastomes in divergence and speciation, and also on the
evolutionary history of A. purpurea and its close relatives.
2 | MATERIALS AND METHODS
2.1 | Taxon sampling
Thirteen individuals representing 13 populations of the three spe-
cies of Actaea L. were sampled in the study, covering all their dis-
tribution area (Table S1– S 5). All voucher specimens were deposited
in Shaanxi Normal University Herbarium (SANU). The latitude, lon-
gitude, and elevation of each sampling site were recorded using a
hand- held eTrex GPS (Garmin). Leaves from each individual were
dried immediately in silica gel, and then stored at room temperature
for further DNA extraction.
2.2 | Sequencing, genome assembly, and
gene annotation
According to the standard protocol provided by Illumina, the silica-
dried leaf material was sent to Novogene (Beijing, China) for library
preparation and sequencing. In short, total genomic DNA was ex-
tract ed from 20 to 30 mg silic a-gel drie d leaves using a modif ied
Cetrimonium bromide (C TAB) method (Doyle & Doyle, 1987 ). DNA
samples were then subjected to ultrasonic treatment, mechanical
cleavage, p urificatio n, end repair, addin g adenylate to the 3′ e nd,
and linker ligation to construct a sequencing library. Quality con-
trol of the library was executed by a Qubit 3.0 fluorometer (Life
Technologies, Shanghai, China). NGS3K/Caliper and q- PCR were
also used to secure the quality of the sequencing library. Sequencing
was performed using an Illumina-Miseq Novaseq 600 0. Double-
ended re ads with a leng th of 150 bp were gene rated. The out put
data was subjected to data quality control by FastP (parameter: - q
30 - u 50) (Chen et al., 2018). The plastomes were de novo assembled
utilizing G etOrganelle v1.7. 5 (Jin et al., 2020), and the parameter was
-R30-J 1-M1 -Fembplant_pt. Theobtainedscaffoldwasvisually
corrected in the Bandage v0.8.1 (Wick et al., 2015) to obt ain the
complete plastome.
After genome assembly, we used Geneious R10 (Biomatters Ltd.,
Auckland, New Zealand) and CPGAVAS2 (Shi et al., 2 019) to per-
form gene annotation. The plastome of Actaea asiatica Hara (Zhai
et al., 2019) was used as the reference for gene annotation. We man-
ually checked and modified the draft genome according to the refer-
ence genome and the result file of CPGAVAS2 to accurately define
the boundaries between start and stop codons, as well as between
gene exons and introns. tRNAscan- SE v1.21 was used to verify
annotated tRNA genes (Schattner et al., 2005). In order to visually
show the structure and genomic content of plastomes of the three

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Actaea species, we made a circular illustration for each plastome
using the Organellar Genome Draw program (OGR AW; https://chlor
obox.mpimp - golm.mpg.de/OGDraw.html) (Greiner et al., 2019).
2.3 | Comparative analysis
WeconductedmultiplesequencealignmentinMAFFTv7(Katoh&
Standley, 2013) with the default parameters (algorithm: Auto; scor-
ing_matrix: 200PAM/k = 2; gap_open _penalt y: 1.53; off set_value:
0.123), and subsequently checked manually in Geneious R10. In
order to identify the interspecific structural variation between A.
purpurea and its relatives, we used mVISTA (Frazer et al., 2004) to
visualize the alignment. We chose the Shuffle- Lagan mode in the
setup and A . biternataasthereference.Inaddition,weusedMauve
v2.3.1 (Darling et al., 2010) to confirm whether gene rearrangement
events occurred among and within species. We also used IRscope
(https://irsco pe.shiny apps.io/irapp/) (Amiryousefi et al., 2018) to
detect the contraction and expansion of these boundaries. In order
to distinguish differences in variation between different regions of
the plastomes, DnaSP v6 (Rozas et al., 2017) was used to estimate
the nucleotide diversity (pi) of all coding and noncoding regions (in-
tergenic regions and introns).
2.4 | Repetitive sequence analysis
Four types of repeats were searched for in the obtained plasto-
mes: tandem repeats, dispersed repeats, palindrome repeats, and
microsatellite sequences (SSR). Tandem Repeats Finder v4.09.1
(Benson, 1999) was used to search for tandem repeats with a length
of at least 10 bp. The alignment parameters (match, mismatch, and
indel) were set to 2, 7, and 7, respectively. We used REPuter soft-
ware (Kurtz et al., 2001) to search for dispersed repeats and palin-
drome repeats, with the minimum repeatlength of 30 bp, and the
minimum interval between repeats of 3 bp. The minimum similarity
betweensequenceswas setto90%. TheMISA-web(https://webbl
ast.ipk- gater sleben.de/misa/index.php?actio n=1) (Beier et al., 2 017)
was used to search for SSRs. The thresholds for single nucleotide,
dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and
dinucleotide were set to 10, 5, 4, 3, 3, and 3, respectively.
2.5 | Phylogenetic and dating analysis
For the phylogenetic and dating analyses, we used the plastome of A.
asiatica (Zhai et al., 2 019) as the outgroup. Based on plastomes of all
populations,maximumlikelihood(ML)treeswereconstructedusing
IQtreev2.1.4(Minhetal.,2020). The optimal nucleotide substitution
model was determinedby jModeltestv2.1(Darribaetal., 2012) as
GTR. Bootstrap values were assessed by ultrafast bootstrap approx-
imation (UFBoot; Hoang et al., 2017) for 1000 rep licates. We used an
uncorrelated relaxed log- normal molecular clock to estimate diver-
gence times using BEAST v1.10 (Suchard et al., 2018). The program
BEAUti was used to set the parameters for analysis. As there is no
reliable fossil record for A . purpurea and its kin, we used a secondary-
calibration method. The separation of A. purpurea between A. japon-
ica + A. biternatawasse tat1.63Mya(95%h ighestposte riordensity:
1.02–2.21 Mya; Chang et al., 2020). We run 100,000,000 genera-
tions of the chain, and sampled parameters every 1000 generations.
The first 20% of the parameters were discarded as burn- in. We then
used Tracer v1.7.1 (Rambaut et al., 2018) to make sure the effective
sampling size (ESS) for each parameter was larger than 20 0. Finally,
Tree Annotator v1.7.1 (Suchard et al., 2018) was used to generate
themaximumcladecredibility(MCC)tree.
2.6 | Detection of signatures of positive selection
Signatures of positive selection could be detected using several
tests. A modest to high amount of sequence variation is often a pre-
requisite for most analyses. As our dat a set had limited sequence
variation, we focused our tests on genes with enough variable sites.
A combination of different tests would provide more reliable results.
We first calculated Tajima's D (Ta ji ma , 19 89) and Fu's FS (Fu, 1997)
for each gene using the program ARLEQUIN v3.5.2.2 (Excoffier
et al., 2005). The significance level was inferred with 1000 simu-
lated samples. These tests cannot distinguish between selection and
demographic dynamics, i.e., population bottlenecks or expansions,
but significant values would indicate non- neutral evolution of se-
quences detected.
The second method we used was the codon- based method that
estimates the ratio of nonsynonymous to synonymous (ω) across a
phylogeny.WeusedEasyCodeMLv1.0(Gao,Chen,etal.,2019 ; Gao,
Liu, et al., 2019) preset mode as the default setting. Then, we utilized
the branch- site model (Yang & Nielsen, 2002) to identify positively
selected loci from genes in the foreground branch. The genes with
p < .05inthechi-squaretestareselectedascandidatepositives.For
both models, we used sequences of A. japonica and A. biternata as
the foreground according to our hypothesis.
It is generally recognized that the codon- based method is conser-
vative, as adaptive sites would be diluted across the entire sequence.
Wethusused the McDonald-Kreitman test (MKT )tocomplement
the above analysis. This test calculates a neutralit y index (NI) by di-
viding the ratio of nonsynonymous to synonymous polymorphisms
within species to the ratio of nonsynonymous to synonymous diver-
gence between species. A less than one value of NI would indicate
positive s electio n. All MKTs were run u sing the MK T-web (h t t p: //
mkt.uab.es/mkt/MKT.asp) (Egea et al., 2008).
2.7 | Environmental analysis
We used a total of 42 sampling sites based on our field collections,
including 22 for A. purpurea, 18 for A. japonica, and one for A. bit-
ernata (Table S3) to conduct the environmental analysis. Nineteen
contemporary environment variables (BIO1- BIO19) were down-
loaded from the WorldClim website (http://world clim.org/) (Hijmans

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et al., 2005). ArcGIS v10.5 was used to extract the values of 19 con-
temporary environmental variables and altitude for each sampling
site. We then performed a Principal Component Analysis (PCA) using
the R pack age FactoMineR (Lê et al., 2008), followed by plotting
using the R package ggplot2 (Wickham, 2016). Data normality was
checked by Shapiro– Wilk's test for each variable in R. For variables
not normally distributed, we took the logarithm for them before the
PCA. For the environmental variables that have a larger contribution
to PCs, we used the Welch's t- test and the Wilcoxon rank sum test in
R to test whether the difference is significant between A. purpurea
and A. japonica + A. biternata.
3 | RESULTS
3.1 | Characteristics of plastomes of A. purpurea
and its relatives
Thirteen complete plastomes of A. purpurea and it s relatives were
sequenced and annotated. These plastomes all possessed a typi-
cal angiosperm quadripartite structure (Figures 1, S1), including the
LSC(88,586–88,984 bp),theSSC(17,490 bp-17,763 bp),andtwoIRs
(26,530 -26,652 bp). Among the 13plastomes, populationPJZof A.
purpurea(159,398 bp)hadthesmallestplastome,andHB11ofA. ja-
ponica(159,821 bp)hadthelargest(Table 1). The total GC content is
38.1%, and it was higher in the IR (43.1– 43.0%) than both the LSC
(36.2– 36.3%) and SSC (32.3– 32.6%) (Table 1).
The number of genes in different Actaea species was also con-
sistent: each plastome comprised 131 predicted genes, 18 of which
were repeated in IRs. The 113 unique genes included 79 PCGs, 30
tRNA genes, and four rRNA genes. The incompletely duplicated cop-
ies of ycf1 and rps19 in IR were two pseudogenes (Table 1; Figures 1,
S1– S 7). A total of 12 genes (excluding three duplicate copies) con-
tained introns, of which nine genes had intron (atpF, ndhA , ndhB,
petB, petD, rpl2, rpl16, rpoC1, rps16 ) and three genes had two introns
(ycf3, clpP, rps12 ) (Table S2).
The comparison of border regions of Actaea plastomes showed
that IRs were relatively stable, and there was no significant expan-
sion or contraction events. The LSC- IRb and IRa- LSC boundaries
were located in two copies of the rps19 gene, respectively, and
no displacement was detec ted. IRb- SSC and SSC- IRa boundaries
were located in two copies of the ycf1 gene (Figure 2). The exac t
location of the boundaries of all populations was constant in A.
japonica and A. biternata. However, in A. purpurea populations, the
IRb- SSC and the IRa- LSC boundary had shifted to varying degrees
(Figure 2).
3.2 | Structural and sequence diversity of
Actaea plastomes
Tak ing A. biternata as the reference, the results of mVISTA showed
that all Actaeaplastomeshavehighsequencesimilarity.Mostofthe
differences existed between the inter- specific divergence of A. ja-
ponica and A. purpurea, while the intraspecific difference was very
small. Mostofthese differenceswerelocatedinthenoncodingre-
gions, and the region with the highest pi value was in the noncoding
region: rp l14- rpl16 (Figure 3).Mauve'smultiplecomparativeanalysis
of 13 chloroplast genomes showed that no genome rearrangement
event had occurred (Figure S2).
The five genes with the highest sequence diversity were psbI,
rpl20, trnG (UCC), ndhG, and ycf1. The corresponding noncoding
regions were rpl14- rp l16, ndhF- trnL(UAG), trnH(GUG)- psbA, ccsA-
ndhD, and ps bT- psbN (Figure 4). Both the noncoding and cod-
ing regions of A. japonica had significant lower genetic diversity
(0.00040 and 0.00002) than A. purpurea (0.001425, p = 4. 8 E- 11 ;
0.00 023, p = 9.1E- 06 ) ( Table 2). In addition, consistent with the
comparison based on the complete plastome (Figure 3), the nu-
cleotide diversity of the noncoding region (0– 0.06527, 0.04381)
was significantly higher than the nucleotide diversity of the coding
region (0– 0.00624, 0.00078, p = 3.7E- 7 ) (Figure 4).Meanwhile,the
nucleotide diversity of the IR region (noncoding region: 0.00089;
coding region: 0.00001) was lower than that of the LSC (0.00471,
p = 5.2E- 5; 0.00081, p = 3.1E- 3) and the SSC (0.00871, p = .012;
0.00165, p = 2.1E- 4) (Table 2). At the whole plastome level, the
nucleotide diversity of A. purpurea is higher than that of A. japonica
(Table 2).
3.3 | Repetitive sequences
The distribution of tandem repeat s, dispersed, palindromic repeats,
and SSR sequences in three Actaea species was analyzed. The re-
petitive sequences were mainly distributed in the L SC, and others
were in the SSC and IRs. The number of dispersed repeat s and pal-
indromic repeats of A. purpurea was significantly higher than that of
A. japonica, and the difference mainly existed in the SSC (Figures 5,
S3). Except for SSRs, there was a significant gap between the three
repeats of A. purpurea and A. japonica in the CDS region (Figure S3).
We found 624 SSRs, including mononucleotide, dinucleotide, trinu-
cleotide, tetranucleotide, and pentanucleotide. The numbers of each
type were289,152,76,125,and28,respectively.Mononucleotide
accounted for 43.13% of all SSRs. A. purpurea had a unique tetra-
nucleotide type and a unique pentanucleotide t ype (Figure S4). In
addition,therewasa242 bplongrepetitivesequenceinA. purpurea
(Figure S5).
3.4 | Phylogeny and dating analysis
The time tree constructed based on the complete chloroplast ge-
nome showed that A. purpurea diverged from A. japonica and A. bit-
ernataatca.1.58Mya(95%HPD:1.18–1.97Mya)andthelattertwo
formed a clade (Figures 6b, S6). The divergence of A . japonica and
A. biternatawas at ca. 0.12 Mya (95%HPD: 0.0 4–0.23Mya;node
1, Figure 6b), which was at the late Pleistocene. PJZ was the first

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A. purpurea population to separate from other populations about
0.56 Mya (95%highest posteriordensity: 0.29–0.87 Mya; node 2,
Figure 6b).
3.5 | Positive selection analysis
As positive selection tests require a modest to high amount of se-
quence variation between taxa, we only reported test result s in 12
genes with suitable number of variable sites. Our results showed
that no Tajima's D or Fu's Fs values were significantly positive or
negative (Table 3). Branch sites tests indicated that most plastid
genes have evolved in a neutral (ω = 1) way or under purifying selec-
tion (ω < 1).Twogenes(r poC1 and rpoC2) had a global ω > 1,butthe
two- ratio model was not significantly better than model 0 (Table 3).
Insufficient variation in our sequence data resulted in infinite or un-
definedNIvaluesinMKTs.Othertestsyieldednonsignificantvalues
of NI (Table 3). Notably, the rbcLgenehad aNI < 1 witha marginal
significant level (p = .08). In summary, our positive selection analysis
showed no definite evidence for adaptive evolution of plastid genes.
FIGURE 1 GenemapoftheActaea japonica plastome. Outside the circle are genes transcribed in a counter- clockwise direction, whereas
inside the circle are those transcribed in a clockwise direction. In the inner circle, the dark gray area represents GC content and the thick line
indicates the extent of different regions. Different colors for genes show different functional groups. LSC, Large- single- copy; SSC, Small-
single- copy; IR, Inverted repeat.

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TABLE 1 CharacteristicsofplastomesofActaea purpurea and its relatives
Species Population
Size(b p) GC content%
Gene no. PCG tRNA rRNA
Genes
with
introns Pseudo- gene no.Overall LSC SSC IR Overall LSC SSC IR
Actaea biternata JP01 159,761 8 8,930 1 7,7 57 26, 537 38.10% 36.20% 32.30% 43 .10% 131 84 (5) 37 (7) 8 (4) 15 2
A. japonica GZ01 15 9,73 5 88,9 00 17, 762 26,536 38.10% 36.20% 32.30% 43.10% 131 84 (5) 37 ( 7) 8 (4) 15 2
HB11 1 59,821 88,984 1 7,76 3 26,537 38.10% 36.20% 32.30% 43.10% 131 84 (5) 37 ( 7) 8 (4) 15 2
JJZ 159,6 04 8 8, 774 17, 75 6 26,537 38.10% 36.30% 32.30% 4 3.10% 131 8 4 (5) 37 (7 ) 8 (4) 15 2
JP02 15 9,78 6 88,955 17,7 57 26, 537 38.10% 36.20% 32.30% 43.10% 131 84 (5) 37 (7) 8 (4) 15 2
SC02 159,768 88,939 1 7,755 26,537 38.10% 36.20% 32.30% 43.10% 131 84 (5) 37 ( 7) 8 (4) 15 2
ZJ02 159,7 25 88,894 17, 75 7 26,537 38.10% 36.20% 32.30% 43.10% 131 84 (5) 37 (7) 8 (4) 15 2
A. purpurea HB01 159,497 88,703 1 7,4 90 26,652 38.10% 36.30% 32.60% 43.00% 131 8 4 (5) 37 (7) 8 (4) 15 2
HB04 159,385 88,586 17,4 95 26, 652 38.10% 36.20% 32.60% 43.00% 131 8 4 (5) 37 (7) 8 (4) 15 2
HE02 15 9,496 88,893 1 7,5 41 26,531 38.10% 36.20% 32.50% 43.10% 131 84 (5) 37 (7) 8 (4) 15 2
PJZ 1 59,398 8 8,814 17, 561 26,530 38.10% 36.30% 32.50% 43.10 % 131 84 (5) 37 (7) 8 (4) 15 2
PZX 15 9,4 35 88,793 17, 54 3 26,531 38.10% 36.30% 32.50% 43.10% 131 84 (5) 37 (7) 8 (4) 15 2
SC01 159, 574 88,780 17, 49 0 26, 652 38.10% 36.30% 32.60% 43.00% 131 84 (5) 37 (7) 8 (4) 15 2
Note: Numbers in brackets mean no. of duplicated genes.
Abbreviation: PCG, protein coding genes.

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3.6 | Environmental analysis
In the PCA of the 20 environments, PC1 and PC2 explained 77.01%
of the total variation (Figure S7). Actaea purpurea and A. japonica + A.
biternata were divergent in PC1 but not in PC2 (Figure S7). The main
contribution of PC1 (58.62% variation) was from environmental var-
iables related to both precipitation and temperature, with BIO12,
BIO14, and BIO16 as the most important precipitation factors, and
BIO9, BIO6, and BIO11 as the main temperature variables (Table S4).
All these bioclimatic variables were significantly different between
A. purpurea and A. japonica + A. biternata (Table S5). PC2 (18.39% of
the variation) mainly captured the remaining temperature- related
environmental variables (BIO5, BIO8, and BIO10) and altitude
(Table S4). These variables were not significantly different between
the two groups, except BIO10 (Table S5).
4 | DISCUSSION
4.1 | Genome size, gene order and number were
highly conserved among A. purpurea and its relatives
Plastomes are normally conserved in genome size, gene order, and
number, especially in close- related species. The plastomes of the
three Actaea species all show a typical quadripartite structure, and
belong to t ype I of Zhai et al. (2019). Also consistent with the previ-
ous study, a total of 131 genes were annot ated, with 84 PCGs, 30
tRNA genes, 4 rRNA genes, and two pseudogenes. No inversions
were detected among species either. These results demonstrate
the conservation nature of plastomes between sister species. The
overall GC contents of these plastomes are similar to those of other
angiosperms (Palmer, 1991; Wolf et al., 2011), with the IR region pos-
sessing a higher GC content. This is because rRNA genes, which have
high GC content, are located in the IR region (Raman et al., 2017).
IR/SSC and IR/LSC boundary shifts between species are com-
mon in plet hora plant group s (e.g., Ye et al., 2018; Zhao et al., 2020).
Length variation of the IR region is responsible for these differ-
ences, which is very crucial in stabilizing the plastome structure
(Maréchal&Brisson,2010). However, in some Leguminosae spe-
cies such as Trifolium subterraneum L., the IR region was even com-
pletely lost (Cai et al., 20 08; Lavin et al., 1990 ; Palmer et al., 1987).
There were no boundary shifts within A. japonica and A. biternata,
consistent with their low sequence diversity, while in A. purpurea,
SSC-IRbbordershiftsof3–30 bpweredetected.Betweenspecies,
clear shif ts of boundaries were detected at both borders, indicat-
ing that the A. purpurea and its relatives are indeed genetically
divergent.
Gene loss or pseudogenization is common in plasto of seed
plants (Jansen & Ruhlman, 2012). For example, among the six genes
lost or pseudogenized in Ranunculaceae, rpl32 and rps16 were lost or
FIGURE 2 GenelocationsatregionboundariesinplastomesofActaea purpurea and its relatives.

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pseudogenization multiple times in gymnosperms and angiosperms;
accD and infA were also lost or pseudogenization in monocotyle-
don and eudicotyledon, while rps7 was lost in Passiflora L. (Jansen
& Ruhlman, 2012). Only the t rnT- UGU gene has been found to be
lost in Trib. Anemoneae of Ranunculaceae (Zhai et al., 2019). In our
study, the two pseudogenes, ycf1 and r ps19, were located in the IRb/
SSC and IRa/LSC boundary regions, respectively. The formation of
these two pseudogenes may be related to the change in the length
of the IR region.
As revealed by the previous study (Chang et al., 2020), genetic
diversit y (measured by pi in this study) of A. japonica plastomes was
much lower than that of A. purpurea (Figure 3), both in coding and
noncoding regions. In both species, IR s were more conserved and
exhibited lower genetic diversity than the LSC and SSC as previ-
ously reported (Ye et al., 2018; Zhao et al., 2020). The mechanism
accounted for the slower substitution rate of IRs may be copy cor-
rection between IRs and the purging of deleterious mutation by gene
conversion (Khakhlova & Bock, 2006). Predicted by the neutral the-
ory of molecular evolution, noncoding regions of plastomes would
have higher substitution rate. This is also true in our data set. The five
most divergent noncoding regions were rpl14- rpl16, ndhF- trnL (UAG),
trnH(GUG)- psbA, ccsA- ndhD, and psbT- psbN. Among these regions,
trnH- psbA has been a classic plastid marker in many phylogenetic
and phylogeographic studies (Shaw et al., 2014). E xcept that, the
remaining four regions were not listed as the most variable regions
across angiosperm lineages (Shaw et al., 2 014). This shows that there
may not a universally hyper variable region that can be used across
all angiosperm groups. Instead, which region accumulated more sub-
stitutions might be lineage specific. The newly identified noncoding
regions as well as SSR markers could be used to study phylogenetic
relationships and population genetics within the genus Actaea.
4.2 | Nonadaptive molecular evolution of plastome
during speciation
We found that during the divergence and speciation of A. purpurea
and A. japonica + A. biternata, the genetic variation of plastomes was
FIGURE 3 VisualizationofthealignmentofActaea plastomes by mVISTA. Actaea biternata was set as the reference. The gray arrows
above represent genes. Different colors represent different regions (coding and noncoding). Position in the genome is shown on the
horizontal axis at the bot tom of each block. Alignment similarity percentages are shown on the right side of the graph (the vertical axis). Two
black frames indicate the two IR regions.

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ZHANG et al.
indeed maintained by the mutation-drift balance. Most molecular
evolution tests failed to reject neutral evolution of the plastome in the
divergence of A. purpurea and its relatives. Functional genes related to
photosynthesis, translation, and other functions are all under strong
purif ying selecti on. This is in contra st to other recent st udies that iden-
tified a few positively selected genes, e.g., rbcL (Lee- Yaw et al., 2019;
Liu et al., 2012) and ndh genes (Zhao et al., 2020). Environmental anal-
ysis indicated that A. purpurea and A. japonica were significantly diver-
gent in most precipitation and temperature- related variables, but we
did not detect any adaptive signals in the plastomes of A. japonica +
A. biternata. It is likely that the adaptive divergence of A. purpurea and
A. japonica + A. biternata was mainly driven by the nuclear genome.
Thus, whether plastid genes contribute to the divergence of popula-
tions and species seems lineage- dependent. Nonadaptive evolution
of plastid genes in our system also indicate that cytoplasmic incom-
patibility may not be the main mechanism of reproductive isolation
between A. purpurea and its relatives.
In our data set, no fixed differences in transfer RNAs or ri-
bosomal DNAs between species were detected, which is not
unexpected giving these genes conserved function. The popula-
tion genetic- based and dN/dS- based methods yielded inconsistent
results. No positively selected genes were detected in the dN/dS-
based method, as in all cases, the alternative models were not sig-
nificantly better than the null models. Insufficient variation in our
sequencedataresultedin infiniteor undefinedNI valuesin MKTs.
TherewerefourgeneswithNI < 1inMKTs,butnoneofthemwere
significant. We note that the rbcLgenehadanNI < 1withamarginal
significant level (p = .08). Thus, it is possible that this gene may par-
ticipate in the adaptation to different conditions for photosynthesis
between A. purpurea and its relatives, considering their large distri-
bution range.
Actaea purpurea is distributed in the northern part of the
group's distribution, while A. japonica is in the south, and A. bit-
ernata only in Japan (Figure 6a). The distribution pattern is largely
parapatric: there appears to be a barrier corresponding to the
Sichuan Basin, theYangtzeRiver,andtheDabieMountains sepa-
rating the two groups, while both A. purpurea and A. japonica can
be found in Hubei and Anhui Provinces (Chang et al., 2020). If the
FIGURE 4 ComparisonofnucleotidepolymorphismsacrossActaea plastomes. (a) Coding regions; (b) noncoding regions, i.e., intergenic
regions and introns
TABLE 2 Nucleotidediversity(pi) across the 13 plastomes from Actaea purpurea and its relatives
LSC SSC IR LSC + SSC + IR
Noncoding
region
All species 0.00471 (0– 0.06527) 0.00871 (0– 0.03559) 0.00842 (0– 0.00496) 0.00438 (0– 0.06527)
Actaea japonica 0.00041 (0– 0.02667) 0.00095 (0– 0.01054) 0.00089 (0– 0.00 062) 0.00040 (0– 0.02667)
Actaea purpurea 0.00154 (0– 0.02667) 0.00249 (0– 0.01406) 0.00235 (0– 0.0 0332) 0.00142 (0– 0.02667)
Coding region All species 0.00081 (0– 0.00624) 0.00165 (0– 0.00403) 0.00001 (0– 0.00015) 0.00078 (0– 0.00624)
Actaea japonica 0.00002 (0– 0.00079) 0.00006 (0– 0.00034) 0.00000 0.00002 (0– 0.00079)
Actaea purpurea 0.00026 (0– 0.00301) 0.00041 (0– 0.00132) 0.00002 (0– 0.00033) 0.00024 (0– 0.00301)
All regions All species 0.00227 0.00361 0.00022 0.00211
Actaea japonica 0.00010 0.00026 0.00001 0.00010
Actaea purpurea 0.00069 0.00118 0.00014 0.00065

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FIGURE 5 Thenumberoftandem,
palindromic, dispersed repeats and SSR
in plastomes of Actaea purpurea and its
relatives. (a) The number of four types of
repeats; (b) the number of different SSR
types.
FIGURE 6 Sampledistributionand
divergence time of Actaea purpurea and
its relatives based on the plastome dat a.
(a) Sampling sites of the 13 populations;
(b) divergence time estimation. Blue bars
indicate the 95% highest posterior densit y
intervals. The number on each node
represents the posterior probability, and
only values less than 1 are shown. Scale
bar =5 mm.

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ZHANG et al.
lack of genetic variation in plastid markers of A. japonica + A. bit-
ernata (all with the same haplotype) is not caused by a historical
selective sweep, then a recent demographic expansion would be
the only explanation. This means that the current parapatric distri-
bution was formed by expansion of A. japonica + A. biternata from
a possible previously sympatric population. In other words, the ini-
tial phase of divergence bet ween A. purpurea and A. japonica + A .
biternata most probably occurred in sympatry. This is consistent
with the results of demographic modeling, showing that there
has been continuous gene flow after the divergence of the two
species (Chang et al., 2020). The expansion of A. japonica and A.
biternata populations might be very recent, as the time for drift
or selection to accumulate substitutions was limited. It probably
occurred in the Pleistocene, when the climatic oscillations often
drove changes in geographic distribution changes of many plant
species (Hewitt, 2000, 2004; Qiu et al., 2011).
AUTHOR CONTRIBUTIONS
Danqing Zhang: Data curation (lead); formal analysis (lead); investiga-
tion (lead); writing – original draft (equal). Yi Ren: Conceptualization
(supporting); super vision (equal). Jian- Qiang Zhang: Conceptualization
(lead); funding acquisition (lead); writing – original draft (equal).
ACKNOWLEDGEMENTS
We thank the associate editor Fang Du and the two anonymous
reviewers for their constructive suggestions. We thank Xiao- Peng
Chang, Yuan-Zhen Zha ng, Da-Lv Zho ng, Meng Han, and Chen-Yu
Niu for their help in sample collections. This work was supported by
the National Natural Science Foundation of China (Nos. 31870194,
32070236), and the Fundamental Research Funds for the Central
Universities (No. GK202103077 to J.Q. Zhang).
CONFLICT OF INTEREST
All authors claim no conflict of interest.
DATA AVAIL ABILI TY STATEMENT
DNA sequences: newly generated plastomes were deposited in the
GenBankdatabasewithaccessionnumbersOM460061–OM460073.
ORCID
Jian- Qiang Zhang https://orcid.org/0000-0003-2019-1185
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TABLE 3 Geneticvariationinselected
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of selection.

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How to cite this article: Zhang, D.-Q., Ren, Y., & Zhang, J.-Q.
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