Accepted Article View metadata,citationandsimilarpapersatcore.ac.uk Running head: DIVERSIFICATIONWITHIN-SPECIES IN VOLCANIC ISLANDS Keywords: ancestral areas, extinction, oceanic islands,paleo-islands, volcanic refugia. 3 2 1 M. The casethe of widespread endemic as refugia andsources Paleo-islands of ge Article type : Original Article Accepted Date : 17-Jun-2015 Revised: 16-Jun-2015 Date Received: Date 05-Mar-2015 This article This protectedis by Allcopyright. rights reserved. 10.1111/mec.13282doi: lead to differences betweenversionthis and the Versionof Record. Please cite this article as through thebeen typesetting,copyediting, pa This article hasbeen accepted for publicationand undergone fullpeer review but has not † Tel: +34914203017; Fax: M. University College London, WC1E6BT London, United Kingdom Institut Botànic deBarcelona(IBB-CSIC-ICUB), 08038Barcelona, Spain Real 28014Madrid, JardínSpain Botánico (RJB-CSIC),

These senior authors contributed equally contributed authors senior These Correspondencetobe sent to: RealJardínBotánico (RJB-CSIC), 28014 Madrid, Spain;

A M LARCÓN AIRAL *1 2 , I. S †

ANMARTÍN +34914200157; e-mail: [email protected] 1 † , J.

J.

A Canarina canariensis LDASORO

netic volcanicdiversity within archipelagos: gination and process, which proofreading may 2 , V. CULSHAW (Campanulaceae). 1 , I.

MANOLOPOULOU

3 AND

brought toyouby provided byUCLDiscovery CORE Accepted Article genetic variation, with intra-island levels of levels intra-island with variation, genetic structuring in role lesser contrast,clustering.tohavein appearplayed a Oceanic barriers, or islands us nearbythe Tenerife within thewere also as ancestralinferred of migrant location areas towards alleles other disturbed areas these andRoquedelConde; of Anaga, Teno "paleo-islands" stable geologically in the divides Canarian populations into eastern andwe and axis as itshas central Tenerife that Ma 0.8 around divergence a first with structure, laurel the forestendemic of Canary astrong Islands. found geographicWe population in variation genetic (cpDNAchloroplast haplotypes) andthenucle diversification within-island of ingeological time. study This employed data thefrom studies haveeffectfew focused thatvolcanic the catastrophic have events on onpatterns had as some of the factorsmain driving diversification within volcanic archipelagos. However, A This article This protectedis by Allcopyright. rights reserved. Gille 1984; &Templeton (Carson diversification the of ecological role to examine adaptations driversgeographicversus isolationas of heterogeneity buffered and climates, oceanic is INTRODUCTION sources of genetic diversityto other areaswithin the archipelago. paleo-islandsTenerife, the with refugia acting as against extinction, andascradles and million years 3.5 islands played ago genetic rolesingenerating key withinboundaries a eruptions volcanic that argue We islands. BSTRACT Geographical isolation by barriers Geographical a oceanic Due to their small size, small geographic discrete substantial boundaries, to their environmentalDue

Canarina canariensis , an iconic plant species associated with the ing aBayesianapproach tophylogeographic nd landslides after the paleo- merging the the after nd landslides of genetic diversity larger than thosebetween- geneticdiversity largerthan ar (AFLPs) genomes to examine patterns of examine genomes to ar (AFLPs) lands represent excellent natural laboratories spie 2004). Patterns ofspie Patterns are 2004). variation genetic stern stern Geneticclades. diversity wasgreatest nd climatic stability has been postulated Accepted Article eruptions, landslides,eruptions, merging paleo-isla of is usually by followed a large numberofde systems (Gillespie & However,Claguethe development 2009). of birth and volcanic islands insular atpromotingin thantopographic generally isolation barriers effective are more al. andtheypopulations; ultimately drive differen al. for plant diversity plant 40%ofCanarianfor &Quézel vascular plants 1997): (Médail approximately al. biodiversitycradles ofmultiple events in-situ where diversification have place taken (Juan continental lineages that have survived theclimatic changes of Latethe Cenozoic, but also as connected to the mainland. The Canary Islandshave long been considered refugia for al. (Carracedoplume mantle of chronological toa pattern due emergence the in last islandsformed The were 1855). Lyell inter-population differentiation in comparison incomparison differentiation inter-population is flora and the 2001) (Santos-Guerra endemics are located 110km the from of north-westerncoast geological highly located history Their Africa. and (allopatric speciation, extirpations and recolonisations). temporal levels scales andwithin-islands)(between- over spatial different andalso different at diversification of patterns studying frameworkfor ideal an archipelagos represent volcanic within-islands (Bottin than islands, hierarchical be with expected to in This article This protectedis by Allcopyright. rights reserved. since the scientists early19 have biota interested endemic 1990; Gillespie & Roderick 2002, 2014; 2002, 2014; Macías-Hernández &Roderick 1990;Gillespie 2011). These events habitat promote fragmentation; thesubsequent genetic isolation of 2000;Francisco-Ortega 2015) (Figure They 1a). areseparated by deep oceanic trenches and have never been The Canary Islands are a volcanic archipelago formed by a chain of seven islands, et al. et al. 2005; García-Verdugo 2005; 2000).In theseaddition, islands are regarded as ahotspot nds, etc (Carracedo 1994; Fernández-Palacios1994; (Carracedo nds, etc between-island genetic differentiation strongerdifferentiation between-island genetic tiation and speciation within-islands (Carson to archipelagos (Francisco-Ortega other structive events in the form ofsecondary 21 million years (Ma) with aneasttowest with years (Ma) 21 million generally characterised by generally characterised of high levels et al. th century 1814; Humboldt (von 2010), because2010), oceanic barriers et al. et et al. 2013). Therefore, 1998; Zaczek 1998; et al et et et et et . Accepted Article 2008; Vitales 2008; Vitales position inposition the lineage's phylogeny. position This arguedagainst these theidea of massifs as Trusty orclimate- human-induced extinction (Harter mighthaveand variety micro-climates that of stability, the (volcanic) three paleo-islands of Tenerife topographic exhibit also complexity a sharethey severalendemicrestricted and species Table (see S1).Besidestheir geological preservedforests, laurel consideredancient,as an flora unique restricted to Macaronesia, and CanarianArchipelagothe (Reyes-Betancourt highest phy the home to are presently – Pliocene the since stable geologically remained has also – which LaGomera with together Tenerife, of 1999; Guillou (Ancochea Ma 0.13 tobeactivecontinued until Tenerife Fig. 1b). paleo-islands The remained thereafter relatively stable, whereas thecentral part of islandsthese 3.5 Ma and gave riseto volcanic central episodes Eruptive Ma) inthe northeast. Anagafused and (4.9-3.9 northwest, thein (6.2-5.6 Ma) southwest, Teno the Ma) in (11.9-8.9 RoqueMiocene: Conde Late del the dating asthreeseparate Itexisted at backto islands. islands, of all the first history complex within-island diversification (Juan secondaryavalanches and manyof of eruptions diversification, particularly at particularly at diversification, sources ofgenetic diversity(García-Verdugo de Paz&Caujapé-Castells2000; 2013). Recently, This article This protectedis by Allcopyright. rights reserved. et al In reviewing the role of Tenerife paleo-islands as refugia across several plant lineages,plant several across refugia as paleo-islands Tenerife of role reviewing the In Most studies on Most the Canarian florahave focused onthepattern of inter-island . (2005) found that species endemic to the paleo-islands aderived occupied species tothe often endemic that (2005) found . et al. et al . 2004; Carracedo 2014; see Fig. 1b). Intesee2004; 2014; Fig.1b). . Carracedo 2014a, topographies b).Nevertheless, complex and the of long histories the species levelspecies (Francisco-Ortega the et al. the present (Ancochea island ofTenerife 2000; Brown et al et al. et al favoured their role as micro-refugia against micro-refugia as role their favoured logenetic diversity and endemic richness diversity andendemic of logenetic . 2015; Patiño these islands are likely to have favoured islandsto likely are these . 2015). 2008). These areas also harbour the best- the 2008).alsoThese areas harbour they have been proposed as reservoirs and et al et al et restingly, the three paleo-islandstherestingly, three . 2006). Tenerife has themost Tenerife has 2006). . . 1990, 1999; Cantagrel . 1990, et al . 2015). et al. et 2002; Kim 2002; et al . 1990; et al et al et . . Accepted Article 2013; Puppo infra-species level (Juan divergence events between taxa these to endemic paleo-islands,thespecies either at the or at refugia.ancient However studies, other especi This article This protectedis by Allcopyright. rights reserved. (Gómez years paleo-islands million ago(Ancochea precursor 3.5 within- butbetween-islands. within- areasas reservoi thathave acted undisturbed or endemics" areespeciallyMIEs) relevant geological island that Species the history. presentare island inmultipleislands ("multiple of lineageestimates patterns which to times, divergence necessary within-island is torelate variation Canariangenetic for widespread endemics, and noneof them have provided a wide, continental-scale disjunction of 7000 km spanning across the Sahara. Mairal Sahara. the 7000 kmspanning across of continental-scale disjunction wide, a onnearbyclimbs pollinated by itis plants; Afromontane forests of Eastern Africa, Eastern of forests Afromontane 2015). In addition to Campanulaceae (Olesen 2008). Genus fleshy2011) andits dispersed are (Valido fruits byvertebrates and ElHierro. La Palma Gomera, La Tenerife, Canaria, Gran Islands: Canary western and the central in occurs presently It in cleared that growsmostly herbaceous plant Canary Islands, elected asits “national multiple island of endemic, a Here, we study patterns geneticof diversit et al. et al 2003;García-Verdugo Canarina . 2014;) that are contemporaneous orpredate the age merging of the of C. canariensis et al et al belongs to tribe Platycodoneae, a basal group family basal within a Platycodoneae, to tribe belongs Canarina canariensisCanarina . 2012; Mansion . 1996, 2000; Dimitrov 2000; 1996, . C. canariensis , the, genus comprises twoother species inhabiting the et al. C.eminii flower” (Kunkel 1991),is a (2n= diploid 34) generalist birds (Rodríg birds generalist 2010) have onpatternsfocused within-island of areas surrounding the endemic laurisilva forest. laurisilva endemic the surrounding areas rs and sources of genetic diversity not only of genetic only diversityrs andsources not et al. to understand the role understand the to and ally in animals, haveagesanimals, ally in reported for y demographic and spatialthe and history is a herbaceous plant that occasionallyplant that a herbaceous is (L.) Vatke. This "flagship" species of the the of species "flagship" This Vatke. (L.) 2012;Wang C. abyssinica et al et alet . 2008;Macías-Hernández et al. . 1990). Few plant studies , beingan of this , example et al. uez-Rodríguez Valido & 2003, Rodríguez 2003, 2013; Mairal of paleo-islands as et al. et al. et al et al. .

Accepted Article distribution of genetic within variation distribution thegeographic history Our geological thearchipelago. to:aims i)determine of were main tocandidate evaluate patterns of within-island diversification in relation tothe recent (Mairal Tenerife within vicariance Pleistocene) Mainvolved east-west (Late and isdatedonly 1 divergence an at around population event of population: the earliest African North closer and geographically canariensis distribution.widespread of colonization the The Canary Islandsby theancestors of climate-drivenvicariance and resultingfragmentation the extinction in ofanancient spatial disjunctionwas and resultofThis extraordinary explained temporal asthe Ma). the phylogeny(2015) recently reconstructed spatiotemporal evolution of and This article This protectedis by Allcopyright. rights reserved. unobserved events such as local population grow ofislands Tenerife as refugiaof genetic diversity, relictualboth andrecent. paleo- the of putativeiv) inter-island role the events, migration reconstruct examine and togeological events related be may that processes diversification and that inferred evaluation of alternative phylogeographic scenarios of alternativeevaluation phylogeographic (Bloomquist associated touncertainty network inference, the mutation of differingevents among haplotypes. al et Templeton (SP, Parsimony Statistical using inferred present.are often is These networks flow gene especially tree, branching when reticulatea than between picturepools clearer relationships genetic of the a provide . 2000),which allows estimationthehapl of This age and the presencethe This age and of Haplotype networks areHaplotype networks commonly usedin apparently much later, occurred in the Pleistocene, probablya nowextinct from C. canariensis divergeditsAfricanrelatives from ofMiocene the the (c.at end 7 et al Canarina canariensis et al . 2015). Canarina canariensis . 1992) implemented in the software TCS (Clement TCS software the in 1992) implemented . However, this fails method theto incorporate otype whilenetwork minimising thenumber and therefore does not allow for statistical th or past extinction of haplotypes may population-level studiesbecause they in several islands makes it an ideal an it makes islands several in , ii) find evidence of evidence extinction , ii) find et al. , iii) find ancestral areas iii)findancestral 2010). Moreover, 2010). Canarina . They C. Accepted Article 2015). We 432generated sequences: new and Ecological Clustering (BPEC, Manolopoulou and EcologicalClustering (BPEC, those obtained amodel-based, Bayesianfrom statistical method, Bayesian Phylogeographic inference in parsimony-basedmislead methods This article This protectedis by Allcopyright. rights reserved. sampling (Caujapé-Castells 2010),samples were biased inflationin geneto descriptors due To ofsamples 10 reduce perpopulation. minimum 2012: 4 in 2 inLG,GC, 8 and in 2 TF, LP possible,1 inEH.Where in we collected a Seventeen populations of (LG), La with Gomera Palma (LP) La ElHierroin (EH). (TF), and comparison Tenerife Population sampling and DNA extraction have proven to be useful for intraspecific analyses intraspecific usefulto be proven population for have structure of (Mairal Chloroplast DNA sequencing expeditions. the field leaves 20-25 mg ofsilica-gelfrom dried obtaine was gel extracted silica USA), California, Inc., (QIAGEN Kit Mini Plant DNeasy using the whole occupancythe areaof each population. (144 sequences) and M useto this formethod island phylogeography. study first this the is our knowledge, To 2012). & Emerson (Manolopoulou model mutation tree haplotype for probabilities posterior ATERIALS AND We selectedWe three cpDNA intergenic spacers regions for sequencing; these markers Canarina canariensis M ETHODS pet B C. canariensis

1365 has a significantly greater onGran Canariapresence has (GC)and asignificantly greater – pet D 738 (144sequences). PCR and sequencingprotocols were sampled in several fieldtrips between 2009 and several fieldtrips between sampled in were rpl networks underacoalescent-based migration- 32- DNA from 160 individuals and preserved in preservedindividuals 160 and DNA from . . Here, we compare results TCS from with d from the fresh plant trn collected individualsfrom scattered across et al. et L UAG 2011), allowswhich 2011), estimating the

(144 sequences), (144 sequences), tissue collectedfrom trn S GCU – trn et al G UCC .

Accepted Article parsimony in order to reduce the number of candidate trees to a manageable set. The BPEC set. amanageable candidate to trees of number the to reduce parsimony inorder population clusters distinct geographical ancestral locations.and Like on TCS, BPECrelies toidentify prep.) genetically Hille, in inthe(Manolopoulou Rpackage & implemented data. haplotypes was calculated 95%confidence with al.et parsimony (Templeton statistical algorithm sequenced regions. Genealogical relationships thethree andexamining populations different analysis, by individuals from using 6-12 each population usingDnaSP (version was used to determine the best fitting model of sequence evolution of each data partition. partition. data each of evolution sequence of model fitting best the determine to used was toperform (Nylander, MrModeltest phylogenetic 2004) software v.2.2 partitions analyses. describedrules inKelchner We(2000). analysed the three sequenced regions as three data Sequences were checked andmanually adjustedwhere necessary by following alignment region for were each Sequences Pro5.4.softwarethe Geneious in implemented analysesHaplotype accession numbers and full references are detailedinTable S2. Mairal of those followed This article This protectedis by Allcopyright. rights reserved. number of haplotypes H(n), H(n), haplotypes of number diversity nucleotide diversity haplotype (Hd), heterozygosity heterozygosity 2000). number The ofmutational steps resulting from single substitutions among The relationshipsThe amongst were lineages investigated through haplotype network The BPEC method (Manolopoulou BPEC The method Summary statistics for within-population gene within-population for statistics Summary θ , G ST and thenumber of migrants generation per (Nm) were estimated for et al . 2015.. The sources of the material examined, the GenBank 5.10;Librado &Rozas 2009). usingaligned MAFFT 6.814b(Katoh et al. et al. 4. Ltd., (Biomatters Auckland, New Zealand). viatheamonghaplotypes were inferred 2011; Manolopoulou &Emerson 2012) was limits, gaps were represented as as represented missing were gaps limits, 1992) implemented in TCS 1.21 (Clement inTCS 1.21 1992) implemented tic as: diversity werecalculated the π et al et , nucleotide . 2002), Accepted Article details see details see into exploration,wedividedthe dataset further upon ahaplotype tree, and used to ancestral estimate locations migration for events. As a phyloge The 5–cluster model. converging a to migrations onthehaving anyabove of and similarly, tree, inferred number 4 noeffect runwere chains MCMC ds>3 with stable, were resultsThe iterations. million three for of sequences of distance ds}{1,…, nucleotides will consideredbe as the “missing Twopath”. missing anunobservedhave intermediate seque isincreased, value is parsimonythe assumption ds As time. the greater computation much modelsbutrequire more include values Larger of migration 1). + hence(MaxMig ofevents number clusters number and maximum the thefor bound upper set the to user the allows MaxMig the software). as in (denoted “ds” software) and parameterthe parsimony relaxation the in (denotedas migrations number “MaxMig” of maximum inputs: user-defined the main assumes the that migration andthe popula rate method The corresponding clusters. events and migration of number trees, probability haplotype) migrates to a new geographical cluster. MCMC simultaneously estimates high occurwere assumed to parent (with orwithout amutation its whenahaplotype from by Sanmartín by Sanmartín explored throughare Markov Monte chain Ca to theled observed population substructure. Different scenarios of trees and migration events (Knowles 2008). Each possible defines settreepossible a that of may eventsmigration have uncertainty inhaplotypefor which relationships, one is of main the criticisms of TCS fu in a withhigh posterior probability trees unlikemethod, Standard Parsimony, afits prior over all possible trees in orderto identify This article This protectedis by Allcopyright. rights reserved. coalescent dating et al . (2008) for estimating rates of inter-island dispersal.events Migration inter-island rates (2008) of estimating for . section in results). Haplotypes sampled from Roque del results). Conde in Haplotypes Roquedel sampled from section llymodel-based framework, thus accommodating ographic clustering obtained was superimposed was clustering obtained ographic rlo (MCMC), similar to the method proposed the method to similar rlo (MCMC), two groups (eastern and western cladeswestern twogroups –for (eastern and tion constant. growthare two BPEC requires nce (an unobserved mutation), then pair any (anunobservednce mutation), relaxed: if twoobserved sets ofsequences used to reconstruct th to used reconstruct e set of possibletreesof set e Accepted Article (eastern and(eastern western groups with without and Roque del Conde). thetwo locationfrom this groups. ran BPEC We analysis ofon each thesefour datasets separate analyses divergent; thus, were quite This article This protectedis by Allcopyright. rights reserved. the for distribution accommodate the in change ratemutation from species to populations, with uniforma S3 to see results from exploratory analyses to analyses from exploratory see results S3 to Factor comparisons usingpath the ucld.stdev reference to these settings. Two MCMC chains were run for 50 million generations, sampling generations, 50 million run for were chains Two MCMC these settings. to reference constant population tree usingprior, a secondary (Mairal age estimate a Rambaut We 2007). out first carried analysis “ thenode of root the to 0.327 Ma)calibrate (SD)= deviation standard Ma, =0.76 mean prior: (Mairal (Mairal datasetpopulation-level stochasticbirth- and a al higher-level wasdataset calibrated with fossil-derived secondary age estimates (see Mairal canariensis ( dataset population-level linked a of rate clock the wasused inform outgroup to taxa including datasetthreespecieswhich ahigher-level representatives of of all second analysis applyingthe“nested da presencepoint.(see and Results) calibration single a level of population out aWe carried age posterior estimates,ESS for due probably at the content information low to the gaveusanalysis very large95%HPD Density) (High credibility intervals Posterior and poor C. canariensis C. . 2015), while. 2015), the tree was unlinked toprior apply acoalescent constant sizemodelthe to Haplotype divergence times were were es times Haplotype divergence et al ; the substitution; the model was set GTR+G; to choice wasbased ofpriors onBayes ) under a ) under a . 2015). dataset”; this included all haplotypes detected in our sampling (N = 10). This This 10). = sampling (N haplotypesin our all dataset”; detected included this mixed model Yule-coalescent ucld.mean

The clock priorto log-normal settoanuncorrelated The clock was model (10 sampling method in BEAST (Baele sampling method BEAST in -4 -10 ting approach” described in ting in described Mairal approach” -1 ) and a default distribution) andadefault theexponential for were run in which we included andexcluded were runinwhichweincluded assess the reliability of our date estimates with ourdateestimates the reliability of assess timated intimated and(Drummond BEAST v.1.7 under a strict clock model underastrictclock anda coalescent death (Yule) prior to the species-level one (Ho (Ho et al et . 2005; Pokorny 2005; . et al. et al. 2012); see Table et al et al Canarina 2015; normal 2015; . 2011).The . . (2015),in . and 9 C. et Accepted Article Tajima´s Tajima´s expansion, – a neutrality test we out carried Fu1996) Li, 1993; & Li´s tests (Fu Fu and and were (including groups populations). approximateThree panmitic geneticallyless structured results), (see our dataset in dem performed we among populations of panmixia orlack structure Demographic history. ones reported here. theare and in first thanthe narrower 95%HPD and intervals values ESSlarger considerably The2009). Drummond resulting estimates age second this from analysis exhibited intervalscredible for nodal andages, visualisedwere in FigTree (Rambaut 1.3.1 and clade usingmaximum credibility tree v.1.6.1, meanTreeAnnotator with values and 95% sampled populations was (5million).discarded sample sizes (ESS effective that were values) astationary whether wasdistribution there was attained; amongstconvergence if chains; and parameters generation.every 1000 used (Rambaut v1.6 Tracer We This article This protectedis by Allcopyright. rights reserved. of structure genetic groups. discludedthe from ET-GC andTENO-GO groups. Overall weran the analyseson five th above, as described reasons same the p (including andLP-EH and La Gomera) Teno in populations (including in east Tenerife and used:BEAST ET-GC analyses networkand were bypreviously haplotype the recognised We used used threeWe different approaches toinfer the demographic processes shaping the usedStatistics todescribe demographic patterns may bebiased by astronggenetic D (Tajima, We group. test eachpopulation used DNAsp the 1989)– for program, C. canariensis . First of all, to test for evidence totestfor population First of of all, . e Roque del Conde population was included andConde population wasincluded Roquedel e Gran(including TENO-GO populations Canaria), > 200 for all parameters.all the >200for A 10%burn-inof ographic analyses insubsetted datasets that . Since wedetected stronggenetic structure Post-burn-in trees were into were aPost-burn-in trees summarised opulations in La Palma and El Hierro). For Hierro). El and La Palma in opulations et al. 2013) to verify: to verify: 2013) Accepted Article Extended Bayesian Skyline Plot (EBSP), implemented in BEAST, for each population group in BEAST, each population (EBSP),implemented for Bayesian Plot Skyline Extended AFLP fingerprinting intervals obtainedcredible thisanalysis. from (HPD) usingcalibrated anormalpriorwithmean age 95% High and Densityestimate Posterior were set identical to those described above the nestedfor dating analysis; was therootnode parametersother wasused,model whereasall astrict sampling1000 generations; everyclock ii): twoindependent chains and were run group i) from each groups. For 16population of the each for EBSPanalyses performed we ii) with theraggedness index ( was tested size model population undera constant thetheoretical distribution to distribution software v.3.0 (Excoffier ofnumber observed differences between the coalescent simulations. we distribution plottedthe Secondly, groupfor each mismatch using (Librado &Rozasversion 2009)and 5.0 assessed thesignificance with 10,000 of each test This article This protectedis by Allcopyright. rights reserved. 1- primers: were sample ca of per number thealleles and Reproducibility contamination. reproducibility and for test to sample blind asa duplicated comb 32primer island), usingin total per (one individuals enzymes five and restriction geographically combining fluorophores distant for Biosystems®). (Applied theTo select appropriate AFLPthe analysis (Vos El Hierro. La andonefrom protocols Palma from for two LaboratoryLa Gomera, molecular one from of from Tenerife, theislands: five all onepopulation Gran Canaria, covered from For the AFLP analysis we used a total of 97 atotalof used we analysis AFLP For the whichindividuals 10populations, from Eco RI 6-FAM -ACT/ et alet et al et HRag . 1995)were implemented using the AFLP plant mapping kit Mse . 2005). goodness-of-fitThe of theobserved mismatch I-CAA, 2- I-CAA, ) (Harpending Eco simultaneously 150 generations,for million inations. One sample from each individual was all pairs of sequences with ARLEQUINof all pairs the lculated by combinationsoflculated choosingthree RI primers, we primers, first out carried a pilotstudy et al VIC -AGG/ . (1994)). Thirdly,. i) thewe created Mse I-CTA, and 3- I-CTA, and Eco RI VIC - , Accepted Article 3’/5’AATTGGTACGCAGTCTAC-3’) (5’- and MseI GACGATGAGTCCTGAC-3’/5’- and linked to theprimers EcoRI phase, digestion samples of DNArestriction with the weredigested enzymes EcoRIandMseI5’-CTCGTAGACTGCGTACC- producedscattered bands, AFLP polymorphic and fragments.clear and profiles the In showed pairs primer These three USA). CA, homogeneously and high reproducibility bound of inprevious5% indicated reports (Bonin the critical below were rates These were estimated. rates the error and scored were profiles (Whitlock package1.4a R AFLPScore the loaded version into and replicbetween andunreliableloci (variation peaks shoulder binsto exclude of editingby manual followed binning wasemployed peak and fragments (Vekemans discarded because the majority thissizeof software. recorded were Peaks in100 500baseto ranges. pairs Shorter were fragments analyses data AFLP selective). PCR productschecked were on 1%agarose gels. in ligation a single reaction; (ii) and (iii) PCR consecutive amplifications (preselective and and restriction i) reactions,were: AFLP different three The ATCTCAGGACTCAT-3’). AGG/ This article This protectedis by Allcopyright. rights reserved. statistics such Nei´s as diversity gene (Hj) AFLPSURVThe v.1.0 software (Vekemans analysedresultingmatrix AFLP was presence/absence of selection using analyses.different a per population. Data were converted intobinary presence reliabilityData was assessed throughcomparison duplicates, of one from or twoindividuals Mse The resulting AFLP fragments were analysedAFLPusing fragments resulting The 3.7 GeneMapper the I-CTT and using the Genemapper v3.7 using Genemapper soft the and I-CTT et al . 2002). For each primer combination, an automated an sizedetection2002). . primercombination, Foreach class have ahigh chance beingof non-homologous , pairwise differentiation among pairwise differentiation , subpopulations et al . 2002) was used to estimate demographic ates). Peak height data were then exported et al et ware (Applied Biosystems, Foster City, Foster Biosystems, (Applied ware . 2004) . primer combination. each for ⁄ absence scores⁄ locus. for each et al et . 2008) and theAFLP 2008) and . The Accepted Article was determined by the method from Evanno from by method the was determined 100,000) wererun for burn-inof generations allele a (plus MCMC between groups.frequencies correlated 500,000 populations. within equilibrium conditionsAnalysesunder admixture were performed and approach assumes linkagemodel-based lociarethat in Hardy-Weinberg and equilibrium 2000; Falush calculate the F a non-uniform prior distribution (Zhivotovsky 1999) allelicusedAFLPSURV toestimate through of was also in frequencies employment method biology of reproductive onthe study previous based ona self-fertilisation, orpartial Weinberg equilibrium This under andMichigan, 1994). wasdone Lynch the assumptionsof either the Hardy- Bayesian clusteringv.2.3(Pritchard STRUCTURE implemented methods in distribution of genetic variance attributable to oceanic barriers. islands asge alternately, paleo-islands and (Excoffier software v.3.0 ahierarchicalsubdivision, analysis mole of amount of quantify differentiation andpopulation the genetic geographic attributable to using analysis useof Neighbour-Net through the visualised samples v.4.10 among relationships (HusonandBryant inSplitsTree 2006) were genetic1998).Next, (Rohlf v.2.1 in Ntsys (PCA) AnalysisCoordinates implemented Principal to subjecteda matrix the resulting and was constructed, distance similarity coefficient theDice’s using as matrix for all individuals similarity pairwise a AFLP dataset, the within individuals of clustering calculated. geographicgenetic locate groupsand To were (F This article This protectedis by Allcopyright. rights reserved. ST ) and the percentage of polymorphic fragments per population (P) (Nei and Li,1979; (P) (Nei and polymorphic fragments ) andthepercentage population per of et al ST parameter from which genetic distances between individuals, populationsindividuals, between genetic distances which from parameter . 2007) were used to assess the genetic structure of populations. This to of structure 2007)used genetic assess populations. . were the K values of 1-10, with 10 repetitions for each. 10repetitions value with TheKlikely 1-10, for of values most C. canariensis et al . 2005). Exploratory analyses were performed considering,performed 2005). Exploratory were analyses . (Rodríguez-RodríguezA Valido Bayesian2011). and cular variance was performed wasperformed using cular variance ARLEQUIN ographical units in order to investigate theographical inorder units investigate to the split decomposition decomposition tomethod. thesplit Finally, et al et . Ten permutations. thousand run to were . (2005), which is implemented in implemented is which . (2005), et al . Accepted Article and east Tenerife (ET). Haplotype Tenerife H9east (ET). wasrestricted (WT)to West andLG. Tenerife Haplotype The most observedfrequently haplotype was haplotypes dominated that andwere geographicallyclearly H1, delimited: H9 andH3. ha with Figure 2a) H10, haplotypes (H1to DNA plastid observedwe 10 different populations, 16 the sampledfrom 144 individuals sequences and2249 nucleotide which sites, of H3 was present in the H3 was present westernin the islandsof LP as F between of thepopulations, structure genetic onthe (measureddistance correlations genetic of substructure genetic especiallypopulations, in explored We 2012). (Earl STRUCTURE HARVESTER This article This protectedis by Allcopyright. rights reserved. haplotypes rarest (frequencies Haplotype networkHaplotype analysis and coalescent dating R withOnlybarriers support greater than96% were asconsidered significant. byexamined themean 1000 of bootstrapped distance matrices obtained using AFLPsurv. algorithm Monmonier´s in BARRIERv.2.2 (Manni canariensis addition, to identifygeographic possible locations asmajor genetic acting amongbarriers population. collected In each geographic the absolute distancesfor between coordinates the presence/absencegebased on the matrix; was used matrix genetic The distance 2.1. v. NTSYS in procedure implemented permutation ESULTS ST trn ) and spatial distances between) andspatial pairswerepopulations determined of usingthe Mantel S The GCU

pet – populations, we usinga Delaunaytriangulation barrierson computed trn B G 1365 UCC – pet of658 144of data consisted concatenated matrix final sites.The D 738 738 regionconsisted 937 of sites, ≤ 2%) were in the ruggedmost areas of Tenerife: the paleo- and EH. Populations with andEH.Populations thecommonest and threewere 0.6632.There of plotype diversity H1 (52.08%), which was dominant inH1 (52.08%), GC and ographicthe distancematrix was basedon 9 S4). werepolymorphic (Table Among the Tenerife. To test the effect of the spatial et al et other values of K to detect of further othervalues K todetect . 2004). The significance was rpl 32– trn L UAG o 64 sites, 654 of C. Accepted Article with La Gomera; and La Palma with El Hierro El with La Palma and LaGomera; with highlywere populations w connected Tenerife Tenerife. G Tenerife. areaspopulation were found in Gran Canaria and outside of the paleo-islands regions of diversity nucleotide H(d), diversity (Conde))Conde showed high the paleo-isla three the situatedin Populations H4) andEH (H6). H7).exclusive presented unique haplotype, LP small each: islands – one The western (LP-Lt H10 alleleallele exclusive H8and – Ten-Ep of islands Anaga (An-W, An-C, An-E– exclusive haplotypes H2 and (Ten-A, H5), Teno This article This protectedis by Allcopyright. rights reserved. Adeje massifs. massifs. Adeje some genetic inwestin particular theTenoand with cohesion with populations Tenerife, Tenerifeshowed east that in This isthe Anagathe exception only population of An-W. with lowest values Nm and found between situated populations were east Tenerife, west and of divergence andnumerous divergence mutations, separate hidden which the haplotype clusters. The geneticflow,gene high for allowed 8, we to equal migrations of number maximum the byfour mutationalsteps H1 from threeand from H9. Haplotypehaplotype H3, H10asintermediate. each side ofTenerife (East H1 andWestH9) were separated steps, by fivemutational with coalescent criteria onhaplotype frequency (Templeton to according haplotype root ancestral as the H1haplotypes,haplotype with dominant Summary statistics for within-population genetic diversity are diversityshown genetic are Summary statistics in 1.for within-population Table haplotype constructedwith network The BPEC results are summarised in Table 2. With mutational step Table 2. limit mutational With are summarised in results BPEC equal to10and ST andN m values between indicated cohesion islands: high genetic eastsome est the values for haplotypesof H(n), number haplotype π adncetd heterozygosity andnucleotide nds (Anaga (An-W), Teno (Ten-A) and Roque del and Roque (Ten-A) Teno (An-W), (Anaga nds shared with LG) and Roque del Conde (Conde –Conde del Roque LG) and with shared ith Gran Canaria populations; west Tenerife present in the westernpresent inthe islands, wasseparated TCS (Fig. 2b) shows a loop involving 2b)shows TCS (Fig. a three (Table S5). (Table G highest the Interestingly, et al . 1992). Dominant haplotypes at θ . The least diverse. Theleast ST

Accepted Article divergence eventamong with thecongruent groups by detected TCS andBPEC crown The age 4). (Figure or first most the An-W probable 2, S1). Figure (Table thefor Western clade; three andthe ofTen-A populations the Anaga clade, withfor Eastern Without probability. Conde, otherpaleo-island po highestpopulation Conde, theancestral withthe always area as this including recovered (pp =0.13), An-W=Conde and (pp 0.10) LP-Lt The subsetting analysis, (pp=0.086). migration and events ancestral within- for source likely The most 2). (Table probabilities mass posterior significant carried H8also H2, Haplotypes H1, ahigh uncertainty. carried 3b), and Fig. as missinginferred (H11– was node wereconsistent node root the of (Fig. haplotype 3b). ofLocation the ancestral populations eventsinference migration for and 3a).in Figure regions (contour (pp probabilities clustering posterior withhigh This article This protectedis by Allcopyright. rights reserved. in Table S6. andLa Gomera. Mean age values and95% HPD intervals for the BEAST analysis are shown older: (95% separa0.522 Ma HPD = 0.161-0.967), at inthe western clade that was dated while = 0.041-0.633), (95%HPD Ma 0.255 eastern western and clade.Thefirst divergence in event the easternclade was estimated 0.452-1.365), an into it divided haplotypes period; Mid-Pleistocene corresponding tothe The Bayesian chronogram of haplotypes showed haplotypes a chronogram of Bayesian divergenceThe pattern of geographical Canarina across exploratory analyses of the complete dataset. The root

The total number of haplotypes was10 missing ofhaplotypes plus one total number The haplotypes was estimated to be 0.878 Ma (95% HPD = between-islands were populations on:located

= theexistence 1) showed of clusters five pulations were areas:as ancestral recoveredpulations ting La Palma Teno from and El Palma tingHierro La Accepted Article were consistent withthewere consistent obtained results Nm), andshowed the same genetic cohesions between-islands andpaleo-islands. from GranCanaria from and Roque del Conde, which 97 572 locifrom and 10 individuals populations. For AFLP analyses we removed populations AFLP polymorphism,genetic diversityand structure. (results not shown). S2). EBSP each for of the individual populations also obtained a constant size population size (Figure population constant a indicated expansion. EBSP population recent of hypothesis null the were thus failedto reject notsignificantthe and mismatch distribution from derived theEastgroup, inobtained Conde of we second peak.small raggednessa The statistics consis thatwere distributions frequenciesof pairwise differencesmism the in ofvaluesEastwereTheonly thetest although these S7). theFu´sgroup significant (Table for Demographic analyses This article This protectedis by Allcopyright. rights reserved. (CTAB)the tostandardise method rigorous) fragments) and two in the Teno massif (Table 1). (Table 1). massifF Teno the two in and fragments) werefragments detected Anaga in the (specificallymassif – An-W population four inthe islandsasingle frag andEl LaHierro Gomera Tenerife. The largest numberof private fragments wasdetected in Tenerife (7 fragments). In Lain the (GO) Gomera population, byfollowed (Ten-A)population theTeno inwest

These results1. summarisedThese are Table in Thedataset, final afterscoring, comprised of values Negative recentindicated Fu'sandTajima's a population expansion, tent with an expansion model. When we include the populationWhen the include an we model. expansion with tent from thechloroplast markers (see above G

quality of extracted DNA.quality extracted Hj andPwerehigher of atch distribution analysis resulted in unimodal in analysisresulted atch distribution ment was detected. In Tenerife, fivement wasdetected. private InTenerife, ST had been extracted with a different (less different extracted with a been had values with the nuclear data (Table S8) nuclear (Table data the values with ST and Accepted Article intermediate populations. Kintermediate Klevels = 4 Kand = latter identified5 (the as K = 6, with 5 eastthe populations from thewest, but also detected the presence of admixture in the 460) number the optimal represented of Bayesian within groups geneticof 2( K clusters mostlikely the number that = STRUCTURE indicated (2005), This article This protectedis by Allcopyright. rights reserved. composition. Thesevariabilityand mixed groups are also congruent BARRIER withthe genetic high explainedto its betweendue analyses; is this differed OnlyLaanalyses. Gomera The and (K AMOVA STRUCTUREsublevels Tenerife. =4 with the K =5)wereconsistent more andaneven populations, La Gomera), and phenomena Teno (among admixture –ElHierro), An-W, LaPalma Anaga and massif S5b). Overall, resultsthe agenetic revealed stronginter-island Canaria (Gran structure – populations Tenerife using only (F clusters resultingadditional in populations, Guillot with cluster plusone clusters defined "ghost" islands and between-paleo-is and islands within-paleo within-islands and differentiation analysisA further considering islandsand pa observed that differentiation (24. within-islands be among found groups5 (Table3and 4and S9).By analysing island each separately, we the twowell separatedgroups, including the West grouppopulation An-W (Figure 5b). divergencessame between network analysis outlined the split The ii). and between i) position population, demonstrate which An-W an intermediate populations; the west iii) Tenerife ii) A PCA analysisA PCA threegroups differentiated Hierarchical AMOVA genetic to largestHierarchical showed of analyses the variation proportion et al . 2005) revealed structure. complex genetic a more in intermediatethese lands According (16.63%). theto method Evanno of also delimited also Fig. twogroups 2inFig.5a, K (inset = ig. 5a, Figure 5a, S5). ig. STRUCTUREThe analysis no individuals so wasignored, assigned see it 27%) is greaterbetween-islands27%) than is (3.62%). -islands loweris (11.30%) between-than leo-islands separateas showed units that complex island substructure complex within the of (Figure S4): i) east Tenerife populations; eastTenerife S4):i) (Figure C. canariensis , separating et al et Δ K= . Accepted Article betweenF pairwise 100% barriers showed values of except LP-EH boundariesrevealed(Fig. which andK K=2 results, major =5).Allfor 5a twoorfour This article This protectedis by Allcopyright. rights reserved. Paleo-islands as refugia asPaleo-islands againstvolcanic(catastrophic) extinctions D 1990; Gübitz geographicalamongare them isolation andextinction-recolonisation processes (Carson interconnected biotic factors(Stuessy physicalrespond toseveral and 2003; García-Verdugo geographical distribution of geneticdiversity in of distribution geographical our BARRIER analyses (Fig. 5a Kin = 5), considering paleo-islands the as separate reversedareas pattern the (TableTogether 3). with guanchica pattern has been found in a Canarianfew other endemics ( This 3). islands different (Table in located thosethan within Tenerife among populations variation genetic showed AFLP wasthat notablyhigherAMOVAdata on the analysis based amongisolation Barrowcl populations (Zink& (AFLPs nuclear 2)and Fig. (cpDNA, chloroplast Hierro Teno; to and GranCanaria toAnaga. The fact that these patterns sharedare between were islandsother genetically associatedto these lineages:two La Palma, ElLa Gomeraand in east. Anaga Populations inthe inthe west and Teno Tenerife: form that currently islands in variation genetic of levels ISCUSSION The strongThe structure genetic detected often and

et al Pinus canariensis . 2005; Macías-Hernández ST andgeographical distance thewith Mantelanalysis ( et al et . 2010; see review in . 2010; García-Verdugo C. canariensis ), but usually marked notas as here (< 10%:Gómez were detected between two ofthe paleo- ancient et al these results support thehypothesis thethat C. canariensis ough 2008). Furthermore, thehierarchical ough2008). Furthermore, , Fig. 5) markers supports a long history of markers supports along history of Fig. 5) , (96.8%). No linearrelationship(96.8%). was found . 2013). The strongest genetic structure andgeneticstructure strongest The . 2013). in has island endemics postulated been to is structured paleo- around the et al et al . 2014). Interestingly, . r =0.42, . 2014); foremost. 2014); Olea europaea p = 1). =1). et al et et al et . . Accepted Article (Juan Canarian plant and taxaanimal include sister lineages endemic to the Tenerife paleo-islands divergence of these two haplotypes in the BEAST tree (Fig. 4) can be traced back to theto be traced back BEAST(Fig.two the haplotypes4)these can in divergence of tree the while clades, respectively, eastern the within andwestern dominant frequent most or the hand, the other two most frequent haplotypes, H1 andH9, in the Conde population are also theOn isthis unlikely. 5), (Fig. populations split east/west among remaining marked the thegiven – thoughanalyses STRUCTURE lower the values in and Kadmixture genetic tohigher of analysislevels led wouldhave in our population this of that inclusion possible Given themixed composition of the chloroplast thethan oceanic barriers separatingtheislands (García-Verdugo structuringspeciesgeneticdifferentiation patterns in within more important of probably been islands – within example,barriers for associated those to catastrophicvolcanic have – events analyses, phylogeographic w asortof "islands that these ancient considered massifsislands, unitsand separate in as could be This article This protectedis by Allcopyright. rights reserved. for Tenerife within diversity genetic of levels decreased than rather increased origin. Itis possiblethus that including populationthis inthe AFLP analysis would have of recent than delCondeis ancient rather Roque for chloroplast the compartment in detected westward and eastward migration events thein BPEC analyses, suggests that the admixture (H7) population the that fact the haplotype and identifiedConde is as the sourceancestral of basalmost split in et al et Manygeneticstructure? within-island of level this caused might have Which Unfortunately, we we Unfortunately, could AFLPnot obtain for inRoquepopulation the data Conde.del . 2000; Báez C. canariensis C. et al et . 2001),. species especiallylaurel forest among Teno and from (0.8 Ma). This, together with the presence of a uniqueof a presence the with together This, Ma). (0.8 compartment in this population (Fig.2), inthis population compartment itis ithin-islands". Itindicates geological ithin-islands". that et al . 2014). C. canariensis . Accepted Article al ( the paleo-islands origin of the predates found between thefound and westerneasternclusters of divergence haplotype anddeeptemporal diversity levels similar monophyly, reciprocal The divergence east-west 3.5 Ma) in Tenerife found explain the ‘young’(< This might reconnection. or no low with distribution widespread more allopatric speciation by driven (vicariance) ca patches Alternatively,could it by beexplained byforest followed diversification. in-situ divergence dispersal explained events by might‘young’ east-west between be historical Ancochea paleo-islands Ma, (c.3.5 the ageof mergingpostdatingof the the between the basal westerneastern and lineages was dated as Ma 0.878 (Fig. 4)substantially, genetic variation has been long lasting. In long lasting. been has variation genetic paleo-islands role Tenerife contemporary acrosstaxa of the suggests instructuring that abaxoidesCalathus and/or postdates merg 2000), the species level(e.g., 4).Anaga (Table While insomecases, the divergence between these taxa is found theat This article This protectedis by Allcopyright. rights reserved. w driving diversification factors important stud Several populations. among differentiation associated species within flow (Boulesteix eventsvolcanic have affected north of the to summit Tenerife, the coast the extending from vicariant, Inthe last scenario. the allopatric that extended across the northern flank of the island (Moya of theisland flank thenorthern across extended that landslides (Fig. 1b).These events could have the fragmented laurelancient forest corridor . 2008); in others, it is observed within species (e.g., species within observed is it others, in 2008); . et al .

2012, Trechus ,

Emerson

2013; Carracedo 2014), Carracedo thee.g. GüimarandLa 2014), 2013; mega- Orotava , Contreras-Díaz Contreras-Díaz , et al. to this laurel forest (e.g., laurel forest to this ing of the paleo-islands ( the ing of 1999). The fact thatthe pattern ofis divergence not Micromeria ithin terrestrial Canarian organisms (Juan Canarianorganisms ithin terrestrial one million landslides one major years, and several million et al tastrophic volcanictastrophic events within apreviously Canarina Canarina . 2007; . 2007; ies have supporteddebris avalanches as C. canariensis , Puppo Pericallis

Tarentola delalandii canariensis , and seen in other taxa (Table 4). 4). (Table taxa other in seen and , et al. Eutrichopus C. canariensisC. et al et (Fig. in Tenerife 4), favour 2014; , . 2004), geneinterrupting

Jones the basal divergence thebasal Pholcus ,

Moya et al et ) andpromoting et al et , . 2014) and/or

Gübitz , . 1990). This 1990). .

et al Dimitrov Dimitrov . 2004; . et al et al. et .

Accepted Article 2000; Tzedakis 2000; Tzedakis the for areasandgenerally other in extinct be higher inareas that have acted as refugia for ofthe preservation genotypes that went hypothesiscentral-marginal (Eckert genetic the from Support for analysis. suggestion comes population this According tothe activity.volcanic bythat wereaffected of the island disappearedparts inother otherwise re areasthese have actedsuggests as could that This 1). Fig. (see geologically mid-Pliocene the Conde,stable remained del since Roque as recently as 0.13 Ma (Ancochea haplotypes (Posada & Krandall 2001). Additionally, haplotyperoot a is expected to have a recent onespresent for andmore long time, a derived rare thecommonest and are from Theoretical predictions of coalescent theorystates high frequency that haplotypes been have similarin a way tothe paleo-islands of Tenerife. 1), DNAnuclear suggesting acted might island (Table this have fragments polymorphic that since the Pliocene (Carracedo &Day, 2002), shows the highest Hj and percentage of extinction. Interestingly,induced La an Gomera, that hasisland geologically been quiescent asreservoirs acted massifs ancientgenetic of (Tab Tenerife paleo-islands of of the populations alleles,and endemic private fragments, largerand the by exhibited heterozygosity levels landslide (Ancochea in onefound the divergence within northern Tenerife (Thorpe (Thorpe Tenerife northern within divergence 2000; Brown This article This protectedis by Allcopyright. rights reserved. Whereas centralWhereas Tenerife wasalmost completely by covered untilevents catastrophic Further support for the supportfor extinction hypothesisFurther comes from the BPECanalysis. et al et al C. canariensis C. . 2006; Macías-Hernández . 2013; Gavin et al . 1990; Boulesteix and corresponds well with the time of theLa mega- time of Orotava the with well andcorresponds et al et al et al . 2014; Feliner 2014). The higher number of ancestral of higher number 2014). The 2014; Feliner . . 1999),. the three paleo-islands of Anaga, Teno, and . 2008), spatial structure and genetic diversity should et al et al. fugia, allowing populations survivalthe of that et al long-term persistence of persistence long-term diversity andas volcanicallyagainst refugia . 2013). le 1), are congruent with the idea that thesethat the idea with congruent are le 1), 1996; . 2013);others have reported a temporal

Moya et al. et 2004) that is similar to similar is 2004) that populations (Hewitt populations Accepted Article to other, disturbed regions (Gavin regions disturbed other, to environmentally stableareas act can as sources Have paleo-islands acted as sources of genetic diversity within and towards other islands? H8 are also associated with most netw probablerootofthe haplotype the H11 BPEC as missing, estimates extinct haplotype haplotypeuncertainty the inthe rooting. theto incorporate thisunderlying method of -allows the existence model migration (probability)but di finite a Parsimony, Statistical The that BPEC task. fact notprovide of asingle thehaplotypedoes estimate like network so accuratelybecoming identifying is prevalent, most challenging roothaplotype the a the extinctionpast However, of haplotypes canobscure inference, with the younger haplotypes haplotypehigher numberof inthe being rather than network, connections tothe tips. close This article This protectedis by Allcopyright. rights reserved. 2003; Sanmartín studies plant (Francisco-Ortega animal describedother and in been patterns withthe central Canaries as centres of dispersal events the within archipelago have dispersal event from LaPalma to El Hierro is inferredby the BPEC analysis (Fig. 3). Similar fourth and a 4), (Fig. tree BEAST by the supported is La Palma to Gomera Tenerife/La Anagaor from to Gran(Fig. Canaria 3a,b, areas ancestralof migration events islands, adjacent to other such as TenotoLafrom Gomera in identifying populations hypothesis, the this changes the climatic be responsiblefor might than events geological catastrophic rather althoughthis case archipelagos, volcanic in in spatio-temporal pattern of colonization comparable to The theoryThe of Pleistocene climate refugia (Hewitt 2000) states that historically et al . 2008; Puppo 2008; . high posterior probabilities. et al et al et . 2014). Paleo-islands . could have played role thesame . 2015; see also Mairal see . 2015; ork (Fig. 3b), althoughork (Fig. haplotypes 3b), H1,H2 and Table Athird 2). dispersalevent westfrom observed patterns. BPECobserved provides patterns. supportto of geneticdiversityof alleles exporting migrant the paleo-islands of Tenerife as the sourcethe as Tenerife of paleo-islands the stribution of haplotype trees-asthe ofhaplotype well as stribution Canarina , showing Tenerife as the as showing , Tenerife et al et al. ., 2015).Moreover, a 2002; Gómez et al. et

Accepted Article of of have summits,faci connecting attheir could "barrancos") as (locally ravines known of where anetwork Canaria, Gran of topography explaindi extinction might genetic present lowits (24eruption events;activity Rodríguez-GonzálezHolocene during the acatastrophic even after colonisation recent by a Figs3). (H1, 2, Low divers Tenerife haplotypic dispersal events –but also of the concentration of crossroad fora acting as the archipelago – within Tenerife positionof geographic central 2011), and Vitales of lineage center of dispersal events toadjacent inthe lastislands be can 1Ma, found inthe Canarian This article This protectedis by Allcopyright. rights reserved. forest gaps and edges of roads as dispersal corridors (Delgado (Delgado corridors dispersal as of roads edges and gaps forest populations are located (La Orotava Valley and Güimar Valley, respectively) Valley, andGüimar Valley located (La Orotava beenare have populations haplotype, which agrees the ideaof with a recent colonisation. The where areas these thesetw size; Fig.S2), effective population should taken Although result be this with caution (theEBSPanalysis supported a constant Te of east expansionin two populations population areas. analysesgeologically arecent Ourdisturbed unstable, indicated demographic of genetic acted diversity with have sources as to connectpopulations inthe highly altered laurel ofGranforest Canaria. Canarina The case ofThe case Gran Canaria is especially interesting. Ithaplotype same shares the with In addition todispersal In addition between-is events et al Gallotia galloti Cistus

.

canariensis 2014a), (0.33 (0.88-0.07) Ma; (0.33 Vargas(0.88-0.07) Guzmán 2010), & Cistus monspeliensis seeds by seeds (0.8-0-9 Ma; Cox Cox Ma; (0.8-0-9 Gallotia (0.93-0.20 Ma; Fernández-Mazuecos & Vargas Vargas & Fernández-Mazuecos Ma; (0.93-0.20 lizards (Rodríguez litated among Dispersal populations. geneflow et al o populations exhibited also a single cpDNA single exhibitedalso a o populations in Tenerife, exporting migrant alleles to other allelesto exporting migrant in Tenerife, ity (Hn and Hd inTable 1) could be explained t. The island was subject to intense volcanic wassubjectThe island tointense t. plantgenetic diversity inpaleo-islands,. the . 2010).This might be a consequence of the versity. Another possibility is related to the to related is possibility Another versity. lands, the paleo-islands of Tenerife might Tenerife of lands, paleo-islands the nerife close to Anaga (TF-Br and TF-Bj). and to Anaga(TF-Br close nerife et al. et al et 2007), might helped have . 2008),probably using Cheirolophus et al . 2009), so 2009), . (1 Ma; (1Ma; Accepted Article Rodríguez such dispersedfleshy byvertebrates as are fruits pollinatedbywhich is birds generalist (R transported via pollenor,transported by alternatively, the small size andhaploid nature of chloroplastthe someconnectivityis to extent lostin the cpDNA be explainednot cpDNA bythe beingmight canariensis ( pollinator main the of migration haveconfirmed techniques ringing patches(e.g. nectar-feedingforest birdsbetween by carrying by pollen of the explained be Admixture could 5a). (Fig. LaTeno and west-Anaga, Gomera between admixture and those areasother from supportedis by nuclear the genome, which shows genetic inevents populations to other eastern Tenerife and Gran Canaria 3, (Fig. Table2). our BPEC analyses, in found suggest which delalandii Tenerife migrations of the reptiles to explain fringe of coastal al et subjected to catastrophic volcanic even This article This protectedis by Allcopyright. rights reserved. as in (García-Verdugo islandendemics withinwidespread speciation preventing 2007; Padilla birds have been cited as vectors important the dispersalfor of fleshy fruits (Arevalo bysupports someamong gene seed driven populations flow In dispersal. Canarythe Islands, of some cpDNAdistribution 2a) haplotypes Fig. acrossand H9, H3archipelago the (H1, markerschloroplast (Avise 2000; Jakob & Blattner 2006).On the other hand, the widespread which imply genome, shortercoalescent times . 2000)have proposedthe existence ofacorri Canarina Ongoing genetic connectivity between the populations in the paleo-islands of Tenerife paleo-islands populationsin the of the genetic connectivity between Ongoing from Anaga to the west. These dispersal events could also explain the patterns the also explain dispersaleventscould These west. the to Anaga from et al. ) between Teno) between and Anaga; Alejandro González, comm.). pers. The fact thisthat . This fits with what is known on the reproductive biology of on isknown biology of This the reproductive withwhat fits . et al 2008). An additional factor to explain fr explain to factor 2008). additional An . 2012), latter andthe gene associated. flow has with frequent been ts (Fig. 1b). Some (Thorpe authors odríguez-Rodríguez &Valido 2011),whileits Anaga populations as sources of migration of sources Anagaas populations and less time to fix novel mutations novelforand lessto time fix mutations dor of suitable habitatdor of northern the along Gallotia equent dispersalequent between-islandsare lizards (Valido Gallotia galloti et al et al C. canariensis and . 1996; Gübitz. 1996; Phylloscopus Phylloscopus . 2014), such et al. Tarentola 2003, et al , . Accepted Article C distances separate the Canary Islands, they migh Canary they the separate distances Islands, (2010) & Schaefer Carine barriers. oceanic Tenepaleo-islands around the of structured are volcanic historical In events. be as strong as or st differentiation. within-island than Here, we show betw with barriers, aroundoceanic structured be canariensis flow (Macías-Hernández gene to barrier geographical as acting 1b), (Fig. Taganana of arc volcanic the and ravines andeast west forest ranges Anaga within have been explained bythe deepexistence of differentiation massifs.within these Forexample, found populations divergences between in 2007). of rugged nature The pa the Tenerife high within-islandsexplaining (Whittaker speciation and speciesrichness rates geneticis one novel diversity.Maximum of topographic of the complexity factors main th with agree 1) in (Table areasthese populations gene andnucleotide diversityand higherhigherNei's haplotype diversity Hj The exhibited by diversity. newor sources as genetic cradles might acted have of Tenerife paleo-islands the distance between-islands (Rijsdijk geographic decreased have might which Pleistocene, the during shifts sea-level eustatic the This article This protectedis by Allcopyright. rights reserved. ONCLUSIONS

Traditionally, the distribution of genetic distribution of the di Traditionally, Finally, in addition to Finally, exporting alleles migrant to other islands disturbed and areas, .

ronger thanthoseobserved between-islands when they are associated with et al. Canarina canariensis 2013). These 2013). divergences were also detected in et al . 2014). argued that although relatively short oceanic although short relatively argued that een-island divergences expected to be divergences larger to expected een-island t be responsible for the high the for responsible levels diversity t be eir role as ancient refugia assources also role asancient but eir rife, with a minor secondary effect due to due secondary minor rife, effect with a ed that within-island genetic within-island might patterns that ed leo-islands has likely promoted genetic has promoted likely leo-islands versity within archipelagos is assumed to assumed is archipelagos versitywithin , geographic patterns variation genetic of et al C. . Accepted Article CGL2009-1332-C03-01 andCGL2012- discussions. work Ministryfruitful Science This through by of wasfunded projects Spanish Bar Rubén Arrigo, Nils Álvarez, Nadir Soto, Castells, Lisa Pokorny, Candelaria Rodríguez-Rodríguez, María Moisés Marta Martínez, Alfredo AurelioÁguedo Carlos Valido, Martín, García-Verdugo, Marrero, Juli Caujapé- thank manuscript. also We the of a review critical Nieto-Felinerto Gonzalo for grateful helping samplingfor with andaccommodation during field permits expeditions. We are thanked Hierro are and El Cabildo Tenerife of the and LaPalma, Cabildo of the from Medina Clavijo (Gran JacintoCanaria); Leralta and withoutconducted been cooperation of staffthe the the Jardín at Botánico Viera y Canario Cano and FatimaEmilio Duránfor their laboratory assistance. Field work nothave could Herrero, thanklike to Alberto Wewould improve significantlymanuscript. helped to our A reservoirs of unique genetic diversity, w 2009) andDiadema & (Médail “phylogeographical potential them hotspots” makes islands genotypes became that extinct everywhere else the and topographic complexity of the paleo- refugia and sources of diversitynew within- andbetween-islands. preservation The of of The paleo-islands thishypothesis. confirm MIEs on other studies Phylogeographic islands. volcanicwithin relief topographic than a barrier less of ocean apparently are of stretches withspecies widespread acrossdistributions several islands (e.g., speciation. However, ourresultsthis suggestthat not be hypothesis might for endemicvalid promotingallopatric and dispersal to the archipelago,barriers actingin as effective found This article This protectedis by Allcopyright. rights reserved. CKNOWLEDGEMENTS We areWe grateful toMitchell Cruzan and

40129-C02-01). M. Alarcón M. byJAE- was 40129-C02-01). the funded hose conservation should be prioritised. one, Xavier Picó and YulánXavierone, and Picó Úbeda forthe all Manuel Félix (La Gomera); Ángel Fernández Tenerife have probably actedasbothgeneticTenerife four four anonymous reviewers, whose comments (multiple island endemics)(multiple are needed to Canarina ), for whom for ), Accepted Article Baele G, Lemey P, Bedford T, Rambaut A, Suchard MA, Alekseyenko AV (2012) Improving AlekseyenkoImproving A,Rambaut Suchard MA, G, AV(2012) Bedford LemeyT, Baele P, Avise JC (2000) Phylogeography: thehistory and Arevalo JR, Delgado JD, Fernández-Palacios E, Huertas MJ, Ancochea FústerCantagrel JM, Coello JM, J, E (1999) ArnaudN, Ibarrola F, A, J, Coello Hernan Cendrero E, Cantagrel JamondIbarrola C JM, J, E, Fuster Ancochea REFERENCES (BES-2010-037261 fellowships BES-2013-065389,and respectively). MM andVCwere Doc program(CSIC/FSE). 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DNA markers. polymorphism. Islands. pr Nannofossils 2011ElHierroeruptive in “diversity begets diversity” model. an ofisland pulse model tothealternative immaturity–speciation evolution: island The error. methodreproducible for scoring AFLP peak Codonopsis (Campanulaceaestr.) s. special with reference to thecircumscription of new aAFLP: technique forDNA fingerprinting. Data. AFLP from Inferred T (2014)Key Processes for Biology Evolutionary of explosive radiation 151. homoplasy between fragmentrelationship of degree and size. ofahomoplasy size and of show indication markers (AFLP) polymorphism length Herpetology galloti Gallotia Europe. northern 1177-1189. palaeo Molecular phylogenetics Macaronesianof the adaptation. both lacertid Tenerife the elucidate historical causesof and morphological estimation. Cladogram III. endonuclease restriction from inferred andDNAmapping data. sequence with haplotypes Linnean Society genetic variationof in endemic plantspecies of islands.oceanic phylogeography. Molecular Ecology Resources ‐ islands, ecological shifts and interisland colonizations. islands, ecological shiftsandinterisland colonizations. Scientific Reports Systematic Biology . , 741-747. Taxon Molecular Ecology , (Lacertidae) in a xeric habitat of the island of Tenerife. Genetics 174 Molecular Ecology Trends& inEcology Evolution , 62 , 276-288. , 141 , 498-504. , . Cheirolophus Cheirolophus , 118. 123 PloS one Genetics Cheirolophus A (1996) Matrix correspondence tests on the DNA phylogenyon theDNA tests correspondence A(1996)Matrix , 585-595. ‐ os-Guerra A,Sá-Fontinha S, Francisco-Ortega J (2005) , Sepúlveda P, Crawford DJ (2014) Interpretation ofP, patterns Crawford DJ(2014) Interpretation Sepúlveda 45 tt GM (2013) Cryptic or Glacial mystic? tree refugia in , 335-343. , Ecography , 8 , , 9 , 8 , 907-913. 132 17 , e113207. , 725-735. , 2107-2121. J, Santos-Guerra A, , 619-633. (Asteraceae, Cardueae) inMacaronesia. Deegan F, Carracedo JC,S (2015) Carracedo ...Burchardt F, Deegan (Asteraceae) Diversification on Oceanic Islands Diversification onOceanic (Asteraceae) n de Lee T, HornesdeLee T, M,(1995)n ...M Zabeau ‐ s J, Santos-Guerra A,RS, Cowan Garnatje Santos-Guerra s ... J, Ruiz I (2002) Data from amplified fragment amplified from Ruiz I(2002)Data , oducts reinstate plume model for Canary reinstate for model plume oducts ‐ - 30 endemic genusendemic Palacios Palacios JM, Delgado J, J Arévalo (2007) adisticphenotypic analysis associations of ‐ , Nucleic acids research acids Nucleic height data height that minimizes genotyping 28 , 321-327. , 696-704. Molecular Ecology Bystropogon Sanmartín I (2014a) TheSanmartín I (2014a) Botanical Journal the of Molecular Ecology , 23 , 4407-4414. (Lamiaceae): Journal of , 11 , 139- , BMC BMC , 14 , Accepted Article TABLES contributed withauthors comments andapproved the final version. wroteM.M.paper the withhelp I.S,from M.A J.J.A. and M.M. andI.S. revised the paper. All the supervision ofM.A. and I.S.;I.M., V.C., I.S.,M.M. and performedthe BPECanalyses; J.J.A. contributed some samples; M.M. performedthe researchand theanalyzeddata under study the designed I.S. and J.J.A. M.A., M.M.,

AUTHOR canariensis NEXUS files for the concatenated cpDNAdataset and original AFLPs Matrix of supplemental material. GenBank accessions, sampling locations and/ sequences:DNA Genbank accessions KP797991KP798432. to DATA ACCESSIBILITY. This article This protectedis by Allcopyright. rights reserved.

CONTRIBUTIONS , Dryad doi:10.5061/dryad.j4103. , Dryad ; M.M.; carried outthe fieldwork, M.A. and or online-only appendices uploaded as online uploaded appendices or online-only Canarina Table 1. Descriptors of within-population genetic diversity in the cpDNA haplotypes and AFLP markers for each population studied of C. canariensis. Abbreviations: H(n): number of haplotypes;, H(d): haplotype diversity; π: nucleotide diversity; θ: nucleotide heterozygosity; Hj (se): Nei's gene diversity (standard error). Geographical locations for population codes are shown in Figure 2.

Haplotypes AFLPs Number of Nº of polymorphic Nº Nº Hj private Island (Paleo-island) Population Haplotype H (n) H (d) π θ fragments samples samples (se) fragments (% in brackets)

Article Gran Canaria GC-Cc 8 H1 1 0 0 0 8 409 (68.7) 0.2344 (0.0085) 0 Gran Canaria GC-Tm 11 H1 1 0 0 0 NA NA NA NA Gran Canaria GC-Es 10 H1 1 0 0 0 NA NA NA NA Gran Canaria GC-Ag 11 H1 1 0 0 0 NA NA NA NA Tenerife (Anaga) An-W 6 H1, H2, H5, H9 4 0.8 0.00112 0.00136 7 401 (67.4) 0.2677 (0.0088) 4 Tenerife (Anaga) An-E 10 H1, H2 2 0.2 0.00018 0.00031 10 345 (58.0) 0.2303 (0.0080) 0 Tenerife (Anaga) An-C 11 H1, H5 2 0.327 0.00015 0.00015 11 375 (63.0) 0.2453 (0.0078) 1 Tenerife TF-Bj 4 H1 1 0 0 0 NA NA NA NA Tenerife TF-Br 6 H1 1 0 0 0 NA NA NA NA Tenerife (Roque del Conde 11 H1, H9, H7 3 0.618 0.00121 0.00091 NA NA NA NA Conde) Tenerife (Teno) Ten-Ep 8 H9 1 0 0 0 9 351 (59.0) 0.2449 (0.0082) 0 Tenerife (Teno) Ten-A 7 H8, H9, H10 3 0.524 0.00038 0.00054 7 415 (69.7) 0.2997 (0.0088) 2 Gomera GO 12 H8, H9 2 0.303 0.00013 0.00015 12 473 (79.5) 0.3012 (0.0077) 1 La Palma LP-Ba 10 H3 1 0 0 0 9 285 (47.9) 0.1923 (0.0080) 0 La Palma LP-Lt 10 H3, H4 2 0.2 0.00009 0.00016 10 348 (58.5) 0.2309 (0.0079) 0

El Hierro EH 9 H3, H6 2 0.5 0.00022 0.00016 9 303 (50.9) 0.2115 (0.0082) 1

opyright. All rights reserved. This article is protected by c

Accepted Accepted Article [Ten-A] [GO] [LP] [EH] [An-E[GC] An- + C] [An-W] [Ten-Ep] + LP +EH] [GC+ An-E] [An-C] [Ten-Ep] [An-W +Ten-A] [GO [GC Anaga] + [An-W + Ten-A] [Ten-Ep] A migration eventsshown are with theirassociated probabilities. posterior andds (relaxedparsimony assumption) are s analyses. Selected(BPEC) valuesfor parameters(maximum of number MaxMig migrations) Table 2. B C Fsc Fst Fct This article This protectedis by Allcopyright. rights reserved. [GC ][TF] [GO] [LP] [EH] [GO + LP +EH] and(C) within populations. Geographical locati allelic variation at different levels:(A) am Table 3.

Western Clade + Conde Eastern Clade + Conde Conde + Clade Eastern (WT + +LP GO+ EH) Western Clade Eastern Clade Clade Eastern (ET + GC) +GC) (ET TOTAL Summary ofresults the from BayesianPhylogeographic andEcological Clustering Hierarchical analysisof molecularvariance (AMOVA)for AMOVA groups Nº of groups (K) Levels F-statistics F-statistics Levels (K) of groups Nº groups Maxmig ds ds Maxmig 4 1 1 8 4

10 3 3 3 3

Anc. Loc. Loc. Anc. (0.416) (0.416) (0.212) (0.212) Conde Conde Conde Conde Conde Ten-A Ten-A An-W (0.23) (0.13) (0.40) 1 ong groups,(B) amongpopulations within groups Anc. Loc. 8 16.63 8 16.63 11.30 5 18.92 72.07 11.21 0.13558 69.88 0.27934 0.13819 0.16631 0.30123 0.18918 5 4 (0.197) (0.197) (0.122) (0.122) Ten-A Ten-A An-W An-W An-W (0.17) (0.10) (0.31) Lp-Lt Lp-Lt An-C An-C hown.Ancestral locations (Anc. Loc.) for 2

ons of populations are shown inFigure2. Anc. Loc. 17.44 17.44 3.62 (0.190) (0.190) (0.086) (0.086) (0.116) (0.086) (0.18) (0.18) LP-Lt LP-Lt An-C An-C An-E An-E GO EH EH 3 24.27 24.27 13.06 (missing) (missing) (missing) (missing) (missing) (missing) (missing) (missing) node Root H11 H11 H11 H11 H11 H11 H11 H11 72.10 72.10 69.5 C. canariensis probability Root node 0.18 3 0.16 0.16 0.17 0.18 0.25186 0.25186 0.15820

0.27897 0.27897 0.305 clusters nº of based on on based 3 2 5 3

0.03624 0.03624 0.17439 Accepted Article of each haplotype, sizecircle is proportional to a) Pie charts show the geographical location 2. Figure Boulesteix Boulesteix Plant Plant brunneri Arminda Grasshoper Beetle Eutrichopus Beetles delalandii Gecko abaxoides Beetle verneaui Spider Spider beetle species complex in carlosi Mite viridanus Skink galloti Lizard Pimelia beetle darking Species complex in ofpaleo-islands Tenerife. Table 4. This article This protectedis by Allcopyright. rights reserved. Carracedo the text; numbersinthelegend refer to those in theAges map. and maps adapted from: graphshowson the right Tenerife mainthe catastrophic events that are theincommented directionsArrowsof indicatevolcano. the LasCañadas the post-erosional landslides. The ofedges the island. Lines show valleyscreated after landslides.Dashed indicatelines the series correspondingto the paleo-islands aresh main Pleistocene landslides and other geological events cited in the text. The old basaltic used in the textand their of b)showingyears,(millionage Map Ma). emergence ofTenerife 1.Figure Captions Figure Plant Organism Phylogeographical Phylogeographical Organism disjunction Steganacarus Steganacarus Pericallis Micromeria Chalcides

Pholcus Tarphius Tarphius

Calathus Trechus Tarentola Tarentola Gallotia Gallotia Dysdera Dysdera

Phylogeographic breaks with divergence times reportedPhylogeographicthe in betweenbreaks times divergence with literature the Haplotype distribution Haplotype network and the inferred chloroplastfor markersby TCS.

a) Geographic map of the Canary Island archipelago, with the code for each island

et al et al.

Anaga ( Anaga Conde Conde del +Roque /Teno + Güimar Anaga Clade 1. Anaga + Teno ( 350.000 years ( Teno del Conde Conde/Anaga . Clade Teno/Roque2. del + Roque Teno 1. Clade Teno /Anaga martinezii teneriffae ( Teno Teno Anaga / tenerifensis Teno ( Anaga Teno Anaga / Anaga / Conde del Roque 2. Clade Teno/Anaga. 1. Clade Teno/ Anaga Western/ North-eastern lineages East Tenerife / West Tenerife 5.5 Ma Ma 5.5 Tenerife West / Tenerife East Anaga ( Anaga ( Teno Tenerife Clade 2. fortunatus tenoensis 1998; Ancochea 2012;Zaczek (P. intricatus (P. E. gonzalezi M. densiflora P. echinata P. malpaisensis P. E. canariensis P. tussilaginis , , ) + Central group group +Central ) M. M. glomerata ) T.felix T.flavocintus and and and groups groups and P. roquensis P. ) Anaga/ ( ) / )/ ) / , ) / Anaga ( P. mascaensis ) ) , ; Teno / East /East ; Teno et al. and and T.antonii P. knoeseli P. et al. T. ) M. rivas- M. M. M. 2015. , / 1999; Cantagrel T, T, P. P. ) / 2. Ma 7.6 2. Clade Ma 1. 9–10 Clade Clade 2. 25 - 3.6 Ma Clade1. 3.2 Ma Clade 2. aprox 0.75 Ma Ma 0.75 2. aprox Clade .7M HD .547)ncerIS Jones nuclear ITS 2.87 Ma (HPD: 1.55–4.76) 3.93 Ma (HPD: 2.2–5.88) 3.94 Ma (HPD: 5.1-2.7) of populations and the frequency ofoccurrence Divergence (Ma) Markers Reference Reference Markers (Ma) Divergence (HPD:1.48–2.01) Clade 1. 1.73 Ma Clade population size.population codesgiven Population are in 1.2(1-1.4) Ma own in the three massifscontoured theat 1 –0.17Ma 0.7 Ma 1.1 Ma 0.7 Ma 5.2 Ma et al. 1999;Guillou marker) Mitochondrial (COII b Mitochondrial cytochrome and COII) COII) and two mitochondrial (COI cytochrome oxidase 1 Mitochondrial and 16S) (12S Two mitochondrial b Mitochondrial cytochrome 8 nuclear loci nuclear8 mitochondrial ( and COII) COII) and (COI Two mitochondrial (28s rRNA, ITS2) nuclear gene fragments two and ND5) rRNA, (12s Two mitochondrial nuclear (ITS-1) Mitochondrial (COI) and Cox2),and nuclear (ITS 2) (ITS 2) nuclear Cox2),and oxidase cytochrome genes Four mitochondrial (CO1, tRNAleu). Morphological Part of mitochondrial of mitochondrial Part I and II (Cox1 and 16S, NADH and and NADH 16S, data. cox 1) et al. 2004; Oromí 2005 2005 Oromí & Emerson Moya 2002 Salomone 2000 Gübitz 2000 Brown 1996 Thorpe Puppo Puppo 2013 Hernández Macías- 2008 Dimitrov Emerson Emerson Goerzig 2009 2009 Goerzig & Hochkirch Juan Juan al. Contreras-Díaz 1999 2007 2007 et al. et al. et et al. et al. et et al. et al. et et al. et et al. et al. et 1996 1996 et al. et 2014 2014 2004 2004 2014 et al.

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Accepted Article maysupporting beAdditional information found intheofversion online this article. INFORMATION SUPPORTING S2. Table coloured according to location, as indicatedin the legend. Codes populationsfor in found percentage, gene andtheir flow as inferre membershipof individual to inferredeach Baye clustering of individuals within populations 5. Figure (H1 toH10) correspond tothe haplotypes shown in Fig. 2. Numbers below branchesindicate Bayesianpos haplotypes,cpDNA showing ages mean (abovebranches) and95% HPDcredible intervals. 4.Figure indicates an unobserved (missing) ancestral haplotype. receiving networkb) Haplotype black thehighestprobability. Thesmall H11 posterior circle composition, with population this as source of these clusters,butwith low probability; in the caseof Conde,itsuggests a mixed concentrationcontours around it. Locations situatedbeyondthe clusters could also belong to at the 'centre' of each population cluster,andthe shaded areasshowradiusthe of 50% ancestral location formigration events (denotedby arrows).The contour regions are centred analyses thefor cpDNA markers. Phylogeographicala) clusters(coloured andblobs) 3 Figure colour; different sizecircle is to proportional its frequency among populations. S2.Table b) Statistical Parsimony network infe This article This protectedis by Allcopyright. rights reserved.

. Results thefrom BayesianPhylogeographic andEcological (BPEC) Clustering Results from the analysis of AFLP markers. a) Histograms showing the Bayesian Bayesian the showing Histograms a) markers. AFLP of analysis the from Results Maximum Clade Credibility (MCC) (MCC) Cladeobtainedtree fromtheMaximum of Credibility BEAST analysis d by BARRIER. b)dpoints Splitnetwork BARRIER. bywith (STRUCTURE); colours re migrant allelesto the eastandwestern clusters. rred byEach TCS. haplotype is shown in a terior clade support values.terior clade Codes tips for sian group. Dotted lines indicate barriers to present the proportion Accepted Article This article This protectedis by Allcopyright. rights reserved.

FIgure 1 Figure 2 6.2 -5.6Ma a) b) massif Teno a) EL HIERRO(EH) EL HIERRO(EH) LA PALMA (LP) EH LA PALMA (LP) LP 1.1 - Lt

1.7 0.175 Ma

Roque delConde NW RIFT NW Valley LP.Ba Icod 11.9 -8.9Ma LP-Ba LA GOMERA (GO)LA GOMERA massif

2

RIFT SOUTH SOUTH L

6 a 7 LA GOMERA(GO)

s Tigaiga 2.2 Ma massif

C 1 a ñ 9.4 a d as 5 GO La Orotava 0.78 Ma Valley 4 TENERIFE (TF) T

en-A NE RIFT 3 11.9 Ten-Ep Conde 0.83 Ma Valley Güimar EP TENERIFE (TF) Taganana Arc TF-Br

GRAN CANARIA(GC) An-W 4.9 -3.9Ma Anaga massif TF-Bj 14.5 An-C An-E Age (Myr) 2.5 2.01.51.00.50.0 GRAN CANARIA(GC) 3 2 1 6 5 4 7 GC-Ag 1 Icod debrisavalanche RoquesdeGarcía debrisavalanche Tigaiga debrisavalanche La Orotava mega-landslide Cordillera Dorsalpeakactivity Güimar landslide Increasing occurrence oflavas eainClbinIonian Calabrian Gelasian GC-Es GC-Cc GC-Tm FUERTEVENTURA 20.6 b) H2 H5 LANZAROTE 2 H7 H3 H6 15.5 3 4 5 H4 H1 H9 Morocco

6 7 H10 H8

Accepted Article

This article This protectedis by Allcopyright. rights reserved. Figure3

)b) a) Figure 4 LA PALMA (LP) Age (Myr) 1.41.21.00.80.60.40.2 0.0 EL HIERRO(EH) LA GOMERA(GO)

Calabrian LP-Lt

0.878 Conde 1 TENERIFE (TF)

0.09 An-W 0.522 0.53 GRAN CANARIA(GC) Ionian 0.255 0.97 0.252 0.63 0.216 0.87 0.107 0.56 0.095 0.089 0.99 0.6 0.067

0.99

H6

4H10 H4 H3

H9 H8 H10 H3 H6 H4 H7 H1 H5 H2

H8

H11 H9 West Tenerife La Palma Gran Canaria El Hierro East Tenerife La Gomera (Teno) + +

+

H7

H1

H5 H2

Accepted Article Figure 5 This article This protectedis by Allcopyright. rights reserved. A EH LP - Lt LP - Ba EH GO LP-Lt en-A - n Te Ten-Ep LP -Ba n-W An A GO n-C An-E T en-A Ten-Ep GC-Cc

K=4 n-W An 100% 100% n-C An EH LP - n-E An Lt LP 96.8% - Ba

GC-

Ten-A Cc GO

K=2 Ten-Ep 100% en-A - n Te An-W 100% Ten-Ep 100% An-C An-W An-E An-C

K=5 An-E GC-Cc B + Occidental Islands West Tenerife+ An-W Ta Pa Pt H G Pa Pt Pa H Pt H H Pt H G H Pt Pa H Pt Tp Pa Pt Pt Pt Ta H Pa H Ta H Pa Pa Ta Ta Aw Pa Pa Aw Aw Ta G G Aw G Aw G Aw G G Ta Aw G G Ta Tp G Tp G G Tp G Tp Tp Pt Tp Tp Tp Tp Ac GC Ac GC Ae Ae GranCanaria + East Tenerife GC Ac Ae Pt Pa G H Ae Ae GC Ta GC GC Ae Ae GC GC Ae Ae LP-Ba EH GO Ac Ten- LP- Ac Ae GC Ac Ac Ac Ac Lt Ac A GC Aw Ae Ac Tp An-W An-E An-C Ten-Ep GC- Cc