This article isThis article by protected copyright. All rightsreserved. 10.1111/mec.15189doi: differences to lead between the of thisversion c and Version Record.Please copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot Phone: +34 954 232340 E Vespucio26,E Avda. Américo Biológica deDoñana,Estación EBD Vanina Tonzo Author forcorrespondence: CyprusNicosia, 2 26 Américo Vespucio 1 Vanina Tonzo agrasshopper in taxonomic designation current supportfor no but revealstructure deepgenetic data Genomic Article :Original type Article DR.JOAQUIN (Orcid ORTEGO ID 0000 : MRS. VANINA (OrcidID TONZO 0000 :

Department ofBiological Sciences, ofUniversity Cyprus; Department Biológica ofIntegrativeEcology,Estación deDoñana (EBD -

Acceptedmail: [email protected] Article 1 , AnnaPapadopoulou

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41092; Seville, Spain 41092; Seville,

- 41092 Seville, Spain 41092 Seville,

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2109; da . This article isThis article by protected copyright. All rightsreserved. approache characterizing studyregion.Overall, thisstudyhighlights the theimportance ofintegrative geographic distance variation incompatible withecological/environmental speciation.Our supportlittle phenotypic results distribution variationof thatdonotmatch genetic current with taxonomic designations andis landscape genetic analysesrevealed popu that integrativephylogenetic reconstructions, Bayesian model from coveringof entireknown the the taxa. populations distribution two Coalescent phenotypic obtained (geometric morphometric boundaries, and understand factorshave the that contributedto Mediterranean quantitative phenotypic differences and distinct habitat assoc frommontane grasshoppers Pyreneanregion the wereoriginallydescribedbased that on sourcesother ofevidence.Specifically, wefocuson data by sequencing nor throughput the quantitative integration ofgenomic taxonomytraditional species to that hasled designations are genomic data developments inthe fieldofmolecular ecology diversity.classify Overthe biological decade,technological last T Abstract

Accepted Articleaxonomy

among popu s toidentify taxonomic unitselucidate and the evolutionary history ofspecies. has traditionallyreliedon has morphological andecological traits to interpret and

and changed - montane To current habitats). validate taxonomic species test designations, s

and lations limited connectivity across population abrupt the landscapes the paradigmthe analysis. biodiversity Herewe how of illustrate and a marked genetic and amarked structure lations assignedtothe two taxashowaspatial s ) and and systematics Omocestus antigai genome

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This article isThis article by protected copyright. All rightsreserved. evidence could resul preferences morphological or diagnosable changes andadhering tothese two sources of therein2018 andreferences Platnick quantitative phenotypic differences (diagnosable species or phenetic concept; mostpopulations, descriptions species beentraditionally have onqualitative based or species concept;biological Queiroz species alternative concepts andthe different properties/criteria todefine used them unitfundamental that biological conforms remains contro aspecies formally described taxa establishment ofefficient conservationpolicies oftenputfocusonthat of the lists red loss rapidaccelerated ofspecies andthe degradation ofecosystems Wiens, 2007 formationspecies new taxa of result as a newtechnological and conceptual advancesthat biologistsallow to discover Dobzhansky, 1937; generatethat it is at the of core Describing 1. phylogenomicgenetics, inference Keywords:

Accepted ArticleIntroduction , ,

2007) 1981; Sokal at at resolution an unprecedented

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2013 In recent years,theadventofIn recent highthroughputDNA sequencing , Burbrink, McGuire, & Moritz, T

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- and identify boundaries amongand identify boundaries recentlydiverged usingspecies ) Leavitt, Johnson,Leavitt, Clair, Goward, &St 2011; based based . More . More recent developments have incorporated thepossibility Fujita et Fujita et al. (Fujita et(Fujita al. and resolutionthe of species delimitation

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This article isThis article by protected copyright. All rightsreserved. environmentabetween differentiation the and phenotypicdifferences often have data supported etc.)salinity, divergent naturalselection contrasting under presumed represent to ( description of practices and, ultimately, leadto misguided conservation polic compromise ourability to solid reach conclusions ontheoriginanddynamics ofbiodiversity can in turn, have anegative impact onsubsequent ecological and evolutionary studies, species populationto detect stru Sukumaran Knowles & with species boundaries ofresolution genomic can data lead 2018) delimitationimpact onspecies inferences withsexuallydeal dimorphic traits ( However, integrative speciesdelimitation approaches not are exempt oflimitations Acceptede.g., Article Sukumaran &Knowles recognized

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This article isThis article by protected copyright. All rightsreserved. populations mostlyare Accepted ofbothspecies followthewesttoeastorientation ofranges the Romera, Presa,Arnaldos, & 1999 quantitative andphenotypic traits asbodysize forewing such shape speciesThese two were describedoriginally based ondistinct habitat associa and Catalan Pre distributedthrough1908 are Pyrenees,including some the sisterputative speciespair elevationsoften isolatedathigh in different mountain systems Cordero,Navas, Noguerales, &Ortego, species) and complexis a diversified ofnine recently distributed species Peninsula in Iberian the (6 continuum Article allows phenomena and ecology populations experiencing contrasting & Bradburd manyfound in studies association 2014 not led to at selection a fewgenesor incipient speciation ) .

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environmental dissimilarity and phenotypic generally deserve tobe protecteddeserve (

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(Shafer & (Shafer & Wolf subgenus pattern pattern Omocestus genomic ecological interpreted interpreted as ( in the context ofresolving taxonomic uncertainties Poniatowski, Defaut, Llucia . of

; Olmo Omocestus antigai Dreuxius

progressive islands involved local islands in processesadaptation ( environmental e.g.,

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Mason 2015; &Taylor, speciation

Defaut, 1988(genus ( a signal

Cigliano, Braun,Eades, & 2019 Otte, . Most taxahave. Most (Bolívar Moritz . , genetic and phenotypic

Thus, the studyThus, the of 2002; Puissant

conditions

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, 2002

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populations populations ). -

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This article isThis article by protected copyright. All rightsreserved. phenotypic sequencing (ddRADseq; design determine the of conservation status differentthe taxonomic within units divergenceecological boundaries and thefactors understand 2016b). progressive areas the sizebeing small andhighdegree of fragmentation ofitspopulations, overgrazing human and impacts high theandqualityofcontinuous decline in (Braud extend habitats al.,2016a).Incontrast, et the “ different as the taxonomy com ofthe bellmanninavasi speciesdistinct status(Clemente al.,1999)and et even describe subspecific anew taxon ( synonymize twotaxa the differences morphologicaland subtle variation Pomares is m) with2350 or grasslands associated alpine subalpine above tree the line, whereas endangered” dueto

Accepted Article circumscribed toMediterranean scrubby habita - , and the of decline elevation by grasslands occupied two proper In this study, genomicIn this wegenerated usingrestriction data Overall, t , assessment categories 2002; putative putative information abandonment oftraditional on Olmo - ground Puissant 2008) in Puissant et 2008) the decades(Cigliano last al. his complex makes anexcellent this model system totest species species are the included in this this taxon its extremely estimated small ofoccupancy area (60 plex also has has also importantplex implications for conservation - , Vidal, 2002; Vidal, which will through geometric morphometricanalyses the the Peterson, Weber, Kay, Fisher, Weber, Kay, Fisher, & Hoekstra, Peterson, manag ( Ragge & quality of its

was assessed as was assessedas “vulnerable”, main identifiedwith the threats

(Braud al.,2016a,b) et ement in turn

Poniatowski Poniatowski et al. Reynolds, 1998; that havethat contributedto genomic,phenotypic and practices O. antigai grassland grassland provide necessary provide the specific IUCN red list ofIUCN list red threatened species

have motivated differentauthors to .

ts at elevationsts at lower (700 are supposedles are tobe management practices management et practices (Braud al., alpine alpine habitatsand subalpine to due the Reynolds ,

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198 baseline informationbaseline - site , (Bookstein 6

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This article isThis article by protected copyright. All rightsreserved. localities navasi specimensBetween 2012and2015,wecollected of 2.1. 2. study the area. populationsdistance among andresistancedistances defined bythe contraryon the shapingfactors adaptationto and processesoflocal different elevational andclimatic gradients are the mai system. we Inparticular, main Rannala, 2010 lineages them integrating while and inferred ofspatial geneticpatterns structure compared taxonomicofthe the status stud phylogenetic relationships among polymorphisms and data (SNP) genomic andphenotypicdata. hypothesisthe thatthe current taxonomic designation on light

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navasi

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tested tested the hypothesis Bayesian modelBayesian and In particular,weusedgenome ed alternative

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to identify independently of complex the ecological based method (1 population) (1 population) ; Figure1 ,

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is supported by is supported & to infer the the to infer from 16

(7 populations) 2015 Weisrock . First, . First, ; Yang & ; Yang& sampling sampling - evolving , we tested

es, 2018) is

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n This article isThis article by protected copyright. All rightsreserved. d function the using effects of oflocation, size, and rotation of relative positions the landmarksamong specimens performed Core Team,2017) using 1999) correspond to originally usedto those species thetwoputative distinguish ( pronotum, and forewing clusteringBayesian analyses (seeResults section). ( P most the one or two five phenotypic variation if available) Wea maximum selected of10 2.2. for s stored ( populations entire knownacross the distribution ofthe range S i.e., Clemente al., et istances betweenistances landmarks samples across opulations opulations with availablegeometric AcceptedTE Article geometric morphometric REO Geometric morphometric analyses populations

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This article isThis article by protected copyright. All rightsreserved. populationeach PCscores the of thetwoPCs, first whichexplained 80the 16 %and % ofthe 2.4 1km) (c. sec resolution. “ Then, weusedthe of 19 the dissimilarity eachof between pair ( using (Silva & Silva,2018) calculating pairwise Similarly, weestimated phenotypic differentiation betweeneachpairofsampled pop pairwise estimated differentiation genetic between eachpairofsampled populations calculating heterogeneity; and (3) isolation ( We potential analyzedthree thatcould factors have sh 2.7. both anathese (25 populations BPP analyses) sites SAR,QUE, ERR, andTUR were performed populations available for with phenotyp only http://biodiversityinformatics.amnh.org/open_source/gdmg P

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model five P IBPP . 201 ST We geographical calculated the distance between sampled populations ARLEQUIN

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This article isThis article by protected copyright. All rightsreserved. in multiple statistical tests andcalculate we (eight significant explanatory most adequatemodel for thevariability explaining inthe response only variable, where the model toensurethat model significantthe were significant usingabackward were selected were initially constructedexplanatory withall terms fitted geographical, resistance and env phenotypic differentiation ( with geographical, resistanceandenvironmental and pairwisedistances, (ii) population in cell connection scheme. matrix of ofresistancedistances betweenpairs all populations considering aneight weusedlayer, transformed 30arc to Topographic Mission (SRTM Digital Elevation for fromslope eachm cell a90 digital resolution elevationmodel from NASA Shuttle Radar the for eachpairofpopulationsusingEuclideandistances the between environmental variance,respectively. Afterward, wecalculated environmentaldissimilarity R R Accepted Article applied applied “dist” . (Wang traits We

function in variables , us a

for females for six and males) against 2013) Benjamini ed CIRCUITSCAPE

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(starting with(starting thesignificant ones)until least all retained terms within no additional - R sec (c. 1 km) resolution usingsec (c.1km) resolution . To investigate the . Toinvestigate the ofrole topographic complexity, wecalculated the terms - Hochberg false discovery rate discoveryHochberg false rate

M . Then, we tested . Then,wetested P atrix ST

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This article isThis article by protected copyright. All rightsreserved. included). and SNPs number retained ofunlinked in final dataset the obtained with anaveragecontrol, of2.11 ×10 Illumina sequencingreturned anaverage of2.19×10 3.2. Genomicdata 0.0 F F by shapevariationofforewing (PC2: One Wilk’s= 0. λ = significant differences among sex 65% in variation forpronotumshapes ( The twofirstprincipal component scores,PC1andPC2,trait explainedmost foreach 3. 3.

10 10 1 0.0

Accepted Article Results . 13 , ,

are - minCov 40 42 Geometric morphometric analyses ; way ANOVAs multivariatethat revealed differentiation among was driven populations 0 ; PC2: ♂

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This article isThis article by protected copyright. All rightsreserved. FASTSTRUCTURE FASTSTRUCTURE exception of CRE more geographical sense populations same they five replicates. explainused to structure data inthe towas equal replicates four intwo and tofive replicate, equal inone and numberthe ofmodel components complexity that maximizes marginal waslikelihood 22 equal to threein replicates, equal to according to the to individuals and populations assignedtoa were singlegen groups populations for western analyses showed ahierarchical genetic structure, firstwith a split populations withtheirtaxonomicbut not (Figure assignment S butcriterion, probabilities ofthe log (LnPr(X|data STRUCTURE 3.3. 4 populations Accepted Articlea ). Theinferredgenetic Population genetic Population genetic structure - also ERR genetic genetic M into into genetic clusters other at smaller from geographical scales presented aximum - QUEfrom TUR

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analyses wi analyses criterion the ‘optimal’ clustering K populations populations BOR ; (ii) ; (ii) differences

= 2 and subsequent subdivision subdivision of = 2andsubsequent these two main groups weregroups consistent distribution geographic with the of the - SET FASTSTRUCTURE

th results mostlyresults mirroredthose - BAT asdoneby BAT S6

K from

). Assignment values ). Assignmentvalues

> 5 were extremely cases> 5were lowinall ( (Figure (Figure FASTSTRUCTURE - MON and BOIMON S6

analyses for analyses ) : (i) For : For (i) genetic genetic four in18replicatesand to equal seven in K STRUCTURE )) steadily)) increased up to solution by clusters K

etic cluster - FASTSTRUCTURE

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This article isThis article by protected copyright. All rightsreserved. external TUR sister placed asa clade of CRE 3 STRUCTURE which inferred same the clusters (Figure genetic that Population trees 3.4. admixedthe population of whereas PC1 location geographic ( of clusters presence twomain the genetic revealed by 2019) al., S S (Figure non similar result, seeBaiz, &CortésTucker, their thus, respective Figure 2 Figure ). For the east geneticgroup, the ). For 7

Accepted Articleb - ).

Phylogenetic Phylogenetic inference only spatial spatial model (Figure S separated separated CONSTRUCT were In agreement with The spatial model of

different forthe relationships group PC2 separated ; 7 differed in the differed the in

and assignments onlygenetic for Figure a

), butlayers beyond and grouped within the same genetic cluster at the withinthesame clusteratthe grouped genetic lower hierarchical level by the westernmostthe populations with respect to to fivewith respect the CRE easternmost populations FASTSTRUCTURE reconstructed S

analyses showed stronggeneticanalyses showed admixture 8 ) and main clusters genetic . bar virtuallybar were plots identical to forthose obtained

the rest ofwestern the rest inferred

previous TOS at an intermediate position. TOS atan 7

CONSTRUCT a ). Spatial statistical models supportupto hadstrong SNAPP in K relationships amongrelationships

s analyses BPP

= 3 1a analyses, analyses, -

, and ERR and

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than the the than = 3 - BAT.

3 , , ). - 5

This article isThis article by protected copyright. All rightsreserved. randomizingtest dataacrossindividuals phenotypic yielded support inconsistent phenotypic data IBPP few clustersgenetic (CHI the using (Figure SNAPP 2 ( scenario clustering analyses in ( delimitationSpecies with analyses the 3.5. no support hierarchic among inferred by populations populations tobelonging same the clusters (Figure genetic considerable fuzziness for in terms of As expected H ).

Accepted5 Article BPP ), which considered populations), whichconsidered assigned to each SVDQUARTETS

prior combinations prior and specific g Species Species delimitation analyses support analyses

and

analyses 4 the

). All sampled populations were consistentlysupported asdistinct specieswhen subset of1,000 andonlysome loci populations belonging nearby same tothe H al al structure yieldedgenetic by SVDQUARTETS

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QUE) were not supported as distinct notsupportedas underQUE) were taxa a * trees

method strongly a method strongly supported sixspecies model 3 tested -

4 analyses for subsets the of200and500loci

most external nodes most external tions alsotions showed considerable unc ,

but received much lower support but received muchsupport (Table lower demographic scenario , genetic cluster by FASTSTRUCTURE

the are in goodagreement with the

1a

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ertainty indicate indicate

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This article isThis article by protected copyright. All rightsreserved. taxa groups ecological/environmental speciation. showOur results oftwo thepresence doesnot (i) match with populations assigned to ouranalysesCollectively, the rejected 4. multiple testing ( differentiation applying genetic after defined distances resistance bytopographic complexity, environmental dissimilarity or was showed that no trait significantly in correlated anyse final model ( withsignificant relationship geneticdifferentiation variablewas andthis from excluded the complexity ( distancesboth geographic ( genetic indicatedthat differentiationMMRR analyses ( 3.6. and across tested topologies different speciesfor morphological dataset highamountby the ofgenomic data. S 9 Discussion Accepted Article ) , indicating influence that the ofphenotypic traits in Landscape genetic Landscape genetic andphenotypic ( Wallis,Waters, Upton corresponding toaneast p p

= 0.004) (Table 3 = 0.004) = 0.374; = 0.374; 3 Table all q some external - s values

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x with geographical distances, ) was significantly correlated) wassignificantly with

analyses analyses is probablyoverridden 9 ). only

many based onrandomized ty didshow not a

the into split main other Pyreneanother between sexes corrections for

genetic genetic P ST ) This article isThis article by protected copyright. All rightsreserved. do notmatch currenttaxonomic designations and, accordingly, populations sampled inthe structure analyses thatindicate populations split two in our indicate nosupportfor analyses, results the anda phenotypic data comprehensive suiteofphylogenomic andBayesian clustering have agenetic basisof scenario in line witha theirand determine whether specific associationsphenotypic and habitat slight differences now, nomolecular conservation assessment programs et 2016 (Braud al., aretwo taxa validspecies now(Cigliano considered etal. and biometrical analyses by Clemente al.(1999) et resurr and songs), recommended synonymy the of population (1998)Ragge &Reynolds attributedsubtle their morphological diversity and Bolívar ( of differentcriteriaconsidered the to establish the boundaries thetwoputative between taxa and As inmanyspeciesother complexes 4.1. characterizing studyregion the scalesspatial limited reflecting connectivity across population abruptthe landscapes Accepted ArticleClemente al.,1999; et Clemente al. et (1990) O. navasi Integrative species Integrative de

in the transition the between XIX andXXcenturies

geographic isolation,denieddifferences inacoustic communication (

has changed through time withthe conjunction discussionthe in about validity - based analysis hadbeenperformed themonophyly test ofthetwo to taxa Ragge

confirmed their limitation & ( e.g., Reynolds

( Noguerales, Cordero,Noguerales,

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9 the the taxonomy of ion. Using genomic and ) and treated as such in treatedassuch ) and . Phylogenomic andgenetic . However, newacoustic , 18 ,

201 9 to effect the of 7; 6 O. navasi , a

2016) ) Bolívar (east/west) .

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courtship 1908) and the that that

This article isThis article by protected copyright. All rightsreserved. Acceptedpop high powerof numbermarkers alarge of (hundredsof tens to thousands) to fine detect overestimation beparticularlyexacerbated when can vast using genome Leach populations thancapturingrather speciesdivergence fromisolation truespecies boundaries andtend to over such methods not are abletos coalescent model(MSC) (Rannala limitationsthe delimitation ofBayesianspecies approachesbased onthe multi alsoFig.(see 2bin considering the Accordingly FASTSTRUCTURE assigned to thesame groups genetic clusteringby PCABayesian analyses ( and Articleremarkable that many populati nearby assupported distinct km and assupported combinations forgammadistribution fullyanalyses presence supported the of presencethe of nominated taxa, speciesarephylogeneticallyputative inte two distinct habitats (alpine ulation structureulation ( é , Zhu, Ranna Despite phylogenomicDespite recover not reconstructions belong , the species , the delimitati a ing to theing to same cluster genetic distinct distinct species several several highest number ofspecies,

molecular and Leaché la, &la, Yang taxa CONSTRUCT Hime Noguerales etal., 2018; al.,2016; et plausible species.

under under -

et al.,et based vs tatistically distinguish structuredue genetic to population . Mediterranean/montane) supposedly occupied bythetwo and o ,

a few prior combinationsa fewprior and 201 ) were ) were 2018) species delimitation analyses using on bymodel mostsupported & 9 nly . ; Yang, 2013;Yang

ons (BOR I Sukumaran n most cases, . consistently some Recent Recent empirical and have theoretical studies shown 13 rspersed (Figure Independently ofthe

one per geneticcluster inferred by species species s nearby populationsnearby

(CHI - MON, NER & across across - delimited asdistinct species (Figure

ESP and CRE each ( Knowles Huang, 2018; - split genetically differentiated &

ing 1 of the 17 all Rannala, 2010),pointing outthat ).

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the monophyly ofthetwo tested topologies SAR, BOI specific specific ,

number used, ofloci 2017) BFD Sukumaran separated by less separated byless - ERR * sampled Leaché al., et

. BPP analyses wastheone analyses

The problem of species The problem ofspecies topologies. - - - wide data due the wide data to

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TRUCTURE STRUCTURE were not were not * 2018; It It is also than prior

identified identified BPP - -

s grain grain BAT) , 20

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This article isThis article by protected copyright. All rightsreserved. requirements considered tobe di intersecting ofevidence suggestlines multiple that the delineated entitiesdo notmeet the coalescent diagnosesmodel frequently structure ratherthanspecies, different genetic ameliorate this problem by of emp loci Knowles, 2017 framework prevent cannot overestimation species al. et (Noguerales of delimitationspecies approach implemented in seems little or very individuals yielded populations as distinct species and a test validation randomizing dataacross phenotypic between only ( help could to genomic other with data sour Pyron, Hsieh,Lemmon, Hendry,2016 Lemmon, & distance can bedelimited asdistinctwhen hundredsspecies using ofloci( intraspecific populations presentinggenotypicdifferences simply 2017 Pyron et al.,Pyron et 2016;

Accepteddelineated Article BPP ). pres

to be overriddenbyto be In this line, and Beyond thetaxonomic inflation studiesRecent have currently recognized ented verysubtledifferences loyed are are loyed likely tohave BFD influence

species, species, mitigate ). *

and integrative and integrative In sum,t r an ecent empirical studies Sukumaran 2017 Knowles, & indicating

the problem ofconfounding almost on the .

he stronggenetic structure inour highnumberstudy systemand the the highamount data.the ofgenetic suggested that ces ofinformationces morphological,ethological,etc.) (ecological,

outcome of identical result. identical result. taxa stinct taxaaccording toalternative contemporary species that integrating phenotypic and genetic intodata this analyses

( extraordinarily see among of some pairs Figure issue

IBPP implemented in species delimitation approaches integrating have found

linked withlinked thefactthatmultispecies

Thus, incorporating s analyses analyses IBPP

S2 andS3 ).

inflated inflated the numberidentified ofspecies population ) does Although most traitsanalyzed phenotypic .

that geographically continuous geographicallythat continuous and the not

) IBPP , Our our populations populations notdifferand did really really

structure withstructure speci does not does not seem to tohelp impact ofphenotypic traits

IBPP analyses resulted morphological help to the reduce number ,

analyses supported most 2018 suggest ; Sukumaran & Huang, 2018; from isolation by isolation that the traits es

limits has no This article isThis article by protected copyright. All rightsreserved. do not 3) probably populations. hierarchically structured in congruence with clusteringBayesian analyses supported thatgeneticvariation thewithin complex is Phylogenomic reconstructions andspatial patterns of from structure inferred genetic 4.2. conservation intraspecific with by now 1999; Slatkin, ouropinion,thestudiedshould populations 1993).Thus,in betreated 2010), such mostpattern reflects plausibly with a 2007 of indicative eastern and diverging ar low elevation populations ( environmental studied traits overlappingof largerly populations, phenotypesacross taxa,andfor clusters all genetic (see 1in concepts Table de Queiroz, 2007). Theselines ofevidence (i)theinclude: presence

Accepted Articlee always Factors structuring

) the the . Although rapid even

– possibility of

as a single as a allopatric and explains whythe clearly clearly stepping

However, the strong signal strong However, ofisolation the evolutionary significantevolutionary lack of lack and management (Figure S (Figure - driven divergence and the strong forthe signal isolation recover intrinsic speci - stone speciation process stone speciation process (e.g. VanderWerf,Young, Yeung,&Carlon, western desi 2

and S admixture at contact zoneshappensevenbetweenadmixture twomost atcontact the genomic andgenomic phenotypic variation es gnating

ecological the west/eastthe

taxon with genetic strong structure, whichis

reproductive isolation spatial 3 genetic groups strategies strategies ; phenetic species concept the analyses analyses in units units

the presencethe of + main

phyloge (Braud (Braud et al.,2016a, b). split (Moritz, 2002)that should populatio genetic clusters genetic the geographical distribution ofthe

( revealed byphylogenetic reconstructions - genotypic cluster species concept by ne CONSTRUCT CONSTRUCT - distance might bepotentiallycompatible tic tic species concepts - ( n connectivity by biological species

phylogenetically interspersedhigh and - distance (

);

(ii) Noevidence for (e.g. blur

east most structure genetic and r

= 0.71, (Hutchison & Templeton,(Hutchison & - west be ) ; concept

(iii) Genetic (iii) clusters considered not in p genetic groupsgenetic

< 0.0001; < 0.0001; Table ) compat ), which

sampled ( de Queiroz, in future classic classic –

at least at i

ble ble and is )

as

This article isThis article by protected copyright. All rightsreserved. Wallis al. et highrecolonize elevationsinterglacial during periods & Hugues, andtherange presence likely ofice reconstructionsgeological documenting heavily areas theglaciated in central of part the 2010; Valbuena 2016; ( havebeensplits many reported for plant andanimal other from species P the west ofanimaginary line around located Segre (~1ºE). the river Similar east a equilibrium) artifactual consequence geneticof differentiation gradual and fromgenuine resulted historical processes systemstudy evidence suggest that results from made clustering analyses byclassic ( clustering analyses ( a remarkable congruencebetweenthestructure hierarchical genetic inferred bynon by clusters inferred F other analysesall e.g., SNAPP nalyses nalyses showed abasal intwo split clades corresponding topopulations eastlocated and Accepted Article ST

= 0.35) and extraordinarily high andextraordinarilyhigh = 0.35) drift estimates ofgenetic divergence g after forall ,

Alvarez 2018 Charrier, Dupont, Escaravage, Pornon, & , et al In agreement with In agreement with SVDQUARTETS ) 2011) . ( . merely driven bygeographical distance (Bradburd al.,2018). et Overall, Overall, - and 2016) Presas, Mateos, Vila - Ureña et al.

. Ourstudy ahighdegreeof systempresents differentiation genetic (mean indicate indicate that

STRUCTURE for numerous alpine STRUCTURE these might allowed have montane/alpine species tosurvive glaciations and , and STRUCTURE that

results results BPP ,

2018) the

) and PCA,beinga ) and thelatter methodof free , analyses FASTSTRUCTURE strong genetic revealed by structure all other analyses is CONSTRUCT are - . -

Farre, Sluys,Farre, &Riutort,

(Jombart, Devillard, & 2010)Balloux, free refugia at longitudinal its extremes This

and consistent consistent breakswith the transverse proposed taxa

( east F FASTSTRUCTURE - value >

- and thesupport ideathat

west might split bewell explained bydetailed ( make i.e. 2014; ) and ) and strong strong

0. little biological sense our little in

( 5 Mila, Carranza,Mila, Guillaume, Clobert, & Charrier 2014; et al., both ; Pritchardet al., 2000). Also,

(i.e. migration(i.e. and gene isolation

results for results 2012; phylog Bidegaray ) enetic reconstructions

rather than K the the fragmentation of

= 2, . All these linesof the the Valbuena ( -

- yrenean yrenean region Ehlers, Gibbard, all phylogenetic all west genetic Batista et al., assumptions assumptions being specific tic -

spatial drift

by - there there is an Ureña enetic enetic This article isThis article by protected copyright. All rightsreserved. Mediterranean for eitherdriven ecologically divergence the or taxonomic separationbetween supposedly the a populations local/regionalat scalesfollowed alongitudinal cline rather than asegregation of Cordero, landscapes on structur obtainedto those byprevious studies showing the impactand ofsteepslopes complex studiedtaxon,malesthe brachypterous and being females micropterous, comparableand are topographic (Table3 roughness by boththe geographical among distance populations and resistance distances definedb geneticAccordingly, landscape analysesindicatedthat differentiationgenetic wasexplained geneless increased flowthroughthe abrupt landscape admixture ( ata located similar longitudebut higher elevations showednoevidence of genetic ongoing geneflow between the populations TOS (andinto a lesserextent andPER in SAR),contact the located zonebetween populations,nearby suggest astrong effect ofgeographic isolation been assigned cluster,with toaunique genetic onlyafew casesofgenetic admixture between geneticgroupswest andeast fine spatialscales,at with of also mountain ranges distributions duringancestral glacial and presenceperiods the of along refugia Pleistocene Accepted lpine and Mediterranean Article west and east geneticwest andeast groups montane and Genomic alsorevealed data &

e.g., Ortego O. navasi

BOI, whichsuggestsahigher LLE), isolation populations ofalpine and has ,

2016 alpine organisms. played a key role on the diversificationplayed akeyroleonthe ing variation genetic inmontane/alpine grasshoppers

a and the alpineand the - ) montane populations ( up to . Genetic structure analyses showed split that ofthedifferentthe them (Figure (Figure ). T three genetic clusters comprisedthree clusters genetic each within main ofthetwo

presented signatures ofgenetic admixture

(Figure hese results might the low dispersal reflect capabilityof strong genetic differentiation and 2 ). The fact that almost). Thefactthat all

O. antigai s 1and

e.g., 2). It 2). It isnotew . This was corroborated . Thiswascorroborated by

SET nosupport andBAT), indicating s characterizingthe foothills. Pyrenean

not o orthy that orthy populations other sampled nly (Wa a deep genetic a deep structure of ng lowland speciesbut

populations populations ,

, 2013)

(Noguerales, (Noguerales, evidencing our landscape our landscape . Only have y This article isThis article by protected copyright. All rightsreserved. Accepted of Europeanconservation status grasshoppers has assigned the twotaxato different IUCN for can have species conservation andmanagement. Therecent assessment ofthe S1) Table synonymized unique a into taxon taxonomic ofthe status complex that andindicate delimitationspecies and analyses, distinctivenessecological ofinformationsources to delimit speciesthathave beendescribed solelymorphology by or divergedallopatrictaxaandrecently highlightsthe exemplifiesThis study problems the associated with 4.3. 2016b& Ortego, Article Billman,Ingley, Johnson, Belk, & 2014; Laiolo risk,predation microhabitat structure or adaptations by ecologicalandevolutionary aspects consideredin not Makinen, & 2006;Merila, Leinonen, O'Hara, Cano,&Merila, 2008 selectiondriven differences foundamong populations not are a consequenceof gen orenvironmentaldistances dissimilarity. Overall, suggest this found by populations among differentiation, notexplained were genetic geographical variationgenetic inthecomplex ( analyses,which genetic no revealed effect ofenvironmental dissimilarity on structuring Conclusions O ur geometric morphometric analyses showedthatthe subtle pheno .

Our study also Our study ( )

Keller, Alexander, Holderegger, &Edwards,2013;Leinonen, Cano, .

illustrates implications the taxonomic that incorrect designations ( e.g.,

Jones : i.e., phenotypic and did ecologicaldata notsupport the current O. antigai

isolation &

Weisrock

(Bolívar, 1897) - by ,

Illera, Illera, & Obeso, - O. navasi , environment;

importance ofintegrating different 2018)

to different resources feeding species validationtests involving this this study selection, assexual such

. Phylogenetic inferences,. Phylogenetic Bayesian

and

(for list ofsynonyms a s

that that the Shafer O. antigai 2013; etic driftetic orenvironmental )

andmight beexplained weak & Noguerales

Wolf

typic typic differences must phenotypic ,

2013) be

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see .

- This article isThis article by protected copyright. All rightsreserved. this of version earlier an on comments constructive and helpful for referees anonymous three sequencing. Illumina for Genomics) Applied for Centre (The Pereira Sergio Rodríguez Francisco libraries genomic Ampa to grateful are We ACKNOWLEDGEMENTS Knowles, 2016). distribution variationof genetic could 2011 analysesthat genetic studies genetic structure, strong phenotypiclittle variation, environmentalniche. andawide Future 2002). significant units populations bealsoshould considered conservationpotential infuture as plans evolutionary b ofthe status shouldbeprobablydownlistedto“ taxon ecologicalthe andhabitat requirements assessmentsRed List theresults, conservation ofthe status complex totalreconsider requires et (Braud al. categories Red List ) .

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This article isThis article by protected copyright. All rightsreserved. Accepted for download fr BioProject Raw DATA ACCESSIBILITY manuscriptthe samples. AP VT, AUTHORCONTRIBU Article fellowship,research respectively. (SEV Ochoa Severo a by supported were JO and AP work, this During Competitividad. y Economía de Ministerio from 73159) CGL2017 and Fund Development Regional European the and Competitiveness This resources. computer to (CESGA) Doñana. de Biológica Estación (LAST Teledetección y Geográfica Información de Sistemas de Laboratorio and article. I llumina have reads beendepositedat NCBIthe

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Systematic Biology, 59 (2002). phylogenomic andmorphologicalphylogenomic datatoassess speciescandidate - espece (Caelifera,Acrididae, Gomphocerinae).]. - model species. , B.,Llucia , . N., Kay, E. H., Fisher, H. S., & Hoekstra, H.E. . N.,Kay,H.,Fisher, Double H. S., E. (2012). &Hoekstra, Generalitat Catalunya, de Departament de AmbientMedi i Atlas Dels Ortòpters de Dels i VermellAtlas Ortòpters Catalunya Llibre 2002/ Systematic Biology, 55

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, 1908inFrance with

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[Sur la

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Proceedings the of Royal Society B ion, migrationion, and patterns, ocksnail, ocksnail,

Molecular 23 Ecology, Leptoxis ampla.Molecular

- Biological (23), (23), This article isThis article by protected copyright. All rightsreserved. (legendpriors bottom). at represent the meanprobability (PP)for posterior differentcombinations of demographic (gamma, (200, 500,and1000loci three alternative topologies (a: Figure asin 1. codes Table which at and(triangles) Figure 3 1. Table blackvertical separate individuals lines from populations. different segmentscolored probability ofbelonging representing the to cluster the with that color.Thin 2 Figure STRU map antigai and 6) Figure ( Figure 1 legendsFigure

Accepted Articleto K indicate the main the cluster populations genetic which at indicate were assignedaccording to CTURE

= 6) and= 6)

4 2 (triangles) and

individual α, β α, Results ofspecies delimitationResults analyses in Principal component analysis (PCAs) ofgenetic Genetic assignment ofindividuals (a) Bayesian

analyses for analyses )

for ancestral population (θ)andage size root (τ)

b) map of distribution showingthe sampled the populations of O. navasi ( b ) CONSTRUCT s

were assigned were assigned phylogenetic O. navasi ; two individuals/population); two K (dots). (dots). Node support in Node support termspro ofposterior

= 6. Population codes asinTable = 6.Population 1.

BPP ( C from from (dots) the Pyrenees. in Colorsof trianglesanddots olors ; b: according to tree tree reconstructed with K SNAPP

of indicate trianglesanddots = 2 to = 2to

based of on theresults ; c: ; c: K

SVDQUARTETS STRUCTURE = 4). Individuals Individuals are = 4). BPP , and . Analyses were performed were . Analyses

variation for four gamma com prior SNAPP

analyses for analyses ), three different subsets), three of loci . Coloredboxesateach node

babilities (

(node support support (node

( Population codes asin a

) the the partitioned into STRUCTURE Omocestus antigai main geneticcluster K

for = 6. Omocestus SNAPP Population Population presented binations binations

( using using from on the

tree K

K )

in = This article isThis article by protected copyright. All rightsreserved. Accepted Article asin 1. codes Table demog of combinations (PP) fordifferent (θ)androotage size (τ). Colored boxesateachnode mean represent the probability posterior SVDQUARTETS were performedanalyses three using alternativ phenotypic forseparately sex each both combining genomic (500loci and5individuals/population) and Figure Population codes inTable 1. as and bootstrapping values ( 5

Results ofspecies delimitationResults analyses in data (treesondata left) the and onlyusing ) and four) and gamma combinations prior (gamma,for populationα, β) ancestral

for

SVDQUARTETS

raphic priors (legend priors bottom at raphic right). Population

tree e topologies(a: ) is phenotypic indicated IBPP . Analyses were performed were . Analyses

data (treesondata right).All the

in parentheses node. foreach BPP ; b: ; b: SNAPP ; c: ; c: This article isThis article by protected copyright. All rightsreserved. sampled Table 1 AcceptedO. femoralis O. navasi O. navasi O. navasi O. navasi O. navasi ArticleO. navasi O. navasi O. navasi O. navasi O. antigai O. antigai O. O. antigai O. antigai O. antigai O. antigai Species antigai

Geographical location, Geographical andnumberelevation ofgenotypedindividuals ( population

bellmanni navasi navasi navasi navasi navasi navasi navasi navasi

(locality) Sierra de Espuña Sierra de Batère de Tour Tost de Montan Perves Espés Chía Saravillo Nerín de Monrepós Puerto Borau Setcases l'Home de Turó Queralbs Err laCreueta de Coll Llessui Taüll Boí Locality

of the

three

putative taxa ESP CHI SAR NER MON BOR SET TUR QUE ERR CRE LLE BOI Code ESP BAT TOS PER

42.44307 42.55418 42.56089 42.59047 42.35067 42.67388 42.42756 41.77167 42.36627 42.38830 42.30131 42.44554 42.47945 37.86515 42.50785 42.23790 42.35250 Latitude

and the outgroup

Longitude - - - 0.58389 0.43703 0.23046 0.01062 2.26630 2.44335 2.14902 2.08812 1.99350 1.03668 0.86732 2.57641 1.38052 0.83709 0.39762 0.57932 1.57115

.

Elevation (m)Elevation 1361 1691 1313 1623 1267 1357 2145 1622 2070 2150 1928 1979 2046 1514 1430 1182 1370 n ) for each ) foreach

6 7 2 7 5 7 7 7 6 3 6 6 5 n 6 7 7 6

This article isThis article by protected copyright. All rightsreserved. (calculated factors as 2 defining eachdelimitation alternative species ( hypothesis Table 2 H H H H H Species delimitation hypothesis ( 5 4 3 2 1

Accepted : (BAT+SET) (TOS) (CRE+ERR+QUE+TUR) (PER+SAR+CHI+NER+ESP) (MON+BOR) (BOI+LLE) : (BAT+TOS+CRE+ERR+QUE+TUR+SET) Article: (BAT) (PER+SAR+CHI+TOS+NER+ESP+MON+BOR) (CRE+ERR+BOI+LLE+QUE+TUR+SET) : (BOI+LLE+CRE+ERR+QUE+TUR+SET) (PER+SAR+CHI+NER+ESP+MON+BOR+BAT+TOS) : (BAT+PER+SAR+CHI+TOS+NER+ESP+MON+BOR+CRE+ERR+BOI+LLE+QUE+TUR+SET)

Results of Results BFD * analyses analyses testing thesupportofcompeting* species delimitation Thehypotheses. table shows scheme theclustering ×

ln BF) and their rank. codesln BF)andtheir Population inTable1. as H i )

(PER+SAR+CHI+NER+ESP+MON+BOR+BOI+LLE)

H i ). Forwe hypothesis, marginal each show likelihood estimates (ML

6 2 3 2 1 Species

- - - - - MLE 4,384.48 5,221.06 5,384.07 5,601.22 4,794.85

- 1.67 × 10 2.00 × 10 2.43 × 10 8.21 × 10 2

× lnBF

3 3 3 2

1 3 4 5 2 Rank

E), their E), their Bayes

This article isThis article by protected copyright. All rightsreserved. Accepted Article distances. differentiationgenetic ( Table 3

Multiple Matrix Multiple Matrix with RandomizationRegression (MMRR) for

term Rejected terms Retained Variable

Environmental distance Environmental (slope) distance Resistance distance Geographical Intercept

F

ST ) in relation with geographical, resistance relation withgeographical, ) in and environmental

Coefficient - 0.007 0.255 0.108 -

- - 5.278 6.841 1.061 3.079 t

pairwise population pairwise 0.374 0.004 0.001 0.991 p

This article isThis article by protected copyright. All rightsreserved. Accepted ArticleHochberg significant and geographical, resistance and environmental distances. phenotypic differentiation ( Table 4

Multiple Matrix Multiple Matrix Regressions with Randomization (MMRR) for false discovery rate Ovopositor valve Ovopositor valve Forewing Forewing Pronotum Pronotum Head Head

Geographical distance Geographical distance Geographical distance Geographical distance Geographical distance Geographical distance Geographical distance Geographical distance F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) F Environmental distance Resistance distance (slope) retained into retained ST ST ST ST ST ST ST ST

– –

PC2 PC1 – – – –

PC2 PC1 PC2 PC1

P

final – – ST

PC2 PC1 corrections ) in relationwithgeneticdifferentiation) in (

models

.

of Table shows p

------values toadjust formultipletests. statistical 1.882 0.125 0.989 0.448 0.617 1.401 0.637 1.614 0.210 1.110 3.159 0.388 2.695 2.775 0.868 0.963 2.781 1.423 1.047 1.699 0.257 0.408 0.282 0.222 2.197 0.190 0.735 1.240 0.267 0.960 0.416 1.264 t

Females

No independent variablewas

q 0.791 0.791 0.686 0.950 0.791 0.791 0.919 0.919 0.791 0.919 0.919 0.919 0.686 0.919 0.919 0.791 0.400 0.919 0.919 0.520 0.686 0.919 0.405 0.919 0.791 0.919 0.919 0.791 0.791 0.919 0.791 0.400

q -

values

after after

------0.135 1.223 2.045 0.782 2.185 0.695 1.152 0.318 0.715 0.587 1.713 0.376 1.735 0.429 0.052 2.993 1.106 0.048 1.471 0.031 2.455 0.386 0.054 0.858 – – – – – – – – t

applying

Males F

pairwise population pairwise

ST

) and ) and 0.980 0.973 0.864 0.980 0.288 0.853 0.980 0.720 0.973 0.496 0.840 0.980 0.288 0.973 0.973 0.853 0.980 0.973 0.973 0.973 0.853 0.973 0.973 0.840

q – – – – – – – –

Benjamini

-

This article isThis article by protected copyright. All rightsreserved. Accepted Article 1 Figure (b) (a) BOR

MON NER SAR CHI ESP PER BOI LLE TOS

CRE ERR QUE TUR SET BAT

This article isThis article by protected copyright. All rightsreserved. Accepted Article 2 Figure (b) (a) CONSTRUCT STRUCTURE

K = 4 K = 3 K = 2 K = 6 K = 5 K = 4 K = 3 K = 2

This article isThis article by protected copyright. All rightsreserved. Accepted Article 3 Figure

This article isThis article by protected copyright. All rightsreserved. Accepted Article 4 Figure

This article isThis article by protected copyright. All rightsreserved. Accepted Article 5 Figure