Population Genomic Analysis Reveals That Homoploid Hybrid Speciation Can Speciation Hybrid Homoploid Reveals That Analysis Genomic Population E Origin

Home , Ghost lineage, Picea purpurea, Picea wilsonii

This article isThis article by protected copyright. All rights reserved. 10.1111/mec.14909doi: lea copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot coalescent with combined analysis to referred purpurea P. wilsonii of wilsonii that and Picea w through hybrid study homoploid by originated have to thought speciation are species of number increasing An Abstract *equal 3 Mengla Sciences, 666303,P. R. China 2 ChengduUniversity, 610065,P. 1 : (Orcid 0000 ID LIU JIANQUAN PROF. 0000 ID: (Orcid SUN YONGSHUAI PROF. School of Biology,of School CASK Bio LaboratoryKey for Population genomic analysis reveals that homoploid hybrid speciation can speciation hybrid homoploid reveals that analysis genomic Population e origin

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This article isThis article by protected copyright. All rights reserved. putative Recording 2010; of species hybrid crucial example, For trees. as such times, generation al. when formation ofsuchspecies generations species hybrid homoploid of stabilization genome (Lamichhaney Islands Galapagos wild the (McCarthy of( synthesis artificial on based generations, few a within parents. its from hybrid the of isolation the Rieseberg animalsand plants for reported been have 2011; Orr species speciation hybrid Homoploid Introduction genomics,population Qinghai words Key time. these that show species hybrid homoploid a of origin tbis 15; Grant 1957; Stebbins,

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was the cause of reproductive of cause the was how n h wild the in of homoploid hybrid species species hybrid homoploid of t al et long period of evolutionary evolutionary of period long is missing ( missing is et al et

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This article isThis article by protected copyright. All rights reserved. indicated findings mt al. et cpDNA from approximately between hybridization diploid through originated utilising of that overlaps partially distribution its where purpurea ne in scattered populations few a with QTP northeastern QTP southeastern the in ecological different have to purpurea recently More and of possession joint some Qinghai the to endemic is species purpurea species occurrences contact secondary oc on and drift could remains be not generations is species hybrid the time of period what u ln atr h ita hbi lineage hybrid intial the after long cur Accepted- Article

causes of reproductive isolation reproductive of causes In this study, we aimed to aimed we study, this In the development of development the n cpDN and , 2014 ,

P. wilsonii P. spruce forests. forests. spruce possibly affected

have yet tobe

(paternally inherited through pollen) through inherited (paternally leaky 11 ,

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Lamichhaney Surprising phylogenetic analys phylogenetic

subsequent after flat or nearly flat leaves, flat nearly or flat rapid that

phylogenetic and phylogenetic - Initially, i Initially, remains stage hybridization in the evolution of a putative putative a of evolution the in hybridization stage progeny reported its its

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a . purpurea P.

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et al et

examine

complete owever, both owever, eid of period - , 2 , Ti . likiangensis P. genome coming coming genome ) ., 2018) ., of a hybrid lineage from its parents its from lineage hybrid a of

P. likiangensis P. considered , bet Plateau (QTP) Plateau bet occurs occurs 010; Sun 010; i though in areas of sympatry of areas in though

P. wilsonii P. s of chloroplast (cp) DNA variation variation DNA (cp) chloroplast of s coalescent analyses of analyses coalescent because is a diploid hybrid species species hybrid diploid a is

the diploid hybrid origin of origin hybrid diploid the

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et al. et it targets such species for further analysis analysis further for species such targets it closely related to related closely

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mtDNA genomic composition genomic . wilsoni P. (Ran

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at occurs at occurs omic composition omic , 2014) , from . wilsonii P.

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et al. et where it is it where

ighbouring areas ighbouring

(maternally inherited (maternally olation . likiangensis P. for example during during example for

i . wilsoni P. , 2006) , be A p A . later

high elevations high of leaves leaves of population genomic data. This data. genomic population derived from derived

P. likiangensis P. .

indicated

opulation genetic genetic opulation and established a ahr surprisingly, Rather . P. purpurea P.

dominant differ All three All if reproductive isolation reproductive if a of the action the

of such a lineage a such of . spruce ( . likiangensis P.

(Cheng & Fu, 1978) Fu, & (Cheng

of n otesen QTP northeastern in 1400 If the formation of formation the If . wilsonii P.

n te remainder the and of homoploid the for that indicated that that indicated

P. wilsonii P. - north China north

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This article isThis article by protected copyright. All rights reserved. therefore legacy, a be might all excluded between overlap species stands forest separated spatially from allopatrically of individuals Fr Material and methods Material for easy is relatively analysis transcriptome ( sequences repetitive many conducted was genomes than rather likely most scenarios conducted individuals of transcriptomes divergence bifurcating after admixture help can hypothesis. al. Du I from (introgression) divergence that is scenario alternative popul the contribution occurr likiangensis ntrogression s, mature esh, Accepted Article Ru 2015; ,

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distinguish between distinguish resulting in . wilsonii P. to gntc analysis genetic ation and

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However, Petit & Excoffier, 2009; Excoffier, & Petit 2013 edn suppo lending very large and contains and large very respectively, . wilsonii P.

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This article isThis article by protected copyright. All rights reserved. searches. them identified ran 2.0.1 ver. TransDecoder the by predicted were frame read open the in 2010) non of (Grabherr was quality < 20 trimming (Bolger Trimmomatic used We Read trimming individuals in Hiseq al. Illumina® prepared were individual each for USA). CA, Clara, Santa Inc., Technologies (Agilent Bioanalyzer 2100 DNA TURBO the using treatment DNase a by followed USA) MA, Waltham, Scientific, Fisher extraction. species above, Germany) (German, GIS eTrex an using recorded were

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calculate To investigate To ru nme ( number group

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Accepted ArticleT - he 3 iesoa joint dimensional ing to inferinterspecificrelationships . Maximum withparametric likelihood bootstrap 14 lineage and

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mapped quality mapped After using

to form as

- simulated and observed 2D observed and simulated fold, with average mapping rate of 60.90% 60.90% of rate mapping average with fold, otstrapping approach was used to construct to used was approach otstrapping determined for

outgroup. For each sample, after quality after sample, each For outgroup.

redundancy reduction and open reading open and reduction redundancy the Akaike Info Akaike the

about about SNPs each each bootstrap of de novode

the N the

P. likiangensis P. , 89.9% of raw reads) was reads) raw of 89.9% showing wilsonii P. 50 value of 588, 633, 633, 588, of value 50

The relative fit of the different the of fit relative The filtered reads (Table S1) to the to S1) (Table reads filtered - as the optimal. We assumed a a assumed We optimal. the as RNA

transcr , to - rmation Criterion (AIC) Criterion rmation 146 single nucleotide nucleotide single 146 seq dataset. . MAF > 0.05 and 0.05 > MAF

estimat

iptome assemblies foriptome assemblies and , P. wilsonii P. P. purpurea P. P. likiangensis P. individual - e SFS

h expected the

matrixes and re

and tained H . The . , we , 663 o < o P. P. , , .

This article isThis article by protected copyright. All rights reserved. nucleotide pairwise wilsonii P. that than higher was =0.207) genome Mean traits. correspond group, respective their into approximately ancestry likiangensis of individuals species, parental - ( groups ( analyses PCs two first the by explained variance explain with speciesthree 0.00304). ± 0.00411 to diversity genetic ( ( between SNPs shared of number the speciation, were 5,719 of individuals likiangensis In d Genetic for genome 7705873.06 70,025 86,715

Accepted Articlem the T of Mapping

the C the

t re species hree three the among shared to e is to components two first he

ing ) )

of of P. wilsonii P. K (Fig. S1) (Fig. or of iversity - e.g.

=3) =3) - RNA 15.763

P. abies P. RNA , P. wilsonii P. ( . purpurea P. and ,

( 96,329 D n lws CV lowest and

ST . purpurea P. 60% was 60% the hybrid species, species, hybrid the Malaspinas was lik

- P. wilsonii P. seq =0.127) iangensis seq . likiangensis P. quality

. % and % - and pcfc lses ee identified were clusters specific Picea purpurea Picea

P. wilsonii P. and divergence between species ( species between divergence resolved

optimal Tajima's SNPs dataset dataset - aae, w dataset,

ie differen wide p (

22,213 = 86,350 opulation genetic structureopulation

- derived

7.466 was itrd reads filtered (Table 3, Fig. 3). Similarly, differentiation measured in terms of of terms in measured differentiation Similarly, 3). Fig. 3, (Table -

t l 2016) al. et rm 4 niiul of individuals 34 from

Tbe 2). (Table (Fig.

890 used for 1452 SNPs, 1452 for between - D eie fo te group the from derived

error % of %

(π ) species to

exhibited exhibited w

from identified e

± 0. the the wilsonii 5 and ( P. likiangensis P. as infcn bsd n h Tracy the on based significant

b).

(π . purpurea P. =

the the tiation

761 higher than that between that than higher pururea phylogenetic analysis. N

. wilsonii P. P. wilsonii P. ADMIXTURE showed that a that showed ADMIXTURE P

ing 0.

Fg S1). (Fig. = 0.00392 ± 0.00302) and 0.00302) ± 0.00392 = f these Of - . . total variance, respectively. variance, total RNA from each individual (Table 1) to the chloroplast chloroplast the to 1) (Table individual each from 43

; purpurea of which of ADMIXTURE D a to the three species identified from morphological morphological from identified species three the to ae been have

) = 0.00392 ± 0.00305) contained 0.00305) ± 0.00392 = wil . strong skew toward low toward skew strong between

- W sonii seq 113, hen , ,

d .

= , 1

exhibited

A C

When

,6 aa set data 192 lsee in clustered 188 and -

onsist bout half bout ) . wilsonii P. P 1.111 y h PA f ula SNPs nuclear of PCA the by K 13

showed that that showed .

. likiangensis P. eotd in reported

ersne by represented

= were retained after further filtering to filtering further after retained were purpurea either either SNPs SNPs

K n wt te xetto o hybrid of expectation the with ent 2,

ute sh further ± 0. =3, trees of each species clustered clustered species each of trees =3,

ie acsr drvd from derived ancestry mixed ( individuals of the the of individuals

highest of

P. likiangensis P. 728 P. likiangensis P. were and , o w dsic groups distinct two to rm 0 niiul of individuals 40 from all all

P. likiangensis P. ; d and other other SNPs ( SNPs xy D -

pproximately 40 pproximately - wd that owed Wido pcfc to specific Such

frequency variants frequency pur 10 between

log

either either . likiangensis P. and urea 8 - genomic population ,913 ieiod value likelihood test m 65,765 low

= . wilsonii P. a

( 1.216 . likiangensis P. similar and SNPs P. likiangensis P. F

proportions of proportions three two assumed assumed two ST . purpurea P.

Tbe S3 Table ;

(π of

P. wilsonii P. =0.116)

likiangensis

(Fig.

± 0. 145,135 % of the of % from 40 40 from

level of level , genetic genetic while ,

while 706 in ( 5 F

a) all all P. P. P. P. or

ST ). = = ) ) , ,

This article isThis article by protected copyright. All rights reserved. earlier, much originated 0.56 parameters to backcrossing through formed originally was b followed involving one was T analysis Coalescent a greate between that than (59.19%) probability between relationship reticulate past two of assumption 2a (Fig. together showed purpurea P. support bootstrap 7281 across groups gene orthologous 7762 of total A PhyloNet test 2014) al. S2) (Fig. w several of individuals individuals likiangensi with evident 3,Fig.4),consistent withthe (Table of model. expectation speciation hybrid and

Acceptedmodel fitting best he Article - h t The P. wilsonii P.

1.42 the the of these these of r in a NJ phylogeny constructed from the from constructed phylogeny NJ a in . purpurea P. . , genomic P. purpurea P.

eakly supported subclades represented by represented subclades supported eakly hree hree consistent with previous results based on fewer loci (Lookwood et al. 2013; Sun et Sun 2013; al. et (Lookwood loci fewer on based results previous with consistent Ma), whereas the the whereas Ma),

s y of th of

of lineage. A ML phylogeny phylogeny ML A lineage.

with

introgressi of HHS . purpurea P. ee sd o eeae ee trees gene generate to used were

delimited e . likiangensis P. was significantly was

- model suggest suggest model for all branches branches all for co c). The resulting inferred phylogenetic network network phylogenetic inferred resulting The c). . wilson P.

tiuino ntribution of of alternative speciation patterns

hybrid lineage more closely related to the to related closely more lineage s n sltd ld with clade isolated an as through hybridization hybridization through for the origi the for hybrid events hybrid on genetic . wilsonii P.

~6.4 zto and ization ( and yrd‘hs’ lineage ‘ghost’ hybrid Fig. S4 Fig. ii,

2 32 clustered 1332 that . wilsonii P. constitute Ma . purpurea P. was subjected to subjected was higher groups P. wilsonii n of n , S5 ,

P. purpurea P.

(Fig. 2d) indicated a hybrid origin for origin hybrid a indicated 2d) (Fig.

(95%HPDI: g P. purpurea P.

) a P. purpurea P. than between than backcross . osrce from constructed ve

detected detected The d

lsee into clustered ie to rise between a

m four species species four and ld wt hg bosrp upr, while support, bootstrap high with clade

. purpurea P.

P. purpurea odel odel . concatenated originated very recently ~1 Ma (95%HPDI: (95%HPDI: Ma ~1 recently very originated

. wilsonii P. PhyloNet test PhyloNet ing ttl of total A

1.67 P. purpurea P. y C ad ADMIXTURE and PCA by

between between and

. wilsonii P. ( . purpurea P.

indicates that indicates with the the with P. likiangensis P. P. purpurea P. ( - mo

P. likiangensis P. 6.45

P. wilsonii P. del h C the another another was .

P. likiangensis P. with

3782 gene trees trees gene 3782 and

Ma). Ma). N lowest 15

ing

and - identified - RNA a a ihr inheritance higher a had

) RNA . Of these, 1750 clustered 1750 these, Of . . likiangensis P.

Fg 6). (Fig. . likiangensis P. clade and either of its parents its of either and a ahr hn bifurcation than rather

P. wilsonii P.

The

and y PhyloNet by hybrid ‘ hybrid lineage than to the the to than lineage AIC value AIC -

- seq dataset (Fig. S3) (Fig. dataset seq (40.81%) seq wh , P. wilsonii P.

and estimated

P. purpurea P.

data and T c comprised ich ghost

after filtering after e estimated he ih >= with

respectively e showed set ,

, indicating , . wilsonii P.

were Table S4) Table

clustered ’ and 700 700 and ih an with

, lineage

model which . The . 70% also also P. P.

This article isThis article by protected copyright. All rights reserved. occurred the illuminating coalescent with to ‘ this to that indicates that fact mor are estimates current the think, we And estimates al.’s from (40%) minority that indicated from inherited remainder the that indicated speciation hybrid of analysis genetic su data genomic population of analyses Our Discuss wilsonii direction g wilsonii (95%HPDI: between divergence indicated total, lineage ghost ge nuclear the of 62.84% from indicate also parameters ghost ene flowfrom

Acceptedwilsonii P. Article . wilsonii P.

hypothesis P. likiangensis P. therefore, ’ lineage) existed for a for existed lineage) ’ ion (Table 4). and . , P.

to be to or

purpurea

4.47 P. purpurea P. between between

Or study Our .

following formation of a hybrid between the two parent species, backc species, parent two the between hybrid a of formation following through back through approximately 70% of 70% approximately occurred, maybe

h mjrt (0) of (60%) majority the derived fromderived and indicate and P. wilsonii P. 84 o te ula gnm of genome nuclear the of 38.4% - analysis 14.07 different from

11 , P. likiangensis P.

P. likiangensis P. contains both the mtDNA and cpDNA of cpDNA and mtDNA the both contains and caused by the number of loci of number the by caused . purpurea P.

P. likiangensis P. loci Ma). The current effective population sizes ( sizes population effective current The Ma).

resulting in the origin of origin the in resulting that that

nome of of nome were estimated to be to estimated were for 38.90% from 38.90%

emphasises to stages crossing to crossing s (Sun

P. likiangensis P.

61.10% of the nuclear genome of the ghost lineage ghost the of genome nuclear the of 61.10% l that a hybrid lineage (now extinct and therefore referred to as a as to referred therefore and extinct (now lineage hybrid a that P. purpurea eosrcig the reconstructing ong . wilsonii P.

t al. et the initial population of population initial the period before period novd n uh n origin an such in involved

and and . The difference between the current estimates and Sun et et Sun and estimates current the between difference The P. purpurea P.

and h pwr of power the . purpurea’s P.

P. wilsonii, P. 2014) , P. wilsonii P. P. wilsonii P. . likiangensis P. was estimated to be was estimated to , P. wilsonii P. , the present present the and e accurate than before. than accurate e pr peiu fnig bsd n population a on based findings previous pport

∼ that P. purpurea P. 61.6% from 61.6% rgn of origin 70597, 72700 and 77423 and 72700 70597, ’s genome came from came genome ’s

P. purpurea P. .

was estimatedwas h ita pplto of population intial the

while 37.16% came from came 37.16% while (12095 vs 11 loci) 11 vs (12095 Importantly, it it Importantly, . purpurea P. population

. genome However, whereas However, ,

extensive extensive

and

P. purpurea P. a

originated P. wilsonii P. between between homoploid higher

P. wilsonii P. ae from came Coalescent and genomic

to rgntd hog diploid through originated nuclear was Ne have occurred have

than that than that vr what over This, together with the the with together This, P. likiangensis P.

) of )

used in each analysis. analysis. each in used was derived from the the from derived was through . Under this scenario, this Under . further indicated that indicated further P. likiang P.

yrd species hybrid

analysis our previous study study previous our , respectively, ,

(Sun P. lik P. eoi analysis genomic analysis . purpurea P. P.wi in the opposite in the opposite P. wilsonii P.

backcrossi et al et iangensis lsonii was pe

ensis ~10. combined riod support ., derived rossing

and 2014), 2014), n a and , with , 59

they was was and In . and , Ma ng P. P. P. s

This article isThis article by protected copyright. All rights reserved. formation ofahybrid lineage ( between species 1955) incomplete that noted ecologically from isolated Wi extrinsic aresatisfied, namelycriteria spe homoploid hybrid two parental species, wilsonii say, cannot of whether the course, and that one indicated fastsimcoal simcoalescent homoploid hybrid common with originhybrid of likiangensis approximately F examined variation groupgenetic likiangensis O of origin Hybrid urther ur

Accepted To Article th regard to the tothe latter th regard P. analyses analyses

determine and that wilsonii

more in the wild

was instrumental causing in . P. likiangensis ecological P clustered clustered most spruce species can beintercrossed to 2 hylogenetic analysesof intrinsic

. , and .

ADMIXTURE

showed that

PhyloNet analyses ofthesePhyloNet analyses trees indicated

A total A total in P. wilsonii u , extrinsic 60% of lations were conductedlations onthe

and

P. purpurea the likely the Picea purpureaPicea P. wilsonii phylogenetic

a hybrid . speciation

It isfeasible thattran P. purpurea isolation later backcrossed to postzygotic reproductive isolation of i.e., ciation. its nuclear genomeits nuclear from derived

, 15 alternative and barriers

P. purpurea and to satisfy one of Schumer to satisfyoneofSchumer

P. purpurea evolutionary evolutionary P. wilsonii lineage (now extinct) formedoriginally between ,

P. wilsonii

contain

analysis evidence ofhybridization in theevidence genome of with from versus , 40% shared with 40% shared with However, ourresults indicate that

P. purpurea PCA

are

with as an independent lineage an independent as

individuals initial hybridization event backcrossing and/or to both parent species both orthologous gene

, andADMIXTURE ing key topreventing hybridization and bifurcation , P. purpurea

either either is (eco)geographically is (eco)geographically fromisolated (Fig. 1 (Fig. conducted conducted

evolutionary evolutionary is

scenario and later backcrossing to 1,613

P. wilsonii sgressive segregation following genetically ) adapted toaridenvironments high altitudes at P. wilsonii ; Sun al.2014 et

having, onaverage, species nuclear SNP dataset P. likiangensis divergence divergence

for the on

to become isolatedfrom reproductively its

model et al.et genome leading to origin the of

delimited s produce via -

specific showed

(Sun et 2014 al. is widespread

origin of P. wilsonii

a analyses ’s ’s

s P. likiangensis hybrid originof

(2014) criteria for three were tested were tested followed by - wid ; Wang al.2017 et

. SNPs and that from two its

Schumer e ble ble seeds, P. purpurea P. wilsonii

of 60% SNPs,

of ~ and 81 genome

in the(Wright, genus all three species usingall threespecies ; Wangetal.2017 % of and bestfitting the the secondary homogenization P. purpurea , form

further indicated a further hybridization

of et al. et their ancestry in their respectively P. purpurea P. likiangensis remainder indicating indicating that P. purpurea close

P. likiangensis 3782 - resulted in the , particularly scale scale ing ’s ’s ).

two other

relatives It should It be a gene trees gene trees

gene flow nuclear nuclear distinct distinct P.

and

from .

with .

) We of

, and

, This article isThis article by protected copyright. All rights reserved. 2008; species hybrid homoploid other period a over completed involved species these of history evolutionary genomes Plei change climatic the during extinct became lineage hybrid intermediate proportion greater a of origin lineage hybrid the of period lineage. hybrid a form to hybridized and Pliocene early and 2006; Chamberlain, Miocene late the during QTP northeastern and eastern the observed likiangensis mya. 6 approximately to dated is origin This other. the of cpDNA the and parent one of mtDNA the contained assumemight we which lineage, hybrid intermediate of origin the in involved were stages our to According Formation locati and detailed analysis of difficult,very given the fromboth parent sp which

Accepted Article ons where Abbott al., et 2010; before the second stage occurred in the origin of origin the in occurred stage second the before

P. purpurea P. in the wild. Thus, it is feasible that feasible is it Thus,wild. the in two of

of a

are currently allopatrically currently are . upra P liki P. purpurea, P. phases of hybri of phases

‘ ghost P. purpurea

occurr fastsimcoal Deng & Ding, 2015) Ding, & Deng ’ hybrid , which ,

to f h ncer eoe of genome nuclear the of ing the ofbothparent fitness species and theirhybrid requirement . wilsonii P. ~ Lamichhaney ecies are ecies excluded (Sunthis al.,2014).However, proving et w

6 million years million 6

n h QP ( QTP the in

occurs

contained lineage dization separated by approximately 5 million years, and years, million 5 approximately by separated dization 2

best oe fr hi vr rpd origins rapid very their for noted angensis .

, to undertake long a very - and and

itn coalescent fitting dated dated P. purpurea P. ,

both et al et distributed and no hybrids between them between hybrids no and distributed have

the T the two species came into contact came into species two the eg Dn, 2015 Ding, & Deng , his hybrid his

and which is co is which ., o prxmtl oe mya. one approximately to the mtDNA and cpDNA of of cpDNA and mtDNA the lengthy as a result of aresult as ,

2018). thus

. wilsonii, P. . ,

The fir The origin oforigin

shown . wilsonii P. lineage must have must lineage oe o origin of model nsiderabl P. purpurea P.

tectonic st stage involved the origin of an of origin the involved stage st

- that that P. purpurea P. term study n caecn a coalescent and stocene

. y ).

h oii of origin the and climatic oscillations in in climaticoscillations and Subsequently, longer than than longer D , etailed analysis of the the of analysis etailed Beke Rieseberg & (Buerkle involv possibly , P

P. wilsonii P.

existed

involv hs eutd in resulted This icea progeny Fg 6) (Fig.

during this period thisduring ing

wilsonii ayi o the of nalysis ing

estimated for estimated backcrossin for . purpurea P.

, eas of because at at crossing the

Mlh & (Mulch have been have w main two as well as as well as a lengthy lengthy a

and initial ill

was

the be P. P. g ,

This article isThis article by protected copyright. All rights reserved. can S S and PRJNA301093. T D manuscript. the and D.R. wrote D.R,Y.S. andD.W and Q.H. performed conducted fieldwork, data experiments, analysed etc.J.L.,R.J.A., Y.S. Author contributions Innovation Fund of Un Sichuan (2014CB954100), CAS “Light of West China” Program and Graduate Student’s Research and numbers Key(grant 31590821, for Research 31670665,91731301),National Project Basic development program (2017YFC Acknowledgements with genes frominherited 1999; Farjon,2001) morphologically more similar to genom

upplementary figures S1upplementary figures upplementary Material he sequencing data havehe sequencing data deposited been PRJNA401149in GenBank bioproject: underthe ata accessibility

Accepted Article

Although be found in the onlinebe foundinthe e of e of P. wilsonii P. P. purpurea

our analysisshows that P. likiangensis

Its Its morphology This was by work fromsupported grants National key research and

relative to itsother parent,

J.L. plannedanddesigned research.the D.R., Y.S.,D.W.,Y.C.,T.W.

following -

version ofversion article. this S5, Supplementary tablesS1 , which according tooursimulations iversity (2018YJSY007).iversity P. likiangensis 0505203), National Natural National FoundationScience ofChina0505203), the

is therefore is therefore presumably more influenced by P. purpurea latter’s latter’s origin

(

Cheng &Fu,1978;Farjon,1990;Fu P. likiangensis

contains proportion agreater of the . contributed equally . contributed .

- S4 and perlscripts Supplementary

, P.

continued to purpurea .

is exchange genes

et al. et

, This article isThis article by protected copyright. All rights reserved. Du FK, Peng XL, Liu DePristoMA, Banks E, PoplinR, GarimellaMaguire KV, JR, Hartl C, Philippakis AA, del Angel RivasG, MA, Deng T, Ding L Delhomme N, Sundstrom G, Zamani N € LaDe Torre AR, Li Danecek P, Auton A, Abecasis G, Alber Coyne JA, Orr HA Cheng W, Fu L Chap Capella Camacho C,Coulouris G, Avagyan V, Ma N, Papa Buerkle CA Buerkle CA, Morris RJ Bolger AM, Lohse M, Usadel B Bodare Alexander DH, Lange K Abbott R, Albach D,Ansell S, ArntzenJW, BairdSJ, Bierne N, Abbott RJ, References introgression between two spruce species in the Qinghai s Hanna M, National Science Review of Norway 1363 patterns ofselection among gymnosperms flowering and plants. Sherry ST, Society of London altitude. large architecture and applications. 62 Heredity Bioinformatics spruce ( estimation. R, 1375

Acceptedequencing data. Article , manMA, Hiscock SJ,Filatov DA et al.

266 - - - - S, Stocks M, Yang JC, LascouxM 1386. 1377. scale phylogenetic analyses. Gutiérrez S, Silla – Picea morrisonicola

( , Molecular Biology and Evolution 275. Heg 2013

84 & RiesebergL et al.

spruce (Picea abies).

et al.

BMC Bioinformatics ,

arty MJ, Hiscock SJ,Brennan AC

(1978) 441 (2015) ) ,

Hybridization andspeciation. 30 (2011)

(1998) (2011) Nature Genetics – Z, Van de Peer Y,Ingvarsson PK , ,

451.

Series B

2114 JQ, Lascoux M, Hu FS,Petit RJ

, Asmusse Flora ofChina Paleoaltimetry reconstructions of the Tibetan Plateau: progressand cont

(

- 2011 A framework for variation discovery and genotyping using next

Martínez J, Gabaldón The evolutionary genetics of speciation. The variant call format VCFtools. and H , -

2120. 2

(2008) , ,

). ) 417 353

BMC Bioinformatics Enhancements to the ADMIXTURE algorithm individual for ancestry ( Ecology and Evolution n MA n & RiesebergLH 2014

- , , PLoS ONE

437. , 12 287

The rate of genome stabilization in homoploid hybrid specie Bioinformatics 43 . Science Press ) ,

, (2013) 246. - Trimmomatic: aflexible trimmer for Illumina sequence s CA, Banks E, DePristo MA, HandsakerRE, Lunter G,Marth GT,

305. 491 et al

(2013)

, -

498. . (2015) Serendipitous meta 30 ,

Genomic diver 10

Journal of Evolutionary Biology , T

2553 , e0139080

( Originand demographic history ofthe endemic Taiwan (2009) 2010 dopoulos J, Bealer K, MaddenTL , , 25 , Beijing, China.

(2017) – (2011) , 1972 , ) 256

, 3 (2000) Homoplo

trimAl: atool for 421. , -

TibetanPlateau. 3320 7.

Contrasting rates ofmolecular evolution a – - Direction and extent of organelle DNA

gence during speciation driven by adaptation to 1973.

Thelikelihood of homoploid hybrid speciation. BoughmanJ, Br - 3333. Bioinformatics id hybrid speciation in action. Molecular Biology and Evolution Philosophical Transactions

- transcriptomics: the fungal community

a New Phytologist utomatedalignment trimming in , , elsford A,

27 26 , ,

2156 (2009) 229 - - 246. - generation DNA 2158. Buerkle CA, Buggs

, BLAST+:

192

of the Royal radictions. data.

Taxon s. ,

1024 Evolution ,

34 , nd -

, 59 1033. ,

This article isThis article by protected copyright. All rights reserved. Jiang L,SchlesingerF, Davis ZhangCA, Y, Li R, Salit M, Gingeras TR, Oliver B(2011) Syntheti Hu Heliconius Genome C HansenBr KD, Haas BJ, Papanicolaou A, Yasso BL,Gross Rieseberg LH Gra Grant V GrabherrMG, HaasBJ, YassourM, Levin JZ, Thompson DA, Amit I,Adiconis X, Fan L, Ray Gompert Z, ForisterML, Fordyce JA, Nice CC, WilliamsonRJ, B Gompert Z, Fordyce JA, F Foote AD Fumagalli M, Vi Fu L, Li N, Mill R Finn RD, Coggill Eberhardt P, RY, Eddy SR, Mistry J,Mitchell AL Farjon A F Excoffier L, Dupanloup I,Hue Erlich Y arjon A standards forRNA biological sequences. species. hexamer priming. reference generation andanalysis. M, 96 Nature Biotechnology Zeng Q, butterflies. molecular variance in pyrosequences quantifies population genetic structure across the genome of habitat. ecotypes.whale KorneliussenTS from next database: towards amore sustainable Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix and Picea. genomicSNP and data. callerfor next

Acceptedang Y, Niu B, Gao Y,Fu L, Li W Article nt (1966b)V ,

et al. 241

, Mitra PP, delaBastide M,McCombie WR, Hannon GJ (2008) Alta (1966a)

(2001) (1990) , - Science 252.

Nature Vijay N et al. (2013) - generation se MolecularEcology

enner SE, Dudoit S (2010) Biases inIllumina transcriptome sequencing caused byrandom

eira FG, Linderoth T,NielsenR WorldChecklist Bibliography and ofConifers. Pinaceae. Drawings and descriptions of thegenera Abies,Cedrus, Pseudolarix, Keteleeria, (2011) Selection for andvigor fertility inaspecies cross in The origin ofa newspecies of generation sequencing. , ,

De novo Nature Communications , (1999) 487 , 314

Ávila Martin MD Nucleic Acids Res -

seqexperiments. (2012) , ,

94 Full 1923 Bioinformatics ,

(2005) 29

- quencing data. Pinaceae. Flora of China oristerML, Shapiro AM, Nice CC PLoS Genetics - ArcosMC

98. transcript sequence reconstruction from RNA , -

length transcriptome assembly from RNA -

644 Butterfly genome reveals promiscuous exchange ofmimicry adaptations among 1925.

rta ,

ur M,ur GrabherrM, Blood PD,Bowden J, CougerMB, Eccles D, LiB, Lieber The , et al. - -

652 Sanchez E, Sousa VC, Foll M 19

, Nature Protocols e , (2010)

2455

cological

Baird RW ( earch , Genome Research 2016

future. 26 Nature Methods , Bioinformatics

9 - , ,

, 2473.

680

CD , ) 7 e1003905.

38 Genome , 11693. Nucleic Acids Research

- - g Gilia , ,

HIT Suite: awebserver forclusteringand comparing 682.

(2014) enetics of

e131. Durban JW . Science Press

in a hybridization experiment.

, -

culture coevolution promotes rapiddivergence of killer

, NGSTools: methods for population genetics analyses ,

30 , 1494 5 21 h , 679 ,

omoploid , 1486 (2006) , 1543

Fumagalli M

- Koeltz Scientific Books 1512.

2 – (2013) nd 682. uerkle CA - , Beijing, China.

1487. edn -

et al. 1551. Homoploid hybrid speciation inan extreme

Gilia. Genetics

- h , .

Seq datawithout a reference genome.

Robust demographic inference 44 ybrid Royal Botanic Gardens

seq using theTrinity platform for (2016) The Pfam families protein

, D279

Gibbs RA Cyclic: Aself (2010) s peciation. – 2

85 , Bayes Genetics

, Koenigstein Hanson MB ,

757 Journal of Heredity iananalysis of - optimizing base – ,

775. 54 chowdhury R, , Kew, UK. ,

c spike 1189

, Lycaeides , Germany.

– from 1199. - in

This article isThis article by protected copyright. All rights reserved. McCarthy EM, AsmussenMA & Anderson WW Malaspinas MaoJF, Wang XR Ma XF, Szmidt AE, Wang XR Loytynoja A, Go Lisiecki LE, RaymoME Li Y, Stocks M, Hemmila Kallman S, T, Zhu H,Z Li M, Tian S, YeungCK, Meng X,Tang Q, Niu L, Li M, Tian S,Jin L,Zhou G, Li Y, Zhang Y, Li L,Sun Li L, Abbott RJ,Liu B, Sun Y, Li L Li J,Shi Y, Li B, LiSD Li H, Handsaker B,Wysoker A,Fennell T, Ruan J, Homer N,Marth G, Abecasis G, DurbinR, G Li H Li J,Fang X Lamichh Katoh K,Standley DM Heredity densata 807 densata evolutionary analysis. dynamics. multiple nuclear loci. histories of fourspruce ( (European native pig) provides insigh selection in domesticatedpigs andTibetan wild boars. 661 Piceasmithiana the Qinghai and Plio University Press,L 2078 Data ProcessingS 25 2117 Darwin’s finches. performance usability. and AcceptedNature, Article , Durbin R ,

– - 1754 672. - - 816. 2079. 2124. aney S, Han F, Webster MT, Andersson L, Grant BR, Gran

Y, Y, Zou

- on the inferr , 74, -

538 Pleistocene range expansions within

1760.

(1999) A, A, Quaternary Science Reviews

- Tibet Plateau.

, 207 (2009) 502 ed from thenucleotide variation atseven gene loci. Westaway ldmanN

Tibetan Plateau. J, Yue W, Wang X, Liu J

in theCenter AsianHighlands and Himalayas.

(2011)

– Uplift of theTibetan Plateau and environmental changes.

Science – 509. (2009) an

214 Fast and accurate short read alignment with Burrows

(2013)

zhou, China. Molecular Biology andEvolution (1995)

Science (2007)

(2008)

Distinct niche divergence characterizes Picea

, The seq MC,

Molecular Ecology 359 Molecular Biology and Evolution

MAFFT multiple sequenc

( Uplift ofthe Qinghai

2006 Plio , ) species) theof Qinghai

, Phylogeny Craig 320

The AmericanNaturali 224 , Zou J uence alignment/mapformat SAMtools. and –

) , Pleistocene climate trends evolution: andtransitions in glacial cycle

- Genetic structureand evolutionary 1632 228.

ts into its origin and domestication. M ,

, Wang X,Miehe G,Liu J

26 et al

(2015) - - 1635. aware gap placement prevents errorssequence in alignment and et al. ,

Picealikiangensis , (

- (1995) 2016 22 69.

hou Y, Chen J,LiuJ, Lascoux M (2013) Origin and speciation of et al. (20), –

Xizang (Tibet) Plateauand global change. )

. A genomic history of Aboriginal Australia. Aboriginal of . A genomic history -

A theoretical e alignment software version 7: (2014)

TibetanPlateau and neighboring area Nature Genetics

st 5237 , Genomic analy

27 ,

177 ,

- 1001 , 5255.

, Whole 30

424 Plant Molecular Biology Reporter

the homoploid hybrid speciation of

, (Lijiang spruce), adominant forest tree of

- Molecular Biology and Evolution t PR 772 1014.

– (2013)

assessment of 439. - genome sequencing ofBerkshire -

780 (

ses identify distinct patterns of , history history of a diploid hybrid pine 2018

Picea schrenkiana - Scientific reports

. Wheeler transform. Pliocene intraspecific divergence

, Chinese Science Bulletin 1431 )

Rapid hybrid speciationin Bioinformatics

– (2010) recombinational speciation. 143 8. improvements in

Demographic ,

4 s inferred from and , ,

Bioinformatics

4678. L enome Project 25 anzhou , (1

33 , 6), Pinus

23 ,

, 44

, Pinus

This article isThis article by protected copyright. All rights reserved. Stamatakis (2014) A Shi Y, Li J, Li B, LiL Schume Salazar C,Baxter SW, Pardo Ru D,Mao K,Zhang L,Wang X, Lu Z,Su Robinson JD, Coffman RiesebergLH, Raymond O, Rosenthal DM, Lai Z, RiesebergLH Renaut S, Grassa C, YeamanS RanJH, Wei Purcell S, Ne Price AL, Patterson NJ, Plenge RM, We Petit RJ,Excoffier L Nystedt B, Street NR, NietoFeliner G,Álvarez I, Fuertes Nice CC, Gompert Z, Fordyce JA, Fo Nei M M ulch A, Chamberlain CP AcceptedBioinformatics Cenozoic 1553 evidence for hybrid trait speciation inHeliconius butterflies. PLoS Molecular Ecology interlineage gene flow within complextaxa: a casestudy of spectrum Science Donovan LA, Lexer C speciation insunflowers. 405 for phylogeographical studies using cytoplasmic haplotypes. ArticleAm MJ, corrects forstratification ingenome 497 Alexeyenko A, Heredity JA, Soltis PS, Villa independent evolution in lineages ofalpine butterflies. erican - ,

et al. 419. (1987) 579 - r M, Rosenthalr GG, Andolfatto P 1560. ,

, - 301

- .

584.

based based population genomic inference. 118 (2007)

Journal of Human Genetics Guangdong ale B,Todd XX, Wang XQ

Molecular Evolutionary Genetics. (1997) ,

, 513 1211

et al. 30

PLINK: atool set for whole

, – (2009) RAxML version 8: atoolfor phylogenetic analysis and post - , Hybrid origins of plant species. ‐

1216.

1312 Wetterbom Zuccolo A, A, Lin YC, Scofield DG, Vezzi F, Delhomme N, GiacomelloS, 516. (1998) 25

AJ, AJ, Hickerson MJ, Gutenkunst RN Machío I(2017) Is homoploid hybridspeciation that rare? Anempiricist's view. (2013) -

Science , (2003) Brown K,Thomas L,Ferreira MA, Bender D, Maller Sklar J, Bakker P, de DalyPI,

2373

- (2006) Nature Communications

1313

Gene flowand species delimitati - (2006)

Diaz C, Wu G, Surridge A,Linares M,BerminghamJiggins E,CD (2010) Genetic Uplift andenvironmental changes of Qinghai

The Norway spruce genome sequence and conifer genome evolution. (2013) -

2386. Majorecological transitions in wild sunflowers facilitatedby hybridization.

. andTechnology Press,

The rise and growth of Tibet. ‐

Molecular phylogeny and biogeography of AguilarJ, Heuertz M, Marques I, Moharrek F, PiñeiroR, Riina R, Rosselló rister ML, Lucas LK, Buerkle CA

- Genomic o islands wide association studies. inblatt ME,ShadickReich NA, D ,

81 (2014) n Y ,

559

(2016) -

genome association and population Columbia University Press How common ishomoploid hybridspeciation? - BMC Evolutionary Biology 575. Livingstone K, Nakazato T,Durphy JL, Schw ,

4 Annual Review of Ecology and Systematics

Genomic evidence for polyphyletic origins and

1827. Guangzhou, China. fdivergence are notaffected by geography of Evolution

(2014)

on. on.

Picea brachytyla Molecular Phylogenetics andEvolution Nature Genetics Nature Trends Ecology Evolution

Sampling str ,

Genetics, 6,1 , ,

( 1055 439 2013

(2006) - , New York, USA. TibetanPlateau the in Late

, - 670

) 1068. 14

ategies for frequency Hybrid specia

Picea in the Qinghai , , -

Principal components analysis 38 analysis oflarge phylogenies. 254. 671. –

- , based linkage analyses. 12.

904 (Pinaceae): implications

- 909. ,

24 tion and -

, Tibet Plateau.

386 Evolution , arzbach AE,

28 - 393. , Nature

359 ,

- , 389. ,

68 ,

This article isThis article by protected copyright. All rights reserved. ZouJ, Sun Y, Li L, Wang G, Yue LuZ,W, W Yu Yakimowski SB, Rieseberg LH taxonomy. and to distribution relation in inspruce crossability Species (1955). JW Wright Weir BS, Cockerham CC Wang GH, Li H, Zhao HW, Wang Z, GersteinM, Snyder M Wang GD, Zhai YangWW, HC, FanRX, Cao X,Zhong L, Wang L, LiuF, WuH, Cheng LG, ThanC, Ruth Tajima F Suyama M,Torrents BorkD, P(2006) PAL2NAL: Robust co Sun LiY, L,Li Sun Y, Abbo Suarez Suarez Stebbins GL 1829 pattern and gene flow between Proceedings of the synthesizing genetics andecology. 1358 divergence amongspruce ( Genetics 4 genom evolutionary relationships. Genetics corresponding codonalignments. Biogeography likiangensis 343 in theQinghai Phytologist balsamifera Molecular E adaptive introgression from 336 Science Accepted Article Y, ,

1860 - – Dong - - 359. 340. - - Gonzalez A,Hefer CA, LexerC Gonzalez A,Hefer CA, Christe C, 1844. 1370.

ics of selection ics of in dogs and the parallel evolution between dogs and humans. (1989) .

, , ,

10 123 1

J, tt RJ, LiL, LiL, Zou J,Liu J (1957) s D , 319 ,

, underlies adaptively significant va cology and Liu 217

L, Zou J,Liu J, 57 ,

, -

, NakhlehL (2008) Statisticalmethod fortestingthe neutral mutation hypothesis by DNA polymorphism.

585 Tibet Plateau: homoploid hybrid origin and Pleistocene expansion. 42

- , KJ, andKJ, P. –

63.

416 , The hybrid origin of microspecies inthe 340

475 , National Academy of Sciences of the UnitedStates of America 595. wilsonii

25 - 427.

- , (1984)

484.

2427 Nakhleh Zhang WK (2017) Detecting climatically driven phylogenetic morphological and

Picea BMC Bioinformatics

triggered byclimate change on the Qinghai Populus balsamifera

CarineM (2015) (2014) (2009) 2442.

Estimating Piceawilsonii ) species worldwide.

Nucleic Acids Research L PhyloNet: asoftware package for analyzing and reconstructing reticulate American Journal of Botany

(2014)

The role of homoploid RNA , Dou

et al (2014) F -

glas Seq: a revolutionary tool for transcriptomics. Maximum inference likelihood ofreticulate evolutionary histories. ‐ ang Q, Liu J .

, ( statistics

2016 P. morrisonicola riationand range boundaries in Distributional dynamics interspecific and gene flow in

CJ, &CronkQCB Evolutionary history of purple ,

9 (balsam in poplar) ,

) 322.

Genomic functional and approaches reveala Biogeosciences

for theanalysis of population structure.

Q , 34 Elymus (2013) nversion of protein sequence alignmentsthe into hybridization in evolution: a century of studies , W609 ,

and 101

Population genetic evidence speciationfor

( glaucus 20 P. trichocarpa P. neoveitchii – , , W612. 1247 18 14 - Tibet Plateau. ) , 2307

Introgression

- complex. 1258.

cone spruce ( P.trichocarpa - 2319.

. Molecular Ecology 111

Annals Botany of (black Nature Communications Cytologia

, Journal of

from 16448 Nature Reviews

cottonwood). Picea purpurea . et al.

Populus New – , Evolution 16453. Suppl.Vol., Forest

case of (2013) ,

, Picea 112

, The , 38

)

, ,

This article isThis article by protected copyright. All rights reserved. ZR_11 ZR_10 ZR_08 ZR_07 purpurea P. MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ MSZ likiangensis P. breweriana P. species of 1. Table

Accepted Article breweriana P. ------50 48 47 46 42 40 38 34 33 31 30 25 15 35 09 08 07 06 05 04 03 02 01

Locations of of Locations

, 40of 27.13161 27.19836 27.569 27.93083 28.40706 29.82383 29.8905 29.67392 29.56961 29.46339 29.18475 29.49017 29.55578 29.68322 29.61128 29.72983 30.28694 29.99958 30.27436 30.29497 30.12058 35.93306 latitude 34.45132 34.27802 34.02296 33.07804 27.02508 27.14214 Picea P. likiangensis

individuals sampled for transcriptome analysis (including individual one (including analysis transcriptome sampled for individuals 102.69788 103.00059 102.73741 102.85164 100.20897 100.2335 100.23303 100.27886 100.02383 99.616472 98.987944 95.711528 95.523278 94.720028 94.557972 94.61775 93.978556 96.672278 96.777333 97.931917 98.156944 98.62975 99.519222 100.87139 101.522 101.60944 101.75611 104.15008 longitude , 40 of of , 40

P. purpurea

3920.11 4186.15 4122.95 4104.2 4026.58 4252 4178.94 3589.66 3978.51 4221 1774 Altitude(m) 3132 3556 3526 3568 2845.13 3197.45 2947.51 3260.41 3025.85 3511.8 3882.86 3262.82 2698.53 3663.2 3421.91 2913.14 2988.12

and 34 of 34 and

P. wilsonii P. 2 1 2 2 3 3 1 1 3 1 40 1 n 7 7 5 5 40 2 2 3 1 1 2 1 2 1 3 1 1 1

)

This article isThis article by protected copyright. All rights reserved. for statistics Summary 3. Table d c b a D Tajima’s π SNPs of Number loci of Number 2. Table numbersamples. of the indicate n ZR_26 ZR_24 ZR_16 ZR_15 ZR_09 wilsonii P. ZR_25 ZR_14 Based on the rawN the Basedon rawC Basedon Based on the final N theBased final on finalC Basedon ± Accepted± SD Article SD P. wilsonii wilsonii P. likiangensis P. likiangensis P. Species pair

Summary statistics for N for statistics Summary

1 1 samples A 452 ll - vs. RNA -

RNA 34.1588 33.29427 35.78197 36.95562 34.04897 33.0443 35.53111 -

P. purpurea P. a vs. vs. vs. RNA -

RNA - seq dataset without MAF and and MAF dataset without seq -

seq dataset after MAF and and MAF dataset after seq P. purpureaP. wilsonii P. 1 1 samples A 92 ll -

seq without MAF and and MAF seq without -

seq after MAF and and seq afterMAF

b ST

- RNA

and

6 1 samples A 102.90655 104.47862 104.05484 102.46394 103.2207 103.72414 102.24462 70146 2276 ll - d seq and C seq and 0.127 ± 0.145 0.127 ± 0.104 0.116 ± 0.158 0.207 F xy ST

± SD c

1 samples All 107301 o control. o

H 1410 - RNA H o filtering. o o control. o H

o filtering. o

2769 2389 2304 2306 2390 3497 3085 -

d seq datasets seq

14107 likiangensis P. 0.761 - 0.00304 0.00 113188

0.890 ± 0.890

411 ± 411

0.269 ± 0.130 0.269 ± 0.108 0.276 ± 0.134 0.319 d

± SD d

7 7 7 6 7 34 8 8

12237 purpurea P. 0.706 - 0.00305 0.003 108913 1.216 ± 1.216

92 ± 92

12156 wilsonii P. 0.728 - 0.00302 0.003 96329 1.111 ± 1.111

± This article isThis article by protected copyright. All rights reserved. wilsonii and purpurea T gen per m m and lineage wilsonii between afterhybridization and before generationmigration per indicate m respectively. likiangensis N T T T m m m m m m m m m m N N N N N Parameters best the of parameters demographic Inferred 4. Table ADM1 DIV ADM2 ADM1 e e e e e e 2 2 ghost 1 1 P.likiangensis P.wilsonii P.purpurea P.purpurea P.likiangensis P.likiangensis ------P.likiangensis P.likiangensis P.likiangensis ghost P.wilsonii P.purpurea P.likiangensis ANC

Accepted P.wilsonii ArticleP.wilsonii P. wilsonii P.

- >

indicates time (years) of backcrossing of the ghost lineage to to lineage the ghost of backcrossing of indicates time (years) P.likiangensis eration between eration

- obtained fromobtained m respectively; > - -

> > P.purpurea while T - -

> > P.wilsonii P.likiangensis - - -

P. likiangensis P. likiangensis > > >ghost - -

, , > > P.purpurea P.purpurea N P.wilsonii P.wilsonii P. purpurea P. ghost e . T 1 -

P.purpurea P.likiangensis

- ADM2 DIV > P.likiangensis

, m

indicates the estimated divergence time (years) between time (years) divergence estimated the indicates

indicates time (years) of formation of thebetween of ghost lineage formation of indicates (years) time P.purpurea fastsimcoal2 , Point estimation Point 10,586,400 6,422,150 1,001,200 1.51e 4.84e 6.95e 1.68e 8.80e 1.76e 0.00108 0.0344 7.65e 0.00366 48,999 72,700 77,423 70,597 65342 P. likiangensis P. purpurea P. N - , > e P.wilsonii P. wilsonii P. - P.wilsonii

migration per generation from ghost lineage to to lineage from ghost generation migrationper ------

4 5 5 5 6 7 6 -

P.likiangensis

, m -

>ghost ,

N .

and e P. wilsonii , the ghost intermediate lineage and ancestral population ancestralpopulation and intermediate lineage , the ghost - ghost : migration per generation fromgeneration per : migration

, m P. likiangensis P.

and - P.wilsonii > p. p. likia N 1.43e 3.78e 1.06e 7.02e 5.53e 9.19e 16,238 19,881 32,917 29,918 2,104 bound CILower 95% 4,465,050 1,673,800 564,100 2.04e 1.93e 4.07e 7.29e e - ANC ngensis - > P.purpurea

------

indicate the effective population sizes of indicate theeffectivepopulation -

8 8 8 9 8 9 8 8 8 8 fitting demographic model shown in Fig.6 in model demographic shown fitting

, m

or or

2 P.likiangensis , and m , and P. wilsonii P.

P.purpurea - > P. wilsonii P. P.wilsonii

P. likian P. in both directions, respectively. in directions, respectively. both 5.27e 2.57e 0.00861 0.0494 0.0160 0.0167 137,627 77,336 94,648 75,635 96,147 bound CIUpper 95% 14,068,850 6,447,150 1,416,350 4.62e 2.21e 1.62e 1.64e P. likiangensis P. - > P.wilsonii

and and m P. likiangensis P. ------

5 5 4 4 4 4

P. likiangensis P.

gensis

that gave rise to riseto that gave

2 indicate migration indicate P. wilsoniiP.

to the ghost the to P. likiangensis P.

and and - > . p. p. likiangensis

P. and and P. P.