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Assessing phylogenetic relationships among varieties of Toona ciliata in sympatry with chloroplast genomes

December 2023
13(12):10828

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Toona ciliata is an endangered species due to over-cutting and low natural regen-eration in China. Its genetic conservation is of an increasing concern. However, several varieties are recognized according to the leaf and flower traits, which complicates genetic conservation of T. ciliata. Here, we sequenced the whole chloro-plast genome sequences of three samples for each of four varieties (T. ciliata var. ciliata, T. ciliata var. yunnanensis, T. ciliata var. pubescens, and T. ciliata var. henryi) in sympatry and assessed their phylogenetic relationship at a fine spatial scale. The four varieties had genome sizes ranged from 159,546 to 159,617 bp and had small variations in genome structure. Phylogenomic analysis indicated that the four varieties were genetically well-mixed in branch groups. Genetic diversity from the whole chloroplast genome sequences of 12 samples was low among varieties (average π = 0.0003). Besides, we investigated genetic variation of 58 samples of the four varieties in sympatry using two markers (psaA and trnL-trnF) and showed that genetic differentiation was generally insignificant among varieties (Ф st = 0%-5%). Purifying selection occurred in all protein-coding genes except for the ycf2 gene that was under weak positive selection. Most amino acid sites in all protein-coding genes were under purifying selection except for a few sites that were under positive selection. The chloroplast genome-based phylogeny did not support the morphology-based classification. The overall results implicated that a conservation strategy based on the T. ciliata complex rather than on intraspecific taxon was more appropriate.

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Ecology and Evolution. 2023;13:e10828.  

1 of 17

https://doi.org/10.1002/ece3.10828

www.ecolevol.org

Received:27July2023

Revised:19Oc tober2 023

Accepted :28Novembe r2023

DOI:10.1002 /ece3.10828

RESEARCH ARTICLE

Assessing the phylogenetic relationship among varieties of

Toona ciliata (Meliaceae) in sympatry with chloroplast genomes

Xin- Sheng Hu1,2

Thisisanop enaccessarti cleundertheter msoftheCreativeCommonsAttributionL icense,whichpe rmitsuse,dis tribu tionandreprod uctioninanymed ium,

provide d the original wor k is properly cited.

©2023TheAuthors .Ecology and Evoluti onpublishedbyJo hnWiley&S onsLtd.

1CollegeofForestr yandL andsc ape

Architecture,SouthChinaAgricultural

University, Guangzhou, China

2GuangdongKeyLaboratoryfor

InnovativeDevelopmenta ndUtilization

ofForestPlantGermplasm,Guan gzhou ,

China

3Instit uteofHighlandForestScience,

ChineseAcade myofFores try,Kunming,

China

Correspondence

Xin-ShengHu,CollegeofFores tryand

Lands capeArchitecture,SouthChina

AgriculturalUniver sity,Guangzhou

510642, China.

Email:xinsheng@scau.edu.cn

Funding information

GuangdongBasicandApp liedBasic

ResearchFoundation,Grant/Award

Number :2023A1515012137;Nati onal

NaturalScienceFoundat ionofChina,

Grant /AwardNumber:32171819;South

ChinaAgriculturalUniversity,Grant/

AwardNumber:4 400-K16013

Abstract

Toona ciliata is an endangered species due to over- cutting and low natural regen-

eration in Ch ina. Its genetic cons ervation is of an inc reasing concern. However,

severalvarietiesarerecognizedaccordingtotheleafandflowertraits,whichcom-

plicates geneticconservation of T. ciliata.Here,wesequencedthe wholechloro-

plastgenome sequencesofthree samplesforeachoffourvarieties (T. ciliata var.

ciliata, T. ciliata var. yunnanensis, T. ciliata var. pubescens, and T. ciliata va r. henryi)

in sympatr y and assessed th eir phylogenetic relat ionship at a fine spatial sc ale.

Thefour varieties had genomesizesranged from159,546 to 159,617 bpandhad

small variations in genome structure. Phylogenomic analysis indicated that the

fourvarietiesweregeneticallywell-mixedinbranchgroups.Geneticdiversityfrom

thewholechloroplastgenomesequencesof 12sampleswas lowamong varieties

(average π = 0.0003). Besides, weinvestigatedgenetic variation of 58 samples of

thefourvarietiesinsympatryusingtwomarkers(psaAandtrnL- trnF)andshowed

thatgeneticdifferentiationwasgenerallyinsignificantamongvarieties(Фst = 0%–

5%).Purifying selectionoccurred in all protein-codinggenes except for the ycf2

genethatwasunderweakpositiveselection.Mostaminoacidsites inallprotein-

codinggeneswereunderpurifyingselectionexceptforafewsitesthatwereunder

positive selection.The chloroplast genome-basedphylogeny didnot support the

morphology-basedclassification.Theoverallresultsimplicatedthataconservation

strategybasedontheT. ciliatacomp lexrath ert han oni ntr asp e cif ict a xonwasmore

appropriate.

KEYWORDS

chloroplastgenome,geneticconser vation,purif yingselection,sympatricspeciation,Toona

ciliata

TAXONOMY CLASSIFICATION

Taxonomy

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XIAO et al.

1 | INTRODUC TION

Toona ciliata,aka Chinesemahogany (Edmonds& Staniforth,1998),

belongstotheToonagenusoftheMeliac eaefami lyandisanimpor t-

ant timbe r species bec ause of its high -qua lity wood for i ndustrial

purposes in China (Chen et al., 19 97). The species is naturally dis-

tribute d in India, Malays ia, Indonesi a, and other tro pical and sub-

tropicalregions.Itgrows in thelow-altitudegully forests orinthe

hillside sparse forests in central andsouthern China, ranging from

300to260 0 mabovesealevel.Thespecieshasbeenoverexploited

and was listed as an endangered species at Level IIin China (Fu &

Jin, 1992)although not listedas thespeciesat risk byInternational

Union for Co nservatio n of Nature and Natur al Resources (IUC N).

Populationdensit yinnaturalforestsdeclinesduetothelownatural

regeneration(Liangetal.,2011)andpotentialinbreedingdepression

(Zhouetal.,2020).Becauseofthesereasons,an impor tant issueis

concernedwithgenetic conservationof this endangered speciesin

China.Geneticbreedingprogramshave been setupbythestateto

improvewoodqualityandresistancetoinsect s.

Another issue relevant to genetic conservation is concerned

withintraspecificvariationofT. cil iat a.Cu rre ntl y,fiv ev ari e ti e sw ere 

recognizedaccordingtothe leaf(size,shape, andpubescence)and

flower (petallength andshape)traits(Chenetal.,19 97). They are

T. cilia ta va r. ciliata, T. cil iata var. yunnanensis, T. c il iata var. pubes-

cens, T. cili at a v ar. sublaxiflora, and T. c iliata v ar. henryi (Appendix

Table S1). These varieties are sympatry or partially overlapped

in natural geographic distribution in Yunnan Province in China

(Zhang,2018),andnaturalhybridizationamongthemcannotbeex-

cluded.However,morphologicaltraitsare oftencontinuously dis-

tributedorpo or fo rsub di vi si on ofintr a-an di nter specifi cv ar ia ti on s

(Ferreiraetal.,2021),which makes it difficult to delimit varieties

frommorphologicaltraits.Previousgeneticstudiesmainlyreferred

to the T. c iliata complex, without identifying specific varieties.

These include provenance trials(Lietal., 2017;Wenet al.,2012),

populationgeneticvariationassayedbysequence-relatedamplified

polymorphisms (SRAP) andsimple sequencerepeat (SSR) markers

(Liet al., 2015;Zhanetal.,2019),chloroplastDNA(cpDNA)mark-

ers (Hu, 2 019), a nd nuclear intern al transcribe d spacer (ITS) and

mitochondrialDNAmarkers(Xiaoetal.,2023).Fewstudiesinvesti-

gatedgeneticvariationinaspecificvarietyexceptforT. cili at a v ar.

pubescensisassayedbySSRmarkers(Liuetal.,2009).Allthesepre-

viousstudiesprovidedageneralpictureofgeneticvariationinthe

T. cilia tacomplex.

Classificationofvarietiesconsequentlycomplicatesgeneticcon-

servationofT. ci li ata (Andriamihajaetal.,2020; Ennos et al., 2005;

Panitsaetal.,2011).Thisisbecausegeneticconservationintermsof

single variety neglectsnatural hybrids amongvarieties in the sym-

patricregion and species interactions. Whether geneticconserva-

tion is based on the T. cilia ta complexor on singlevarietyremains

uncer tain. Ther efore, it is of pra ctical sign ificance to c larify the se

concerns for genetic conservation of this endangered species in

China(Federicietal.,2013).

In this study, we examined the phylogenetic relationship of

fourvarietiesofT. ci liataatafinespatialscalebasedonprevious

studies(Xiao et al., 2023;Zhang, 2018).Allthese varieties occur

inYunnanProvinceandaresympatricingeographicaldistribution

(Chen et al. , 1997; Zhang, 2018). The similarity and differences

among these varietiesprovide import antinformation to genetic

conservation.Italsoaidsinourunderstandingthemechanismsof

sympatricspeciationatthefinescale(Xiaoetal.,2022).Here,we

reportedthephylogeneticrelationshipsandthegeneticdifferen-

tiationamongsympatricvarietieswithboththewholechloroplast

genome sequence and markers, and explored the conservation

strateg y of this endangered species. Analysis with nuclear ge-

nomes wil l be present in a sep arate study usin g our recent as-

semblyofnucleargenomesequencesofT. cil ia taasthereference

genome (Wang, Xiao,He, Li, Lv,etal., 2022; Wang, Xiao, He, Li,

Song, etal., 2022). One reviewer suggested inclusion of nuclear

data anal yses, but it i s more approp riate to present t he nuclear

part in a separate study to accompany this worksince more ex-

tensiveanalysesareneededwiththeresequencingdataofthese

varieties.

Itiswell-knownthatchloroplastgenomehasmultipleattributes

forphylogeneticandpopulationstructureanalysis(Hu et al., 2019;

Ravi et al., 20 08), including (i) uniparental inheritance (maternal

inherit ance in most an giosperms but p aternal inher itance in most

gymnosperms), (ii) a relatively small genome size (120–220 kb)

(Palmer,1985),and(iii)a lowmutationratecompared with the mu-

tation r ate of nuclear genom es (Chmielewsk i et al., 2015; Provan

et al., 2001). The chlo roplast genom e sequences o r markers have

beenwidelya ppliedtoass essingbothp hylogenyandpopulationge-

neticvariation(Huetal.,2019).Althoughdiscordancewasreported

in phyloge nyi n terms of orga nelle genome s versus morp hologica l

traitsin the literature (Brown et al., 2010 ; Ebersbach et al., 2020),

thisremainsuncertainregardingthephylogeneticrelationshipofva-

rietiesofT. ci liata(Leliaertetal.,2009).

We addressed the following questions: (1) Could the whole

chloroplast genomesequences resolve the morphology-based de-

limitation of varieties of T. cili at a? (2) How genetic var iation was

distrib uted within and b etween symp atric varietie s of T. cil iata in

ter msofchlo roplastgenom es?Toans werth ef irstque st io n,wecon-

ducted phylogenetic analysesamong four sympatric varietiesusing

thewholechloroplastgenomesequencesandcomparedtheresults

withmorphologic delimitation.Toanswerthesecond question, we

conductedcomparativegenomicanalysesamongvarietiesusingthe

whole chloroplastgenome sequences andinvestigated geneticdif-

ferentiation withexpanded samples using two markers at the fine

scale. Toderiveamoregeneralconclusionassayedbymarkers,we

employed onemarkerincodingregion( psaA)andtheotherin non-

codingregion(trnL- trnF)region.Besides,weevaluatedthepotential

effec ts of natura l selecti on on shaping ch loroplast g enomic diver-

genceamongsympatricvarieties.Theoverallresultswerethensyn-

thesizedtoexploreanappropriatestrategyforgeneticconser vation

ofT. cilia ta .

20457758, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.10828 by South China Agricultural, Wiley Online Library on [13/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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XIAO et al .

2 | MATERIALS AND METHODS

2.1 | Taxonomic sampling and DNA extraction

InviewofapreviousstudyonvarietiesofT. ci liata(Zhang,2018), we

collectedleafsamplesofvarietiesindifferentlocationsinYunnan

Province of Ch ina (Figure 1a). These sampl es were taxonomic ally

identifiedaccordingtoboththefieldobservationsandliteraturere-

cords(Chen et al., 1997;Zhang, 2018).AppendixTable S1 provides

detail ed informatio n of the specim ens of these fou r varieties w ith

vouchernumbers.Wecollected12samplesofT. cili at a v ar. ciliata in

PuerandLijiangcities,13samplesofT. ci li ata v ar. henryiinPuercity,

20samplesofT. ci li ata v ar. pubescens,and13samplesofT. c il iata va r.

yunnanensisinLijiangandKunmingcities (Appendix Table S2).Note

that T. cil ia ta va r. sublaxiflorawasnotfoundinnaturalforestsand

hence was not included in this study. Figure 1bprovidesanimage

ofanadulttreeforeachvarietygrowinginYunnanProvince.Allcol-

lected samples were locatedatmorethan100meters awayamong

individualstoavoidgeneticrelatednessinnaturalforests.Forwhole

chloroplastgenomesequencing,werandomly selected three sam-

ples of eachvariety(Appendix Table S2). To investigate population

genetic di fferentia tion at the fin e spatial sca le, we analyze d all 58

samples with two markers. The sampling had been allowed by the

Chinese Government andcomplied with the laws of the People's

Republic of China.Thefresh sampleleaves were immediately dried

wi ths ili c age la n dth e nt r ans p orte dto lab o rat or yf orD N Ae x t rac t i on.

Totalgenomic DNA of eachsamplewas extracted using CTAB

method (Doyle & Doyle, 1987 ). The quality of extracted DNA

was checked by following analyse s: (i) use of 0.8% agarose elec-

trophoresis to detect DNA samples for degradation and impu-

rity, and to es timate the DN A concentrat ion; (ii) use of Nan odrop

Spectrophotometer(ThermoScientific)todetecttheconcentration

and purit y of sample s; and (iii) use of Q ubit 2.0 Fluo rometer (Life

Technologies)todetecttheconcentrationofsamples.

2.2 | Library construction and

high- throughput sequencing

Three individuals of each variety were sequenced by Science

Corpor ation of Gene (SCG ene), Guangzho u. Tested sa mples were

made according to the standardprocedure of IllumianDNA library,

and a doub le-end seque ncing libr ary with a n insert size of 35 0 bp

was constructed. After the construction of DNA library, qPCR

methodandAlignment2100Bioanalyzer(AlilentTechnologies)were

usedforqualitycontrol.TheDNAlibrarythatpassedthequalityin-

spect ion was sequenced by Illumina Nov aseq 6000 (Il lumination)

high-throughputsequencingplatform.Thesequencingstrateg ywas

pair-end150,andthesequencingdatawerenot<1Gb.

Illumina high-throughput sequencing results were originally

presentedas raw imagedata files, whichwere converted to raw

reads after base calling by the CASAVA software. Raw reads

from Illumina sequencing were subjected to adaptor trimming

andfilteringoflow-qualityreadsbyfastpv0.20.1(https://github.

c o m / O p e n G e n e / f a s t p ; Chen et al., 2018).Themin imumleng thfor

reads aftertrimmingwassetto150nucleotides, andthequalit y

thresholdwassettoQ20.Detailsforqualitycontrolweresumma-

rizedinAppendixTable S3.

2.3 | Chloroplast genome assembly and

annotations

With the c lean reads af ter trimm ing (App endix Table S3),geno me

sequences were de novo assembledusing SPAdesv.3.15.2 (h t tp://

c a b . s p b u . r u / p u b l i c a t i o n / n e w - f r o n t i e r s - o f - g e n o m e - a s s e m b l y - w i t h - 

spade s- 3- 0/ ),withk-mersizesof33,55,79,97,and127.Theassem-

bled conti gs include d a mixture of s equences fr om organel lar and

nuclear genomes. Weidentifiedchloroplastcontigs usingsimilarit y

searchesbyB L ASTN2.13.0 +ag ai ns tN CB In uc le ot id ec ol le ction(nt)

databas e. We had assessed t he assembly q uality of thes e chloro-

plast genomesby mappingrawpaired-end readsto the chloroplast

genome using bowtie2 (v2.3.5.1; Langmead &Salzberg, 2012) and

generate d sequencing dep th and coverage map acco rding to Ni's

protocol(Nietal.,2023).

Genes were annotated using Cp GAVAS (Liu et al., 2012) and

ORFFinder(h t t p s : / / w w w . n c b i . n l m . n i h . g o v / o r f f i n d e r / ).For the pre-

liminar yresultsofannotations,theaccuracyoftheresultswasveri-

fiedbycomparingtheencodedproteinsandrRNAwiththerepor ted

chloroplastgenome ofT. cil ia ta (GenBank access no.: NC_039592)

usingBlastn2.13.0 (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against

NCBI nucleotide collection (nt) database. All proteins had been

verifiedbyusing similaritysearches by Blastp2.13.0 against NCBI

nonredundantproteinsequences(nr) database(Zhangetal., 2000).

ARWEN (Laslett & Canbäck, 2008)was used toannotatetRNA . If

abnormal tRNA occurred, the verification would be carried out

again in combination with tRNAscan-SE 2.0 predictions (Lowe &

Chan, 2016).Thegenemapofchloroplastgenomewasdrawnusing

OGDRAW(Greineretal.,2019).Thefinalgenome sequencesofall

12sampleswere deposited in GenBankunder the accession num-

bersOM135324–OM135327 and OP373439–OP373446.

2.4 | Comparative genome analysis

among varieties

Alignments ofchloroplast genomesofthefour varieties were con-

ducted using ClustalW fromMEGAX(Kumar et al.,2018) with de-

fault parameters. Tandem repeat finder( TRF) (Benson,1999) and

microsatelliteidentificationtool(MISAv2.1)(Beieretal.,2017) were

used to sea rch for repet itive seque nces. To visualize the v ariation

amongthe12 samples,weanalyzedthehomolog yof their genome

sequences using mVIS TA(Frazer et al.,2004) wherethesequence

ofT. ci li ata(GenBank accessno.:NC_039592)was used as the ref-

erence. The nucleotide diversity per site (π) was estimate d using

DnaSPv5(Librado&Rozas,2009).

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2.5 | Phylogenetic analysis and divergent

time estimation

Toev aluate the phyl ogenetic rela tionship amo ng varieties , we se-

lected T. sinensis (GenBank access no.: MW4 01816) and Melia

azedarach(access no.: NC_050650) of the same family (Meliaceae)

as outgro ups. Tod erive a relia ble tree, we us ed the conc atenated

wholegenomesequencesthatwereorthologousamongsixtaxaand

alignedforphylogeneticanalysisusingclustalWalgorithminMEGAX

(Kumaretal.,2018).NotethatonlyoneIRregionwasincluded.The

FIGURE 1 SamplesitesandimagesoffourvarietiesofT. ci li atainYunnanProvince:(a)Samplingsitesof58individuals.Thefourvarieties

are T. c il iata var. ciliatainblue,T. ci li ata var. henryi in red, T. cil ia ta va r. yunnanensis in green, and T. cilia ta va r. pubescensinpurple.(b)The

bottomfourtreeimagesfromlefttorightareT. c iliata var. ciliata, T. cil ia ta var. henryi, T. ci liata va r. yunnanensis, and T. ci liata va r. pubescens,

respectively.

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general time reversible (GTR) modelwith a discrete Gammadistri-

bution (G TR + G) was detected as t he best-fit-mode l according to

Akaike informationcriterion (AIC) and Bayesianinformation crite-

rion (BIC ). Weco nstructed a m aximum likeliho od (ML) tree using

MEGAXwith1000BootstrapreplicationsandtheGTR + Gmodel.

BEAS T v1.10.4 (Suchard et al ., 2018) was used to reconstruct

phylogeneticrelationshipandestimatedivergenttimes,withaYule

process as the prior,the assumptionof an uncorrelated lognormal

relaxedclock,andtheGTR + Gmodel.Threecalibrationtimeswere

selectedforestimatingdivergenttimes.Thedivergenttimebetween

Toona and Meliawas approximately 68.3 Mya with a 95% HPD of

80.5–52.8Mya(Appelhansetal.,2012).Forcalibratingbranchages

containingallmembersoftheToona and Meliagenera,themeanage

ofthe mostrecentcommonancestor (MRCA)wasset at68.3 Mya,

withapriornormaldistributionandastandarddeviationof7.1Mya.

TheageoftheMRCAbetweenT. sinensis and M. azedarach was set

at48.0Mya,withastandarddeviationof0.3anda95%confidence

interval [47.8, 48.9] Mya from the TimeTree database (Grudinski

et al., 2014; Kum ar et al., 2017; Li, Yi, e t al., 2019). The divergent

time between T. cil ia ta and T. sinensis was set at 31.6 Mya, with a

standarddeviationof8.0anda95%confidenceinterval[15.1,46.5]

Mya(Muellneretal.,2 010).

The leng th of Markov chai n Monte Carlo ( MCMC) was set as

5.0 × 108 generations and data were sampled every 1000 gener-

ations. Ef fective sample size (ESS) was ch ecked in Tracer v1.7.2

(Suchard e t al., 2018) and accepte d for further analysis under a

largeESS(>200).WeusedTreeA nnotatorv1.10.4tobuildthema x-

imumcladecredibility(MCC)treewiththefirst20%ofsamplesas

theburn-in,andthetreederivedfromBayesian inference(BI)was

comparedwith the ML tree derived from MEGAX under GTR + G

model. T he phylogen etic trees w ere graphe d using iTOL (h tt p s : //

itol.embl.de).

2.6 | Detection of positive and purifying selection

Bothbranch-andsite-modelswithcodemlfromPAMLv4.9package

wereusedtodetectselectionintermsoftheratioofnonsynonymous

tosynonymoussubstitutions(ω = dN/dS)forallprotein-codinggenes

(Ya ng, 2007;Yang&Bielawski,2000).Thebranchmodelswithone-

ω versus two- ω ratiosweretested using likelihood ratio test(LRT):

2∆ℓ = 2log(L1- L0), where L1 is the log- likelihood under the alternative

hypothesis(i.e.,thetwo-ωratiosmodel)andL0 is the log- likelihood

underthenullhypothesis(i.e.,theone-ωmodel).The statistics2∆ℓ

followsachi-squareddistributionwiththedegreeoffreedombeing

equal to the dif ferentnumber of parameters between nulland al-

ternativehypotheses.Thebranchforeachofthe fourvarietieswas

separatelysetastheforegroundbranchandtheremainingbranches

as the background branches (Figure S1), with the aim at detecting

selection in each variety.

Thesitemodelwasseparatelyappliedtoanalyzingtheconcate-

natedsequencesofprotein-codinggenesinlargesinglecopy(LSC),

smallsinglecopy(SSC),inver tedrepeat (IR),andthewholegenome

regions withoneIRincluded.Twopairs of contrasting sitemodels,

M2a(selection) versusM1a(neutral),M8 (beta&ω > 1)versus M7

(beta), wereused todetect positiveselectionsites.Likelihoodratio

testwasusedtotestthesignificanceofalternativemodels.Posterior

probabilitiesateach amino acidsitewere estimatedfromthreesite

classes underM2a modelusingnaïveempiricalBayes(NEB)proce-

dure,andaprobabilityofnot≤0.95indicatedapositiveselectionsite

(Yang&Swanson,2002).

2.7 | Genetic differentiation among varieties

Toinvestigatepopulationdif ferentiationatthefinescale,wefound

single nucleotide polymorphisms (SNPs) from the alignment of

wholechloroplastgenomesequencesamongthefourvarietiesand

designeda pair ofprimerswithpolymorphisms in psaAregion. The

designedprimerswere5′- A T C G C C G T G T T G T A A C A G A G A - 3 ′ and 5′-

T G A T T C T T C C T G G G T C G A T G C - 3 ′.Fromtheliterature(Taberletetal.,

1991), we selec ted a polymor phic marker in t he intergenic re gion

trnL- trnF,and the designed primers were 5′-CGAA ATCGGTAGACG

C T A C G - 3 ′ and 5′- A T T T G A A C T G G T G A C A C G A G - 3 ′. The PCR was

performedin25 μLvolume,whichcontained1 μLplantDNA,12.5 μL

2 × Es Taq MasterMix (0.1 U Tap polymerase, 500 μmol/L dNTPs,

20 mmol/LTris–HCl,3 mmol/LMgCl2,100 mmol/LKCl),1 μLofeach

primer,and9.5 μLddH2O.ThePCRprotocolw assetbelow:preheat-

ingat95°Cfor4 min,35 cyclesat94°Cfor30 s,annealingat55°Cfor

1 min,andelongationat72°Cfor1 min,followedbyafinalextension

at72°Cfor10 min.ThePCRproductsweresubjectedtoagarosegel

electrophoresisat2%,andtheresultsweredetectedbygelimager.

The single amplified product with clear bandswas sentto Sangon

Biotech(Shanghai)Co.,Ltdforsequencing.

Themultiplesequencealignmentwasconductedwith ClustalW

algorith m from MEGAX with default pa rameter settings. The s e-

quences of 363 bpforpsaAand 969 bpfor trnL- trnFwere used for

downstr eam analyses , and the aligne d sequences we re submitted

to figshare (h t t p s : / / d o i . o r g / 1 0 . 6 0 8 4 / m 9 . f i g s h a r e . 2 4 2 0 3 7 6 3 ). To

exploregeneticvariationamongfourvarieties,analysisofmolecu-

larvariance(AMOVA) was doneseparately for sequences of psaA ,

trnL- trnF a nd their concat enated sequence s (psaA-trnL- trnF ) using

Arlequinv3.0(Excoffieretal.,2005).

Genetic differentiation among varieties was also analyzed by

comparingpopulation differentiation in termsofmarkersequence

divergence (Nst) versus in terms of allele frequency (Fst) (Pons &

Petit,1996).DnaSPv6(Rozasetal.,2 017)wasusedtoestimateNst

and Fst.Adif ferencebetweenFst and Nstwastestedusingpermuta-

tions(Rozasetal.,2017).Alltheseestimateswereobtainedusingthe

concatenatedsequencesofthesetwomarkers.

Isolationbydistance(IBD)atthefinescalewastestedusingthe

regression of Fst/(1−Fst) on the logarithm of geographic distance

(Rousset, 1997 ). Pairwise population differentiation (Fst) was cal-

culated by A rlequin v3. 0 (Excoffi er et al., 2005) using the conc at-

enated sequences. Geographical distance matrix among pairwise

varieti es was calculate d from central sam pling sites (T. c iliat a v ar.

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XIAO et al.

ciliata:26° 24′19″ N, 100°39′17″ E;T. ci lia ta va r. henryi:24°32′60″ N,

100°47 ′39″ E;T. ci liata va r. yunnanensis: 25°3 8′43″ N,102°38′54″ E;

T. cilia ta va r. pubescens:25°43′10″ N,102°39′34″ E).

3 | RESULTS

3.1 | Genome sequences of four varieties

Ahigh-qualityassemblyofchloroplastgenomewasobtainedforeach

variety.Detailsforgenome assembly information were providedin

AppendixTable S3.Forinstance,theQ30valuesrangedfrom89.8%

to93.4%andtheaveragesequencingdepthwas>180×. The cover-

ageratewas100%forall sequencing of12samples.Thecomplete

sequencesof 12sampleswerereposited toNCBI GenBank(access

numbers: OM135324–OM135327 and OP373 439–OP373446).

Theirgenomesizesrangedfrom159,546 bpto159,617 bpinlength.

Therewere334SNPsand96indelsamongthe12samples.Thege-

nomeexhibitedacircularmoleculewithtypicalquadripartitestruc-

ture of angi osperms (LSC , SSC, IRA , and IRB), and harb ored 103

genes in tot al, includ ing 79 protein-cod ing genes, 20 t RNA genes,

and4 rRNAgenes.Figure 2showsthecircularchloroplastgenome

mapofT. ci liata va r. ciliata, and the remaining threevarietieshada

similargenome structure except forsmalldifferencesinsizedueto

lowmutationrates.

Acomparisonindicatedthathighlyconservativechloroplastge-

nomesoccurredamongthefourvarieties( Table 1).LSCandSSCre-

gionsexhibitedrelativelyhigherdivergencethanIRregions.TheSSC

region had t he largest nucleotide diversity p er site (π = 0.00039),

followed by the LSC (π = 0.00 036) and IR regions (π = 0.00004).

Asimilarpatternof nucleotidediversitypersitewasalsoobserved

fortheprotein-codingsequencesinSSC(0.00037), LSC (0.00035),

andIR(0.00023)regions.Aglobalcomparisonofchloroplastgenome

homolog y across th e 12 sample s showed high se quence simil arity

and indic ated that the chloroplast genomes of the four varieties

werehighlyconserved(AppendixFigure S2).

Sequencerepeats,includingtandem,forward,palindromic,com-

plement,andreverserepeats,wereidentified.Figure 3 shows that

these repeats weregenerally conservative among the fourvariet-

ies. The re were 54–56 repetit ive sequence s in total where 11–12

forward repeat s and 19–20 palindromic repeats were separately

detected. Tandem repeat sequences occurred most frequently,

with22inthecpDNAsequencesofT. c iliata v ar. ciliata, T. c il iata var.

henryi, and T. c ilia ta var. yunnanensis,and23inthecpDNAsequence

ofT. c il iata var. pubescens.Fewcomplementaryandreverserepeats

wereobser ved(Figure 3a).

Therepetitivesequencesinthefourvarietieswerebetween30

and 58 bp (Figure 3b). Most repeatswere distributed innoncoding

regions(theintergenicregionsandintrons),andonlyafewincoding

genes. Forinstance, there were seven repeated sequencesin ycf2

gene,butonlyonerepeatinpsaB, psaA ,andndhFgenes(Appendix

Tables S4 and S5).

Table 2summarizesdifferenttypesofSSRsinthefourvariet-

ies. T. cili at a v ar. pubescenshad244SSRs,andthe remaining three

varietieshad243SSRs.Thepredominant typeofSSRs was mono-

nucleotide repeats, with A/T accounting for 74.07% of SSRs in

T. cilia ta va r. ciliata, T. ci li ata var. henryi and T. ci li ata var. yunnanensis,

and73.77% in T. cili at a v ar. pubescens(Appendix Figure S3). Other

typesofSSRs accounted forasmall proportion, with a decreasing

abundancefromdinucleotidestotetranucleotides,totrinucleotides,

and to pent anucleotid es. The chloro plast genome of T. ci lia ta va r.

pubescenshad oneun iqu ehe xan ucl e ot ide SSR tha tw asa bse ntint he

genomesoftheremainingthreevarieties.

A few coding genes, including rpoC2, rbcL, petA, ycf2, ndhF,

ndhD, ycf1, and ycf 2,containedmorethantwotypesofSSRsinT. ci l-

iata va r. ciliata, T. cil iata va r. henryi, and T. c ilia ta va r. yunnanensis. The

codingsequenceshad23.05%ofthetotalnumberof SSRs inthese

threevarieties,but23.36%inT. c iliat a v ar. pubescens.Inaddition,a

hexanucleotideSSRoccurredingenerpoAofT. cili ata var. pubescens.

3.2 | Phylogenetic relationships among varieties

Thetopologyofmaximumcladecredibility(MCC)treederivedfrom

Bayesian inference (BI) generally matched the ML tree using the

concatenatedsequences ofLSC,SSC, and one IR regions. Figure 4

shows that samples ofthe four varieties were not monophyleticin

MCC tree. A ppendix Figure S4 provides the ML tree constructed

byusing the whole-genome sequenceswith a single IR region. For

instan ce, the clade of T. c il iata var. yunnanensis (the firs t sample)

and T. cili at a v ar. pubescens(the twentiethsample)had a Bayesian

posterior probabilityof 100%. The remaining subclades had lower

Bayesia np oster io rp ro babilitie s, rangingfrom21%to52%(Figure 4).

TheMCCtree derivedfromBEASTanalysisprovided the point

estimatesofagesand 95%HPD(Figure 4).Thedivergenttimebe-

tween M. azedarach and genus Toonawasestimatedas48 .02–34.36

Mya.Thelargestdivergenttimeamongvarietieswasapproximately

24.47Mya(95%HPD:39–12Mya).Themostrecentdivergenttime

wasabout0.92Mya(95%HPD:0–6Mya).

3.3 | Test of purifying and positive selections

Natural selection was tested in 79 polymorphic protein-coding

genes. Likelihood ratio tests showed that the two-

𝜔

-ratios mod el

wasnotsignificantlydifferentfromtheone-

𝜔

-ratiomodel(Appendix

Table S6), indicating that the ωestimateswereessentiallyconsistent

amongthefourvarieties.

We removed 21 genes w hose dN or dS values were close to

0, which oth erwise y ielded over flow or zero e stimates of d N/d S

ratios.Thesegenesweremoreconservativeamongvarieties.The

remaining 58 protein-coding genes were used to detect natural

selectionbasedonthephylogenyofthefour varieties(Appendix

Table S6).

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Figure 5 shows that most protein-coding genes were under

strong purifying selection. Weak positive selection was present

only in ycf2gene(ω = 1.07).Pair wiseanalysiswithyn00fromPAML

package between eachofthe four varieties of T. ci liata with T. s in-

ensis or with M. azedarachalsoconfirmedpositive selectionin ycf2

gene(datanotshown here). GenesinIR regionhadlowerω values

(ω = 0.1540 ± 0.0 263), whereas gene s in LSC and SSC re gions had

relatively higher ωvalues (ω = 0.1889 ± 0 .1578 in LSC re gion, and

ω = 0.2282 ± 0.2243inSSCregion).

Random-site models were applied tomapping amino acid sites

under positive selection (Yang, 2006). The naï ve empirical Bayes

procedurewas used tocalculatethe posteriorprobabilities ofposi-

tiveselection(ω > 1)underdifferentmodelassumptions.Appendix

Table S7 shows the LRTs of positive selectionin LSC, SSC, andIR

regions.

In the LSC region (60 genes from rps12 to rpl22 in genome,

Figure 2), LRT between M2a and M1a models was

2Δl

= 7.2247

(AppendixTable S8), greater than

𝜒2

0.05,df

= 5.9914,indicatingthe

FIGURE 2 CircularmapofthedenovoassembledchloroplastgenomeofT. cil iata var. ciliatachloroplastgenome.Grayarrowsindicate

thedirec tionofgenetranscription.Thegenesinsideandoutsidethecirclearetranscribedinclock wiseandcounterclockwisedirections,

respectively.Genesindifferentfunctionalgroupsareshownindifferentcolors.Thedarkergraycolumnsintheinnercirclecorrespondto

GCcontent.Regionsofsmallsinglecopy(SSC),largesinglecopy(LSC),andinvertedrepeats(IRA,IRB)areindicated.

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XIAO et al.

presence of positive selection. Test with M8 (beta & ω > 1) versus

M7(bet a)alsoindicatedthepresence ofpositive selection insome

amino aci d sites (p-value = .0 063; Appendix Table S7). The rpoC2

gene had two amino acid sites with positive selection, 3212 V (va-

line) and 3772 Q ( glutamin e), while genes of cemA , rps2, rps8 and

rpl22 had only o ne amino acid si te with positi ve selecti on, 2314 S

TAB LE 1 PolymorphicsitesandnucleotidediversityamongfourvarietiesofToona ciliata.

Region

Coding and noncoding sequences Protein- coding sequences

Site Polymo rphic site πSite Polymo rphic site π

LSC 87, 23 4 10 8 0.00036 43,42 2 46 0.00035

SSC 18,423 25 0.00039 14, 271 17 0.00037

IR 27, 0 6 6 40.00004 10, 419 60.00023

LSC,IR,andSSC 132,723 137 0.00030 68 ,112 69 0.00 034

FIGURE 3 Comparisonofrepetitivesequencesamongfourvarieties:(a)Fivetypesofrepeats;(b)Frequenciesofdifferentsizesof

repeats(bp).

TAB LE 2 DistributionofSSRsofchloroplastgenomesinfourvarietiesofToona ciliata.

Tax on

T. ciliata var.

ciliata

T. ciliata var.

henryi

T. ciliata var.

yunnanensis

T. ciliata var.

pubescens

GenomeSize(bp) 159, 617 1 59,617 159,615 159, 55 0

SSRtype Mononucleotide 184 184 18 4 184

Dinucleotide 42 42 42 41

Trinucleotide 5 5 5 5

Tetranucleotide 11 11 11 11

Pentanucleotide 1 1 1 2

Hexanucleotide 0 0 0 1

Tot a l 243 24 3 243 24 4

Protein-codingsequence No. 56 56 56 57

%23.05 23.05 23.05 23.36

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XIAO et al .

(serine), 10, 893 H (histidin e), 13,713 L(l eucine) and 14,414 P (pro-

line),respectively(AppendixTable S7).

In the IR A region (7 genes from rps19 to ycf15 in genome,

Figure 2), LRTs with M2a versus M1a or M8 versus M7 showed the

presenceof positive selection (p- value ~10−9; Appendi x Table S8).

Therewasoneaminoacidsiteunderpositiveselectionintherpl23

gene,14,861 G(glycine),andfourintheycf2gene,including16,183 N

(asparagine),16,585R (arginine),16,589 T(threonine),and16,591 L

(AppendixTable S7).

In the SSC region (12 genes from ndhF to ycf1 in genome,

Figure 2), LRTs with M2a versus M1a or M8 versus M7 showed sig-

nificant differences (p- value ~10−9; Appendi x Table S8), indicating

thepresenceofpositiveselectioninsomeaminoacidsites.Twosites

under positive selection were detec ted only in ycf1gene, 21,30 8 G

and21,468 T( AppendixTable S7).

Comparedwiththeresultsin Appendix Table S7, LRTs with the

cate na te dse qu ences ofal l79p ro te in-co dinggen es exhib it edac om-

parablenumberofsitesunderpositiveselec tion(AppendixTable S9).

For instance, estimates with M2a were ω0 = 0.1138, ω

1 = 1.0000,

and ω2 = 13.3938,withpropor tions ofp0 = 0.9046,p1 = 0.0856,and

p2 = 0.0098. LRT withM2a versus M1a showed that 12 siteswere

under positiveselection (Appen dixTable S8). Appendi x Figure S5

showsthedistribution ofamino acid sitesunderpositive selection.

Most ofthese aminoacidsites were consistentwith the preceding

resultsderivedfromanalysesinindividualsegments(LSC ,SSC,and

IR;AppendixTable S7).Forinstance, comparedwiththe analysisof

the SSC regiononly,more positive selection sitesinycf1 were de-

tectedwiththeconc atenatedsequencesof79codinggenes,includ-

ing 21,22 5 T, 21308 G, 21468 T, 21505 E (g lutamic acid ), 21,529 K

(lysine), 21, 862 S, and 22,4 40 T.T he ndhF gene had only one s ite

underpositiveselection,18,391H.Thereweretwositesunderpos-

itive selection in the ycf2genein IRregion,16,183 N and16,589 T.

Both the rpoC2 and rps8genesintheLSCregionhadonlyonepos-

itive selection site,3772Qand13,713 L,respectively.Reasons for

suchdifferencescouldarisefromoffsettingef fect samongpositive

andnegativeaminoacidsiteswhendifferentlengthsofgenomeseg-

mentswereanalyzed.

3.4 | Genetic differentiation among varieties

Sequencesoftwomarkers(psaAandtrnL- trnF)wereanalyzedusing

AMOVA.Theresultsindicatedthatmostvariationwaswithinvarie-

ties: Фst = 0.0569,p-value = .0166for the psaA region; Фst = 0.0310,

p-value = .0675 for the trnL- trnF region; and Фst = 0.0351,

p-value = .0430fortheconcatenatedsequences(Table 3). Generally,

alowlevelandinsignificantgeneticdifferentiationoccurredamong

varieties.

The maximum likelihood tree indicated that taxon samples

were not gro uped into disti nct cluster s in terms of the ca tenated

sequencesofthesetwomarkers,whichwasdiscordantwithvariety

delimitationaccordingtomorphologicaltraits(Figure 6).

Genetic differentiation of pairwise varieties was analyzed

usingtheconcatenatedsequencesofpsaA and trnL- trnFsegments.

T. c ilia ta var. ciliatadi f fer e dfr o mT. c ilia ta v ar. henryi with Фst = 0.0534

(p-value = .0587 ), from T. cili at a v ar. yunnanensis with Фst = 0.0276

(.0841), and from T. c il iata var. pubescens with Фst = 0.0520(.0782).

T. cilia ta va r. henryi differed from T. ci liata va r. yunnanensis with

FIGURE 4 Phylogeneticrelationshipamongvarietiesderivedfromthewholechloroplastgenomeunderthemodelofrelaxedmolecular

clockanduncorrelatedrates.Theboldvalueateachnoderepresentsmeanage(Mya),andthevaluesinparenthesesarethe95%HPD

intervalsaroundpointageestimates.BranchlabelsrepresentBayesianposteriorprobabilities.

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Фst = −0.0114 (.6764), and from T. cilia ta va r. pubescens with

Фst = 0.0284(.1730).GeneticdifferentiationwasФst = 0.0501(.0596)

betweenT. ci liata va r. yunnanensis and T. ci liata va r. pubescens. All

pairwiseanalysesindicatedinsignificantgenetic differentiationbe-

tween varieties.

Withtheconcatenatedsequences( psaA-trnL- trnF),Nst(0.0348)

was not signi ficantly d ifferent fr om Fst(0.0354), indicating absent

phylogeographic structure among varieties. Isolation by distance

effects were not significant among varieties (Fst/(1-Fst) = 0.0353–

0.0074ln(distance), p-value = .2507).

4 | DISCUSSION

In this stu dy,we p rovided evi dence that fou r varietie s of T. ci li ata

were ver y closely related i n terms of the variati on of chloroplast

genomesequences.Thisalsoprovidedevidencethat aconflictbe-

tween organelle genome- and morphology-based delimitations in

sympat ric speciation , and implied dis tinct rates of lin eage sorting

processesbetweenorganelleandnucleargenomes(Huetal.,2019).

Althoughtherewassubstantialpopulationgeneticdifferentiationin

terms of the T. c il iata complexderivedfrom nuclear markers(Xiao

FIGURE 5 Estimatesofd N/dSof58protein-codinggenesinfourvarietiesofToona ciliata.

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et al., 2023),the phylogeneticrelationshipamong varietiesinsym-

patricregionwasunknownatafinescale.Fromthepatter nofmito-

chondrialgenomemarkers,Xiao etal.(2023) showed three distinct

regions in n atural dist ribution of t he T. ci liata comple x. This stud y

investigatedfoursympatricvarietiesofT. ci lia ta in the eastern region

(Zhang,2018). The results partly consolidated the previous study on

populationgeneticstructureoftheT. cil iata complexwherevarieties

werenotidentified( Xiaoetal.,2023).

4.1 | Genome divergence and evolutionary process

Thefourvarietieshadthetypicalstructureofangiospermorganelle

genomes , with LSC, S SC, IRA , and IRB part s. Their geno me sizes

were compa rable to those fou nd in T. ciliata, T. sinensis, T. sureni,

and other s pecies in the M eliaceae fa mily althou gh the estim ated

numbers of genes were unequal among them (Lin et al., 2018;

Mader et al., 2018; Tan et al., 2023; Xin et al., 2018).The four va-

rieties exhibited small variation in chloroplast genome size, gene

arrangement,andgenomicstructure.Thenucleotidediversityper

site (π) wasmuch smaller compared with those of plant species in

monophyly stage(Syring et al., 20 07), such as the per- site nucleo-

tidediversitybetweenspeciesinLagerstroemia and Michelia genera

(Dengetal.,2020; Xu et al., 2017).However,theobservedpattern

ofnucleotide diversitysupported the commonalitythat the muta-

tion rate s are general ly greater in L SC and SSC th an in IR regions

withrepetitiveproperties(Lietal.,2016;Perry&Wolfe,2002;Zhu

et al., 2016).Generally, only a fewmutations were accumulatedin

each region.

Therepetitivesequencesoftenexhibitlarge sequencevariation

among spe cies in the mono phyly stage ( Ahmed et al., 2012). The

fourvarietiesshowedthatnoncodingregionswerelessconservative

thanprotein-codingregions,consistentwithpreviousreports(Wen

et al., 2021).However,the samepat tern ofrepetitivesequencesin

three varieties except for T. c il iata var. pubescensforaslightdiffer-

enceimplicatedthattheywererecentlydivergent.

Besides the lowmutationrates,allprotein-coding genes except

forycf2wereunderpurifyingselection(ω < 1).Chloroplastgenomes

accumula ted deleterio us mutations d ue to small eff ective popu la-

tionsize(Kimura,1962)andalackofrecombination.Strongpurifying

selection removeddetrimental mutations in taxa. This alsoimplied

that the disruptive selection that fixed alternative alleles between

varieti es was effect ively impede d at the gene level. Th us, purify-

ing selec tion could p lay an impor tant role i n conserv ing seque nce

structure amongvarieties in sympatric region. Although there was

positive selectiononlyat a fewamino acid sites,whichcould likely

exhibitalternativegeneexpression,theirjointeffectsweresmall.In

addition,thepatternofaveragestreng thof purif yingselectionwas

consistentwiththepatternofmutationratesindifferentregions(IR,

LSC,andSSC).

4.2 | Evolutionary divergence among varieties

The phylogenomic analysis implied that the four varieties were

relativelyrecentlydivergent.Pointestimatesshowedawiderange

ofdivergenttimes(0.92–24.47 Mya)amongvarieties.Notethatthe

large inter val ofthe estimates of divergenttimes (Figure 4) could

arisefromtheuseofawiderangeofdivergenttimesderivedfrom

TimeTreedat abase for calibrations(Kumar et al., 2017). This was

the same situation for estimating the divergent times between

T. cilia ta and T. sinensis (Wang , Xiao, He, Li, Lv, Hu, et al. , 2022;

Wang, Xiao, He,Li, Song,etal., 2022). The fossil recordsarestill

lackingforthespeciesthatarecloselyrelatedtotheToona genus.

Nevertheless, our estimates of the divergent times between

M. azedarach and genus Toona(48.02 Mya) or between T. sinensis

and T. ci liata (34.36 My a) were consis tent with previous studies

(Appelhansetal.,2012; Grudinski et al., 2014; Kumaretal.,2017;

Li,Yi,etal.,2019).

Chloroplast genomemarkers are widely applied to analyzing

both phylogeny and population structure (Hu et al., 2019; Xie

et al., 2018). Markers in noncoding regions are commonly uti-

lized for inves tigating ge netic dif ferentiati on (Binks et al ., 2021;

Fang et al., 2010; Sh aw et al., 2007), but the re are also coding

regionsthatarepolymorphicanddesignedformarkers,suchasin

Saxifraga sinomontana(Lietal., 2018), Quercus liaotungensis(Yan g

TAB LE 3 Analysisofmolecularvariance(AMOVA)ofchlorotypesamongvarietiesofToona ciliata.

cpDNA segment Source of variation df Sum of square

Variance

component

Percentage of

variance (%) Фst p- Value

psaA Amongvarieties 35.592 0.0605 5.69 0.0569 .0166

Withinvarieties 54 54.0 97 1.0 018 94.31

Tot a l 57 59. 69 0 1.0623

trnL- trnF A mongvarieties 323.958 0.1754 3.10 0.0310 .0675

Withinvarieties 54 296.128 5.4839 96 .90

Tot a l 57 320.086 5.659 2

psaAandtrnL- trnF Amongvarieties 32 9. 55 0 0.2359 3. 51 0.0351 .0430

Withinvarieties 54 350.226 6.4857 96.49

Tot a l 57 379.776 6.7215

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FIGURE 6 Phylogeneticrelationshipderivedfromthemaximumlikelihood(ML)analysisamong58individualsoffourvarietiesofToona

ciliatausingthecatenatedsequencesofpsaAandtrnL- trnFmarkers.Branchlabelsrepresentbootstrapsupportingvaluesof>20%.

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et al., 2018), and Prunus armeniaca(Lietal.,2021).Here,we dis-

coveredapolymorphicmarkerinpsaAfromsequencealignments,

whichisinfrequentlyreportedinotherstudies.TherbcL and matK

sequencesareoftenusedasgeneticbarcodestoidentifyspecies.

In this st udy,p olymorphic sit es were absent in rbcL sequences

among varieties, but present in matK sequences only between

T. cilia ta va r. pubescens and T. ci li ata v ar. yunnanensis. The rbcL se-

quenceis often usedforidentifying deep phylogeneticrelation-

ships due to t he more conse rvative pro perty o f this gene. Both

rbcL and matKb ar co deswerenoteffect ivef orident if y in gt hefour

varieti es. Analysis of ge netic differ entiation supp orted the mix-

tureofgenomic composition amongthefourvarieties(Figure 6).

Weconcludedthatthemorphol ogicaldiversityamongfourvari et-

ieswasnotsupportedbythegeneticdivergenceintermsofchlo-

roplastgenomesequences.

Incongruencecannotbe ruledoutbetween morphological-and

organelle genome-based species delimitations. Sincethe morpho-

logical t raits (e.g., le af and flower trait s) are mainly contro lled by

nuclear ge nes, this incong ruence could mainl y arise from the cy-

tonuclear phylogeneticconflict. Cytonuclear incongruence among

closelyrelatedtaxaiswidelyrecordedintheliterature.Forinstance,

reports show mitochondrial-nuclear discordance in phylogeny of

animals,poplars, and other species (Funk & Omland, 20 03; Huang

et al., 2014; Sl oan et al., 2017). A lso, studies s howed chlorop last-

nuclear discordance in phylogeny of different plant species (Hu

et al., 2019; Le e-Yaw et al., 2019; Mata-Sucre e t al., 2023; Rivas-

Chamorroetal.,2023; Xie et al., 2023).Differentratesofevolution

and different modes of inheritance could contribute to this incon-

gruence.Theresults(Figures 4 and 6)impliedthatgeneticdriftpro-

cesscouldplayaminorroleingeneratingdiscordantchloroplastand

morphologyphylogenies.Thisisbecausecoalescent processbyge-

neticdriftisfastertoreachthereciprocalmonophylyforchloroplast

genomes(1/Ne,haploid) than fornuclear(1/2Ne, diploid) genomes

(Huetal.,2019),which was not the caseamong the fourvarieties.

Naturalhybridizationbetweenvarietiescouldoccurinsympatrybut

likely had sm all effect s on chloropl ast genome dive rgence among

varieti es. This is beca use only seed disp ersal contri butes to gene

flowofmaternallyinheritedgenes.Arecentstudyindicatedthatthe

ratio of po llen to seed fl ow was subst antial in the we stern regio n

in T. c iliat acomplex (Xiaoetal.,2023),whichimplies thateffectsof

seedflowweremuchsmallerthoseofpollenflowamongsympatric

varieties.Afurtherclarification with nucleargenomeswouldaidin

gaininginsightsintonaturalhybridization.Thiscouldalsohelptoex-

plaintheincongruencebetweenchloroplast genome-andmorpho-

logicaltrait-basedspeciesdelimitations.

Analternative explanation is thatdifferent models ofselec tion

likely participated in this conflict. The present results provided

strongevidencethatpurifyingselectionac tedonallprotein-coding

genesexceptfortheycf2gene,whichcouldeffectivelyimpedechlo-

roplastgenomedivergenceamongvarieties.Thislikelydifferedfrom

morpho logical trait s (e.g., the leaf and f lower traits) whe re adap-

tivelydivergentselectioncouldbeinvolvedinandyieldedextensive

morphological diversity. Consequently, this produced discordant

phylogeniesintermsofmorphologicaltraits versuschloroplast ge-

nomesequences.

Onlyone protein-codinggene, ycf2, was detected under weak

positive selection. The ycf2 gene is the largest plastid gene in an-

giosperms (Drescher et al., 2000)and had 6822 bp in the four va-

rieties. Previous studies indicatedthat the ycf2 gene had mu ltiple

positive selectionsites duringangiosperm evolution, and thisgene

was recomm ended for con struct ing angiospe rm phylogeny du e to

its long sequence and alowrate ofnucleotidesubstitution (Huang

et al., 2010; Li, Ma, et al., 2 019). The ycf2 genewas recently found

tobeessentialforcellviabilityandakeyenzymeforATPproduction

bychloroplastinthedarkorinnonphotosyntheticplastids(Drescher

et al., 2000; Kikuchi et al., 2018). Here, we fo und that ycf2 gene

hadtwoorfoursitesunderpositiveselectionfromthetestsonsin-

glegenesequenceoronthe concatenatedsequence,respectively.

Furtherinvestigationof thisgenecouldbeinterested in relationto

speciesdelimitation.

Althoughafewamino acidsites were under positiveselection,

they accounted for only a small proportion of genome (Yang &

Swanson, 2002). Most amino acid sites wereunder purifyingselec-

tion. Thus, compared with the drift process(Freeland et al.,2011;

Hudson & Coy ne, 2002; Palum bi et al., 2001), pu rifying se lection

couldbedominantinshapingchloroplastgenomephylogenyatboth

geneandaminoacidsitelevels,orinproducingtheconflictbetween

morphological-andcpDNA-basedphylogenies.

4.3 | Implications for genetic conservation

Previous s tudies accor ding to the max imum entropy (Ma xEnt) ap-

proach showed that T. ci li ata va r. ciliata would potentially expand as

climate changes in the future, while T. cili ata v ar. pubescens would

shrink inYunnanProvince (Zhangetal.,2018a, 2018 b).Thedemo-

graphicchangesinothertwovarietiesremainunknownalthoughthe

wholerangeofT. ci li atacomplexdidnotshowexpansionafterbot-

tleneckeffects(Xiaoetal.,2023).Astrategyofconser vingmultiple

populationswasrecommendedin the westernregionsfromthere-

sultsassayedbynuclearITSsequences.

This studyidentified four sympatricvarietiesofT. cil ia ta at the

finer scale andfurther investigatedgenetic differentiation in sym-

patry in the western region. Our results sup port the strategy of

conservinggeneticvariationbasedonthe T. cili at a complexrather

than on div idual variet y. This is bec ause a close genet ic relation-

ship exis ted among varieti es of T. cil iata. Be sides, natural hy brid-

ization am ong varieties could not be excluded in sympatr y (Xiao

et al., 2023). The t raditional singl e variety-based ap proach is not

effectivetoconservegeneticvariationofT. ci liat awherehybridsb e-

tweenvarietiesareoverlooked.Instead,theapproachthatmaintains

theevolutionarypotentialoftheT. cil ia tacomplexismoreeffective,

suchastheprocess-basedspeciesconser vationproposedbyEnnos

etal.(2012).

14 of 17

XIAO et al.

5 | CONCLUSION

Weinvestigatedthephylogenetic relationshipamong four varie-

ties of T. ci li ata(T. cilia ta va r. ciliata, T. cil iata var. yunnanensis, T.

ciliata v ar. pubescens, and T. c il iata var. henryi) insympatry using

thewholechloroplastgenomesequencesandtwogeneticmark-

ers. Comparative genomicanalysisshowedthat geneticdiversit y

among varietieswas ver y small(π = 0.0003). These phylogenetic

varieties weregenetically well-mixed, and their divergent times

wereabout0.92–24.47Mya.Analysiswithtwomarkersindicated

thatavery smalllevelofgenetic differentiation occurred among

varieties(

Φst

= 0%–5%).Chloroplastgenome-basedphylogenywas

discordantwiththemorphology-basedspeciesdelimitationatthe

finespatialscale(sympatricspeciation).Strongpurifyingselection

wasdetectedacross all protein-codinggenes exceptfortheycf2

genethatwasunderweakpositiveselection.Asimilarpat tern of

purifyingselectionwasobservedacrossgenome-wideaminoacid

sites.Purifyingselection couldplayanimportant roleinimped-

ingchloroplast genome divergence among varieties. From these

results,aconservationstrategyfocusingontheT. c il iatacomplex

ratherthanindividualvarietyisrecommendedforthisendangered

species in China.

AUTHOR CONTRIBUTIONS

Xin- Sheng Hu:Conceptualization (lead); funding acquisition(lead);

projectadministration(lead);writing–reviewandediting(lead).Yu

Xiao: Conceptualization (supporting); data curation (lead); formal

analysis (lead); investigation (lead); methodology (lead); writing –

original draft(lead).Xi Wang:Data curation(supporting);investiga-

tion(supporting);methodology(supporting);resources(supporting).

Zi- Han He: Data curation (supporting); investigation (supporting);

resources(supporting).Yan- Wen Lv:Datacuration(supporting);in-

vestigation(supporting); resources(supporting).Chun- Hua Zhang:

Data curation (supporting); investigation (suppor ting); resources

(supporting).

ACKNOWLEDGMENTS

We appreciate the associate editor and two anonymous review-

ers for con struct ive comments o n this arti cle, and Jiehu Chen f or

help. This r esearch was fun ded by Guangdong B asic and Applie d

Basic Rese arch Foundati on (2023A1515012137), National Nat ural

Science FoundationofChina(32171819),and the fundingfromthe

SouthChinaAgriculturalUniversity(4400 -K16013).

DATA AVAIL AB I LI T Y STATE MEN T

Thecompletesequencesofchloroplastgenomesoffourvarietiesof

T. cilia ta were deposited inNCBI database. Toona ciliata var. ciliata

(GenBankaccessnumbers:OM135324, OP373439, and OP373 44 0),

T. cilia ta va r. henryi(OM135325 , O P373 4 41, and OP373 442), T. cil-

iata va r. yunnanensis(OM135326, OP373 445, and OP373 446), and

T. cilia ta va r. pubescens(OM135327, OP373443, and OP373444).

The psaAandtrnL- trnFalignmentsequencesweresubmittedtofig-

share(h t t p s : / / d o i . o r g / 1 0 . 6 0 8 4 / m 9 . f i g s h a r e . 2 4 2 0 3 7 6 3 ).

ORCID

Yu Xiao https://orcid.org/0000-0001-9759-5685

Xi Wang https://orcid.org/0000-0002-8780-1331

Zi- Han He https://orcid.org/0009-0003-8076-1749

Yan- Wen Lv https://orcid.org/0009-0002-3956-1713

Chun- Hua Zhang https://orcid.org/0000-0002-2735-623X

Xin- Sheng Hu https://orcid.org/0000-0002-8049-9491

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How to cite this article: Xiao,Y.,Wang,X.,He,Z.-H.,Lv,

Y.-W.,Zhang,C.-H.,&Hu,X.-S.(2023).Assessingthe

phylogeneticrelationshipamongvarietiesofToona ciliata

(Meliaceae)insympatrywithchloroplastgenomes.Ecology

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Feb 2024

Toona ciliata is a deciduous or semi-deciduous tree species and belongs to the Toona genus of the Meliaceae family. Owing to low natural regeneration and over-exploitation, the species is listed as an endangered species at level II in China and its conservation has received increasing concern. Here, we sampled 447 individuals from 29 populations across the range-wide distribution of the T. ciliata complex in China and assessed their genetic variation using two chloroplast DNA markers. The results showed that the overall haplotype diversity and nucleotide diversity per site were high at h = 0.9767 and π = 0.0303 for the psbA-trnH fragment and h= 0.8999 and π = 0.0189 for the trnL-trnL fragment. Phylogenetic analysis supported the division of the natural distribution of T. ciliata complex into western and eastern regions. The genetic diversity was higher in the western region than in the eastern region, showing significant phylogeographic structure. Genetic differentiation among populations was moderate (Φst=42.87%), and the effects of isolation by distance (IBD) were significant. A neutrality test and mismatch distribution analysis indicated that the distribution of the T. ciliata complex generally did not expand, although a few local populations could likely expand after bottleneck effects. The overall results were complementary to and consolidated previous studies using mitochondrial and nuclear DNA markers. We finally discussed strategies for the genetic conservation of the T. ciliata complex.

A Community-Based Framework Integrates Interspecific Interactions into Forest Genetic Conservation

Article

Full-text available

Feb 2024

Forest genetic conservation is typically species-specific and does not integrate interspecific interaction and community structure. It mainly focuses on the theories of population and quantitative genetics. This approach depicts the intraspecific patterns of population genetic structure derived from genetic markers and the genetic differentiation of adaptive quantitative traits in provenance trials. However, it neglects possible interspecific interaction in natural forests and overlooks natural hybridization or subspeciation. We propose that the genetic diversity of a given species in a forest community is shaped by both intraspecific population and interspecific community evolutionary processes, and expand the traditional forest genetic conservation concept under the community ecology framework. We show that a community-specific phylogeny derived from molecular markers would allow us to explore the genetic mechanisms of a tree species interacting with other resident species. It would also facilitate the exploration of a species' ecological role in forest community assembly and the taxonomic relationship of the species with other species specific to its resident forest community. Phylogenetic β-diversity would assess the similarities and differences of a tree species across communities regarding ecological function, the strength of selection pressure, and the nature and extent of its interaction with other species. Our forest genetic conservation proposal that integrates intraspecific population and interspecific community genetic variations is suitable for conserving a taxonomic species complex and maintaining its evolutionary potential in natural forests. This provides complementary information to conventional population and quantitative genetics-based conservation strategies.

Generating Sequencing Depth and Coverage Map for Organelle Genomes v1

Preprint

Full-text available

Apr 2023

Yang Ni

The success in development of molecular markers, phylogenetic analysis, and genetic engineering relies heavily on high quality organelle genomes. Any errors in the assembly of these genomes can result in inaccurate downstream analysis. In this article, we present a complete protocol for assessing the assembly quality of organelle genomes using sequencing depth and coverage plot. The protocol consists of nine steps that can be divided into three sections, allowing users to map sequence reads to the assembly, calculate sequence depth and coverage, and plot the data using a custom script, respectively. We provide detailed instructions for setting up the computational environment and running the analytical software tools. This protocol is particularly suitable for users with little bioinformatic experience and will play a vital role in ensuring the high assembly quality of organelle genomes.

Phylogeography of Toona ciliata (Meliaceae) Complex in China Inferred from Cytonuclear Markers

Article

Full-text available

Dec 2022

Toona ciliata is an important timber species but is recognized as an endangered species at level II in China. Its genetic conservation is of increasing concern. Provenance trials and other breeding programs were conducted to develop seed transfer rules and multiplications. Here, we investigated twenty-nine populations sampled across the natural distribution of the T. ciliata complex using mtDNA and nrDNA ITS (ribosomal internal transcribed spacer) markers. Haplotype diversity was h = 0.190 ± 0.202 and nucleotide diversity was π = 0.000383 ± 0.000536 for mtDNA marker. Nucleotide diversity for ITS sequences was 0.00837 ± 0.000783. Haplotypes exhibited phylogeographic structure in spatial distribution. The extent of genetic differentiation was significant (Fst = 0.6994 ± 0.0079 for ITS and 0.8870 ± 0.0077 for mtDNA marker). Isolation by distance (IBD) and by elevation (IBE) occurred among populations. Phylogenetic relationships from mtDNA marker indicated three genetically distinct regions, each without IBD effects. Compared with pollen flow, seed flow was strongly impeded in the western region, but extensive in the central region, and less impeded in the eastern region. Most populations did not exhibit expansion, with only a few populations showing expansion after bottleneck effects. We discussed a strategy of region-based genetic conservation and proposed to conserve multiple populations in the western and eastern regions and a few populations in the central region.

Phylogenetic Relationships of Tilia (Malvaceae) Inferred from Multiple Nuclear Loci and Plastid Genomes

Article

Full-text available

Nov 2022

Evolutionary Divergence between Toona ciliata and Toona sinensis Assayed with Their Whole Genome Sequences

Article

Full-text available

Oct 2022

Toona ciliata and Toona sinensis belong to the Toona genus of the Meliaceae family and are important timber species in China. T. ciliata is an endangered species at level II due to overcutting and a low rate of natural regeneration. T. sinensis was cultivated as an economic and nutritious tree for more than 2000 years. The two species differ in flower and leaf morphological traits, reproductive systems, and range size of natural distribution. To reveal the potential molecular basis of these divergences, we examined the similarities and differences in their whole genome sequences. Results indicate that T. ciliata had a higher number of expanded gene families than T. sinensis. The whole genome duplication (WGD) occurred before their speciation. The long-terminal repeats (LTRs) insertion was earlier in the T. ciliata genome (3.2985 ± 2.5007 Mya) than in the T. sinensis genome (3.1516 ± 2.2097 Mya). Twenty-five gene families in the T. ciliata genome were detected to be under positive selection compared with background branches of ten different land species. The T. ciliata genome was highly collinear with the T. sinensis genome, but had low collinearity with the genomes of more distant species. These genomic and evolutionary divergences are potentially associated with the differences between T. ciliata and T. sinensis in terms of their reproductive systems and ecological adaptation.

A Chromosome-Level Genome Assembly of Toona ciliata (Meliaceae)

Article

Full-text available

Jul 2022

Toona ciliata Roem is an important timber species in the Toona genus of the Meliaceae family and an endangered species due to over-cutting and a low rate of natural regeneration in China. Although molecular markers have been applied to studying population genetic diversity, the absence of a reliable reference genome limits in-depth genetic conservation and evolutionary studies of this species. Here, we reported a high-quality assembly of the whole genome sequence of T. ciliata. The total assembled genome has 520.64Mb in length anchored on 28 chromosomes (contig N50 = 4.48Mb). A total of 42,159 genes were predicted after the ab initio, homology-based and transcriptome analyses. A total of 41,284 protein-encoding genes (97.92%) were functionally annotated and 1,246 non-coding RNAs were identified in the T. ciliata genome. Phylogenomic analysis showed that T. ciliata was divergent at 15.06 (6∼25) Mya from T. sinensis of the same genus Toona. This whole genome sequence provides a valuable resource to study genetic conservation and molecular evolution of T. ciliata in the future.

Phylogeography of Prunus armeniaca L. revealed by chloroplast DNA and nuclear ribosomal sequences

Article

Full-text available

Jul 2021

To clarify the phytogeography of Prunus armeniaca L., two chloroplast DNA fragments (trnL-trnF and ycf1) and the nuclear ribosomal DNA internal transcribed spacer (ITS) were employed to assess genetic variation across 12 P. armeniaca populations. The results of cpDNA and ITS sequence data analysis showed a high the level of genetic diversity (cpDNA: HT = 0.499; ITS: HT = 0.876) and a low level of genetic differentiation (cpDNA: FST = 0.1628; ITS: FST = 0.0297) in P. armeniaca. Analysis of molecular variance (AMOVA) revealed that most of the genetic variation in P. armeniaca occurred among individuals within populations. The value of interpopulation differentiation (NST) was significantly higher than the number of substitution types (GST), indicating genealogical structure in P. armeniaca. P. armeniaca shared genotypes with related species and may be associated with them through continuous and extensive gene flow. The haplotypes/genotypes of cultivated apricot populations in Xinjiang, North China, and foreign apricot populations were mixed with large numbers of haplotypes/genotypes of wild apricot populations from the Ili River Valley. The wild apricot populations in the Ili River Valley contained the ancestral haplotypes/genotypes with the highest genetic diversity and were located in an area considered a potential glacial refugium for P. armeniaca. Since population expansion occurred 16.53 kyr ago, the area has provided a suitable climate for the population and protected the genetic diversity of P. armeniaca.

Genomic divergence in sympatry indicates strong reproductive barriers and cryptic species within Eucalyptus salubris

Article

Full-text available

Mar 2021

Genetic studies are increasingly detecting cryptic taxa that likely represent a significant component of global biodiversity. However, cryptic taxa are often criticized because they are typically detected serendipitously and may not receive the follow‐up study required to verify their geographic or evolutionary limits. Here, we follow‐up a study of Eucalyptus salubris that unexpectedly detected two divergent lineages but was not sampled sufficiently to make clear interpretations. We undertook comprehensive sampling for an independent genomic analysis (3,605 SNPs) to investigate whether the two purported lineages remain discrete genetic entities or if they intergrade throughout the species’ range. We also assessed morphological and ecological traits, and sequenced chloroplast DNA. SNP results showed strong genome‐wide divergence (FST = 0.252) between two discrete lineages: one dominated the north and one the southern regions of the species’ range. Within lineages, gene flow was high, with low differentiation (mean FST = 0.056) spanning hundreds of kilometers. In the central region, the lineages were interspersed but maintained their genomic distinctiveness: an indirect demonstration of reproductive isolation. Populations of the southern lineage exhibited significantly lower specific leaf area and occurred on soils with lower phosphorus relative to the northern lineage. Finally, two major chloroplast haplotypes were associated with each lineage but were shared between lineages in the central distribution. Together, these results suggest that these lineages have non‐contemporary origins and that ecotypic adaptive processes strengthened their divergence more recently. We conclude that these lineages warrant taxonomic recognition as separate species and provide fascinating insight into eucalypt speciation.

Repeat-based phylogenomics shed light on unclear relationships in the monocentric genus Juncus L. (Juncaceae)

Article

Sep 2023
MOL PHYLOGENET EVOL

Interspecific variation and phylogenetic relationship between mangrove and non-mangrove species of a same family (Meliaceae)-insights from comparative analysis of complete chloroplast genome

Article

Jun 2023

The mahogany family, Meliaceae, contains 58 genera with only one mangrove genus: Xylocarpus. Two of the three species of the genus Xylocarpus are true mangroves (X. granatum and X. moluccensis), and one is a non-mangrove (X. rumphii). In order to resolve the phylogenetic relationship between the mangrove and non-mangrove species, we sequenced chloroplast genomes of these Xylocarpus species along with two non-mangrove species of the Meliaceae family (Carapa guianensis and Swietenia macrophylla) and compared the genome features and variations across the five species. The five Meliaceae species shared 130 genes (85 protein-coding genes, 37 tRNA, and eight rRNA) with identical direction and order, with a few variations in genes and intergenic spacers. The repetitive sequences identified in the rpl22 gene region only occurred in Xylocarpus, while the repetitive sequences in accD were found in X. moluccensis and X. rumphii. The TrnH-GUG and rpl32 gene regions and four non-coding gene regions showed high variabilities between X. granatum and the two non-mangrove species (S. macrophylla and C. guianensis). In addition, among the Xylocarpus species, only two genes (accD and clpP) showed positive selection. Carapa guianensis and S. macrophylla owned unique RNA editing sites. The above genes played an important role in acclimation to different stress factors like heat, low temperature, high UV light, and high salinity. Phylogenetic analysis with 22 species in the order Sapindales supported previous studies, which revealed that the non-mangrove species X. rumphii is closer to X. moluccensis than X. granatum. Overall, our results provided important insights into the variation of genetic structure and adaptation mechanism at interspecific (three Xylocarpus species) and intergeneric (mangrove and non-mangrove genera) levels.

Testing species relationships and delimitation in the Amazonian hyperdominant Astrocaryum section Huicungo (Arecaceae) using chloroplast data from genome skimming

Article

Mar 2023

Hyperdominant trees in Amazonia account for half of the individual trees (>10 cm dbh) in the forest, and thus play a crucial role in ecosystem dynamics. However, several of these widespread hyperdominant species may be complexes hiding cryptic diversity that can affect species richness estimates and conservation priorities. Here, we study the intraspecific variation of Astrocaryum murumuru (Arecaceae), a keystone and hyperdominant species in Amazonia, also known as Astrocaryum sect. Huicungo, a complex of 15 understory to subcanopy palm species. Using chloroplast DNA from genome skimming (>66 kbp alignment) in a Bayesian framework, we present evidence that A. sect. Huicungo represents three separately evolving lineages, suggesting that the section is not a single hyperdominant species, and that the 15 morphology-based species may be an over-representation. Genome skimming chloroplast data did not fully resolve the species-level phylogenetic relationships in A. sect. Huicungo mostly because of gene discordance and the paraphyly of most species. Contrary to a previous nuclear-based phylogenetic analysis, the chloroplast genomic data did not recover A. sect. Huicungo monophyletic, but yielded monophyly in an increased number of species (six) in the complex. Interspecific phylogenetic relationships showed a geographic pattern, and the traditional morphology-based classification was not supported. Our phylogenomic results are discussed in light of earlier phylogeographical studies using Sanger sequencing. Our findings show the utility of genome skimming data in species delimitation analyses to uncover intraspecific variation of hyperdominant species in Amazonia, the largest evergreen tropical forest.

Assessing phylogenetic relationships among varieties of Toona ciliata in sympatry with chloroplast genomes

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