i simposio anual de botánica española

José[email protected]

La secuenciación del plastoma completo en la sección Psammophilae (Silene) revela un proceso de hibridación en poblaciones interiores y divergencia en las poblaciones de las Islas Baleares

ISimposioAnualdeBotánicaEspañola

Morethan20,000plantspeciesà60%(13,000spp.)areendemicofthisregion

Médailetal.1999.ConservationBiology

MediterraneanBasin:hotspotofbiodiversity

LasecuenciacióndelplastomacompletoenlasecciónPsammophilae(Silene)revelaunprocesodehibridaciónenpoblacionesinterioresydivergenciaenlaspoblacionesdelasIslasBaleares

TheBalearicIslandsareespeciallyrichinendemics,makingthemexcellentmodelsforunderstandingspeciation

Chuecaetal.2015JournalofBiogeography

-5.5Ma

30Mya 21Mya 15Mya

10Mya Present


TheisolationofBalearicIslandscontrastswiththecolonizationeventsinotherMediterraneanislands



Reyment1983Oikos



Reyment1983Oikos


Bittkau&Comes2005MolecularEcology

IntheIberianPeninsula,dramaticgeologicalandclimaticchangeshaverepeatedlycausedfragmentation,contraction,andexpansionofspeciesranges


IntheIberianPeninsula,dramaticgeologicalandclimaticchangeshaverepeatedlycausedfragmentation,contraction,andexpansionofspeciesranges

Increasedchancesforhybridizationandintrogression


HybridizationeventsareimportanteventsinthetribeSileneae(Caryophyllaceae)



TheabilityofSilenelatifoliaandS.dioicatohybridizeisoneofthebestexamplesofincompletereproductiveisolationinthisgroup


Hathwayetal.BotanicalJournaloftheLinneanSociety


TheabilityofSilenelatifoliaandS.dioicatohybridizeisoneofthebestexamplesofincompletereproductiveisolationinthisgroup


PhylogeniesinSileneaearefrequentlyincongruentwithmorphology-basedclassifications


PhylogeniesinSileneaearefrequentlyincongruentwithmorphology-basedclassifications

ThetribeSileneaeissubdividedintoeightgeneraàSileneisthemostdiverse,withapproximately470spp.-  SubgenusSilene-  SubgenusBehenantha

ErixonandOxelman2008.MolecularPhylogeneticsandEvolution


0.02

S. adscendens *

S. psammitis * S. williamsii

S. fabaria

S. macrodonta S. lydia

S. germana *

S. reinholdii

S. elisabethae

S. uniflora

S. foetida

S. samia

S. zawadskii

S. akinfievii

S. aegaea

S. quadriloba S. sordida

S. pentelica

S. virginica

S. stockenii *

S. nivea

S. multinervia

S. ammophila

S. dioica

S. nana

S. cryptoneura

S. turkestanica

S. menziesii

S. atocion

S. littorea *

S. conoidea

S. heuffelii S. marizii

S. diclinis

S. tatarinowii S. insularis

S. dichotoma

S. laconica

S. sedoides2

S. thysanodes

S. noctiflora

S. conica

S. viscosa

S. integripetala

S. fedtschenkoana

S. vulgaris

S. baccifera

S. sedoides

S. latifolia

S. haussknechtii

S. pendula

S. viscidula

S. littorea S. cambessedesii *

1

0.92

1

1

1

1

0.99

0.94

1

0.98

0.99

1

1

1

1

1

1

0.94

1

SileneSect.Psammophilae

Casimiro-Soriguer2015

S. stockenii

S. littorea S. adscendens S. cambessedesii

S. psammitis

•  FivespeciesendemictotheIberianPeninsulaandBalearicIslands

•  Self-compatible,mainlypollinatedbyinsects•  Distinctedaphicaffinities


Whataretheaimsofthisstudy?

ToexplainthephylogeographicpatternsofSileneSect.Psammophilae


Whataretheaimsofthisstudy?

ToexplainthephylogeographicpatternsofSileneSect.Psammophilae


How?Byusingagenomeskimmingapproach

Straubetal.2012.AmericanJournalofBotany

DNA

REFERENCE-GUIDEDASSEMBLY

DENOVOASSEMBLY(WITHOUTREFERENCE)

FragmentationAmplification

Reference

DNA

DNA


Sloanetal.2012.GenomeBiologyandEvolution


Whynextgenerationsequencing(NGS)?



Parksetal.2009.BMCBiology

Phylogeneticresolution:

A) 2lociB)Wholechloroplast(increaseof∼60xphylogeneticinformativecharacters)



Parksetal.2009.BMCBiology

Phylogeneticresolution:

A) 2lociB)Wholechloroplast(increaseof∼60xphylogeneticinformativecharacters)

Whittalletal.2010MolecularEcology

LowlevelofdivergenceinPinustorreyana(1polymorphism⁄∼21kb)


Librarypreparation Sequencing Dataanalysis


Librarypreparationandsequencingdetails:

• DNAextractions:5individualsperpopulation• NexteraKitw/barcodes• SequencedonHiSeq@USC’sEpigenomeCenter(CA)

o  Single-end:4sampleso  Paired-end:22samples

Librarypreparation Sequencing Dataanalysis


1.  Phylogeneticreconstruction2.  Genetree-Speciestreereconciliation3.  Populationgeneticanalyses4.  SimilarityofS.cambessedesiiplants

frommainlandandBalearicpopulations

5.  SNPsvalidation






•  Trimofrawdata





•  Trimofrawdata•  Reference-guidedassembly





•  Trimofrawdata•  Reference-guidedassembly•  Extractionofconsensussequence






GTCAATGACCCGGCAACGACT






•  Alignment(MAFFT)







•  Alignment(MAFFT)•  Phylogeneticreconstruction


SequencingresultsandcpDNA,mtDNAandnrDNAassemblies’information:

Averagesequencingdepth(median)

Assemblylength

Recoveredsequence

Variablesites

cpDNA 359.91X(153.95X) 154,199bp ∼99% 6,322

mtDNA* 33.4X(10.5X) 254,270bp ∼38% -

nrDNA 2,765.2X(2,046.7X) 6,415bp 100% 257



Wuetal.2015.PNAS

SequencingresultsandcpDNA,mtDNAandnrDNAassemblies’information:

*Weselectedaconcatenationof5,648bpfromsixmitochondrialgenes(130variablesites)

Averagesequencingdepth(median)

Assemblylength

Recoveredsequence

Variablesites

cpDNA 359.91X(153.95X) 154,199bp ∼99% 6,322

mtDNA* 33.4X(10.5X) 254,270bp ∼38% -

nrDNA 2,765.2X(2,046.7X) 6,415bp 100% 257


Phylogeneticresults:


cpDNA:


cpDNA:

-  MLandBayesiananalysisshowedmostlycongruenttopologies


cpDNA:


-  17of27(63%)nodeswithBS>70


cpDNA:


-  17of27(63%)nodeswithBS>70-  Veryfewgeographicpatterns:

-  BalearicIslands-  ThreepopulationsfromCadiz


mtDNA:


mtDNA:


-  4of27(15%)nodeswithBS>70-  ThemtDNA-basedtreesdidnotshow

anyclearphylogeographicpattern,exceptforpopulationsfromCadiz



nrDNA:


-  4of27(15%)nodeswithBS>70-  Veryfewgeographicpatterns:

-  FivepopulationsfromAlmeria-  ThreepopulationsfromCadiz+

onepopulationfromMalaga


nrDNA:

2.34Ma

1.33Ma


BAPS:Populationstructuring


ThereisjustonegeneticclusterforthecpDNAdata(K1=-37,598.87,K2=−39,489.59,K3=−39,576.70,K4=−40,069.07)



DiscriminantAnalysisofPrincipalComponents(DAPC)

Balearicpopulations

Speciesaspriors:




DiscriminantAnalysisofPrincipalComponents(DAPC)

Balearicpopulations

Speciesaspriors: Locationaspriors:




DAPCresultswhenusingspeciesaspriors:



PartialManteltest:correlationbetweengeneticvariationandgeographicdistance

•  Allpopulations:P<0.001,r=0.35•  Thecorrelationwasnotsignificant

whenanalyseswereconductedseparately



•  Allpopulations:P<0.001,r=0.35•  Thecorrelationwasnotsignificant

whenanalyseswereconductedseparately

•  Allpopulations:FST=0.23;P<0.001.Mostofthegeneticvariationwasconcentratedwithinspecies(77.3%)

•  NogeneticdifferentiationneitheramongmainlandspeciesnoramongBalearicislands



AMOVA:geneticdifferentiationamongspecies

Sangersequencingoffourregions:


Sangersequencingoffourregions:•  trnK:814bp•  ycf1:871bp•  atp1:753bp•  ITS:714bp




•  99.2%ofsequenceswereidentical(theremaining0.8%correspondtoambiguitiesthatwerepartiallyvalidated)




•  MostSNPswerealsopresentinsomepopulationsfromtheSandSEoftheIberianPeninsula.However:




•  MostSNPswerealsopresentinsomepopulationsfromtheSandSEoftheIberianPeninsula.However:o  ITS1/ITS2:2SNPspresentonlyin

Balearicpopulationso  ycf1:1SNPwasexclusivelyfoundin

allS.cambessedesiisamples

ycf1

ITS

•  Verylownumberofinternalbrancheswithstrongsupport


trnK

atp1

ycf1

ITS

•  Verylownumberofinternalbrancheswithstrongsupport

•  Onlyintheycf1-basedtree,theAlmenarapopulationformedaclusterwithBalearicpopulations,butwithbutnodewasonlymoderatelysupported


trnK

atp1

SangersequencingwasperformedinordertovalidateputativeSNPs:

vs



•  PCRofmanysamplesaspossiblefromallpopulations

vs



•  PCRofmanysamplesaspossiblefromallpopulations•  Amplificationandsequencingofspecificregionswithhighnumberof

phylogeneticallyinformativesiteso  trnK:814bp–60SNPso  atp1:753bp–125SNPs

vs


Wesequenced16,648bp(trnK)and15,813bp(atp1)bySangerSeq:

trnK=99.1%confirmed•  3SNPsconfirmedat100%•  47SNPsconfirmedat50%•  10SNPsnotconfirmed

atp1=98.1%confirmed•  11SNPsconfirmedat100%•  98SNPsconfirmedat50%•  16SNPsnotconfirmed


Wesequenced16,648bp(trnK)and15,813bp(atp1)bySangerSeq:

NGS data

NGS data

Sanger-F

Sanger-F

Sanger-R

Sanger-R

T

A

trnK=99.1%confirmed•  3SNPsconfirmedat100%•  47SNPsconfirmedat50%•  10SNPsnotconfirmed

atp1=98.1%confirmed•  11SNPsconfirmedat100%•  98SNPsconfirmedat50%•  16SNPsnotconfirmed


Mainconclusions



①  PhylogeneticrelationshipsandhybridizationinIberianSilene

•  Thereisanincongruencebetweentaxonomyandphylogeographicresults.Infact,geographywasabetterpredictorofrelatednessthaneithermorphology-basedspeciesboundariesoredaphicpreferences




•  IncongruenceamonggenomesandtheintricaterelationshipsamongspeciessuggesthybridizationisactinginSileneSect.Psammophilae





•  Geographicproximityofmorphologicallydistinctspeciesincreasestheprobabilityofinterspecifichybridizationorintrogression



stockeniiadscendens

littoreapsammitis≠




•  Geographicproximityofmorphologicallydistinctspeciesincreasestheprobabilityofinterspecifichybridizationorintrogression

•  ArespeciesofSileneSect.Psammophilaeasinglespecies?




②  GeographicisolationpromotesgeneticdifferentiationofBalearicpopulations


•  ModerategeneticdifferentiationofS.cambessedesiiwithrespecttomainlandspecies



•  ModerategeneticdifferentiationofS.cambessedesiiwithrespecttomainlandspecies

•  ThisgeneticdifferentiationisprobablyduetofoundereffectsfollowingthecolonizationoftheBalearicIslandsbyasmallnumberofindividualsand/orsubsequentbottlenecks


Colonizationevent


•  AbsenceofgeneticdifferentiationbetweenpopulationsfromIbizaandFormentera





•  TheoriginofS.cambessedesiicouldbeexplainedbyalongdistantdispersaleventfrommainlandtotheBalearicislands




•  TheoriginofS.cambessedesiicouldbeexplainedbyalongdistantdispersaleventfrommainlandtotheBalearicislands

•  GenetictracesobservedinDNAsequencesofAlmenaraindividualsaretheresultofatleastonedispersaleventfromtheislandsbacktothemainland,followedbyintrogressionwithmainlandpopulations


③  Ontherelativeaccuracyofreference-guidedassemblyofeachgenome


•  Genomeskimmingisanefficientapproachtogeneratethemajorityofthechloroplastgenome,nrDNAcistron,andsomemitochondrialcodingsequences


X




•  Largerfragmentsofthemitochondrialgenome,includingintronsandintergenicregions,willbenecessaryforfurtherbiogeographicanalyses

X




•  Largerfragmentsofthemitochondrialgenome,includingintronsandintergenicregions,willbenecessaryforfurtherbiogeographicanalyses

•  InSilene,lineagesfromhybridizationhavephylogenetichistoriesthatmaybecannotbeeasilysolved,evenwithhighamountofdatafromNGS

X

CGL2009-08257,CGL2012-37646andCGL2015-63827-P

Acknowledgements


Thankyouforyourhelp:•  JustenB.Whittall–SantaClaraUniversity,SantaClara,CA,USA)•  InésCasimiro-Soriguer–UniversidadPablodeOlavide,Seville,Spain)•  EduardoNarbona–UniversidadPablodeOlavide,Seville,Spain•  MªLuisaBuide–UniversidadPablodeOlavide,Seville,Spain•  DarynBaker–SantaClaraUniversity,SantaClara,CA,USA)•  MarcialEscudero–UniversidaddeSevilla,Seville,Spain)•  Thunder–SantaClaraUniversity,SantaClara,CA,USA)•  ÁgataCardoso…andthankyouforyourattention!

i simposio anual de botánica española

Documents