Genomic, morphological and physiological data support fast ecotypic differentiation and incipient speciation in an alpine diving beetle

Molecular Ecology. 2024;33:e17487.   | 1 of 19 https://doi.org/10.1111/mec.17487 wileyonlinelibrary.com/journal/mec Received:8March2024 | Revised:31May2024 | Accepted:22July2024 DOI: 10.1111/mec.17487 ORIGINAL ARTICLE Genomic, morphological and physiological data support fast ecotypic differentiation and incipient speciation in an alpine diving beetle Susana Pallarés1 | Joaquín Ortego2 | José Antonio Carbonell1 | Eduardo FrancoFuentes1 | David T. Bilton3,4 | Andrés Millán5 | Pedro Abellán1 This is an open access article under the terms of the CreativeCommonsAttribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. ©2024TheAuthor(s).Molecular EcologypublishedbyJohnWiley&SonsLtd. 1Department of Zoology, University of Seville,Seville,Spain 2Department of Ecology and Evolution, EstaciónBiológicadeDoñana,EBD-CSIC, Seville,Spain 3SchoolofBiologicalandMarineSciences, University of Plymouth, Plymouth, UK 4Department of Zoology, University of Johannesburg,Johannesburg,South Africa 5Department of Ecology and Hydrology, UniversityofMurcia,Murcia,Spain Correspondence SusanaPallarés,DepartmentofEcology and Hydrology, University of Murcia, Murcia,Spain. Email: [email protected] Present address JoséAntonioCarbonell,Departmentof Zoology, University of Murcia, Cordoba, Spain Funding information NextGenerationEU;MinisteriodeCiencia eInnovación,Grant/AwardNumber: PID2019-108895GB-I00;Ministeriode Universidades,Grant/AwardNumber: 19868; Consejería de Economía, Conocimiento, Empresas y Universidad, JuntadeAndalucía,Grant/AwardNumber: SP-DOC_01211 Handling Editor: Brent Emerson Abstract An intricate interplay between evolutionary and demographic processes has frequentlyresultedincomplexpatternsofgeneticandphenotypicdiversityinalpine lineages, posing serious challenges to species delimitation and biodiversity conservation planning. Here we integrate genomic data, geometric morphometric analyses and thermaltoleranceexperimentsto exploretherole ofPleistoceneclimaticchanges and adaptation to alpine environments on patterns of genomic and phenotypic variationindivingbeetlesfromthetaxonomicallycomplexAgabus bipustulatus species group.Geneticstructureandphylogenomicanalysesrevealedthepresenceofthree geographicallycohesivelineages,tworepresentingtrans-PalearcticandIberianpopulationsoftheelevation-generalistA. bipustulatus and another corresponding to the strictly-alpineA. nevadensis,anarrow-rangeendemictaxonfromtheSierraNevada mountainrangeinsoutheasternIberia.Thebest-supportedmodeloflineagedivergence,alongwiththeexistenceofpervasivegeneticintrogressionandadmixturein secondary contact zones, is consistent with a scenario of population isolation and connectivity linked to Quaternary climatic oscillations. Our results suggest that A. nevadensis is an alpine ecotype of A. bipustulatus, whose genotypic, morphological and physiological differentiation likely resulted from an interplay between population isolation and local altitudinal adaptation. Remarkably, within the Iberian Peninsula, suchecotypicdifferentiationisuniquetoSierraNevadapopulationsandhasnotbeen replicated in other alpine populations of A. bipustulatus. Collectively, our study supports fast ecotypic differentiation and incipient speciation processes within the study complexandpointstoPleistoceneglaciationsandlocaladaptationalongelevational gradients as key drivers of biodiversity generation in alpine environments. KEYWORDS alpineecosystems,Coleoptera,glacialrefugia,hybridisation,integrativetaxonomy,Pleistocene speciation,sky-islands 2 of 19 | PALLARÉS et al. 1 | INTRODUCTION Understanding the processes that generate and maintain biodiversity is a central issue in evolutionary biology (Avise, 2000), with clearimplicationsforconservationmanagement(Coatesetal.,2018; Seehausen,2006;Stantonetal., 2019).High-elevationtemperate mountains, traditionally considered as centres of lineage diversification or‘speciespumps’ (Schovilleet al., 2012),provideaglobal model for understanding patterns of biodiversity and the evolutionaryprocessesinvolvedinspeciation(Antonellietal.,2018; Flantua et al., 2020). Historical environmental changes during repeated glaciation and deglaciation events in the Pleistocene had a dramatic impact on the patterns of ecological and genetic diversity of both alpine and lowland biotas (Baker, 2008; Hewitt, 2000; Weir & Schluter, 2004). Glacial cycles have induced range expansions during either glacial or interglacial periods – depending on the ecology of the species – followed by range contractions to refugia whenconditionsbecamemoreadverse(Bennett&Provan,2008; Dynesius&Jansson,2000;Stewartetal.,2010).Pleistoceneice ages have often promoted lineage diversification via cycles of allopatry in ecologically divergent refugia, which has been identified as an important driver of the formation of alpine endemics (Tribsch, 2004). Such alpine endemics are subject to repeated cycles of isolation, divergent adaptation and secondary contact(e.g.the‘glacialpulse’modelofalpinediversification;Maier et al., 2019).Secondarycontactcanacceleratespeciationviathe reinforcement of incipient reproductive isolation (Butlin, 1987; Hedrick, 2013)orleadtotheformationofhybridspecies(Mavárez &Linares,2008;Noguerales&Ortego,2022).However,speciation is not always a linear, unidirectional process and divergence may also slow down or even be reversed if lineages that have not evolved reproductive barriers merge when they came into secondarycontact(i.e.‘speciationreversal’or‘lineagefusion’;Garrick et al., 2014; Kearns et al., 2018;Seehausenetal.,2008). In mountain systems, lineage formation associated with glacial cycles can be accompanied by either phenotypic plasticity or local adaptation processes along altitudinal gradients, as these systems are characterised by steep environmental changes over short geographicaldistances(i.e.strongselectiondifferentials;Körner,2007; Steinbaueretal.,2013).Infact,therearenumerouscasesofmontane andalpineformswithin species andspecies complexes forwhich local adaptation and phenotypic plasticity play contrasting roles (Kelleretal., 2013;Stanbrooket al.,2021; Tsuchiya et al., 2012). Theselineagesoftenshowcomplexpatternsofgenetic,ecological andphenotypicdiversity,promptingintensetaxonomicdebates(e.g. Drotz et al., 2012; McCulloch et al., 2019; Ortego et al., 2021; Tonzo et al., 2019),whosesolutionwillrequirefullyintegratedresearch approaches. TheSierraNevadamassifinsoutheasternIberiaisrecognised as a biodiversity and endemicity hotspot for plants (Médail & Quézel,1997, 1999)andanimals(Ruanoetal.,2013).Itsisolation fromothercomparablemountainranges(e.g.thePyrenees)and its location at the southernmost limit of influence of Quaternary glaciations in Europe, have resulted in a high number of evolutionarilyuniquetaxaandspeciesassemblagesinthismountainrange (Zamora&Oliva,2022).TheSierraNevadawascoveredwithglaciers only at elevations >2500 m,withlargeareasremainingfree of glacial ice (Gómez-Ortiz et al., 2013). This, coupled with the largeandrapidaltitudinalgradient(0–3479 min35 km,fromthe coasttothehighestpeak),meansthatmanytaxalikelysurvived glacial cycles locally. As a consequence, this system provides a unique opportunity to understand processes of local adaptation linked to glacial cycles, study how species and populations have evolvedinhigh-altitudeenvironmentsandevaluatethepotential impacts of ongoing climate warming on the conservation of narrowlyendemictaxa. TheSierraNevadahostsasystemofglacialpondsandlakes between approximately 2800 and 3100 m that harbour highly specificassemblagesof cold-adapted macroinvertebrates,some ofthemmicroendemictothisarea(Millánetal.,2013),including the diving beetle Agabus nevadensis Lindberg, 1939. Despite being currentlyrecognisedasavalidspecies(Nilsson&Hájek,2021),its precise taxonomic status in relation to its widespread Western Palearctic congener A. bipustulatus(Linnaeus,1767)hasbeen subjecttomuchdebate(Bergstenetal.,2012; Drotz et al., 2001, 2010, 2012).Whatevertheirtaxonomy,thesebeetlesprovidean excellent model system for exploring lineage diversification in mountain systems and the processes driving phenotypic and genetic divergence along altitudinal gradients, since A. nevadensis is restricted to high altitude waters and is completely surrounded by populations of A. bipustulatus at lower elevations in the region. A. nevadensisdiffersexternallyfromA. bipustulatus on its smaller size, slenderer and more elongate body shape, secondary elytral reticulationpatternandtheshapeofmaleprotarsalclaws(Millán et al., 2014),althoughthesecharactersvarysomewhatinA. bipustulatusandthegenitaliaofthetwotaxaarealmostidentical. Angusetal.(2013)examinedthekaryotypesofseveralDytiscidae species, and found no differences between A. nevadensis and A. bipustulatus. Intraspecific morphological variability across altitudinal gradients is common within the A. bipustulatuscomplex,with numerousformsofuncertaintaxonomicstatusacrossitsdistributionalrange(Drotzetal.,2012).Mostofthesearepresumably alpine,cold-adaptedmorphotypes,suchasthesolieriAubé,1837 or kiesenwetteriiSeidlitz,1887forms,foundindifferentmountainrangesofEurope(Balfour-Browne,1950; Drotz et al., 2001, 2010; Sharp, 1882). In light of this variation, it has been suggested that A. nevadensis might also represent a morphotype of A. bipustulatus(Riberaetal.,1998);differencesbetweenthetaxa reflecting intraspecific altitudinal variation rather than altitudinal speciation. Agabus nevadensis nests deeply within A. bipustulatusingenefragment-basedphylogenies(Bergstenetal.,2012; Drotz et al., 2010),butallozymestudiesofthecomplexsupport the hypothesis of recent reproductive isolation between the two taxa(Drotzetal.,2010),makingitdifficulttodistinguishbetween these possibilities at present. 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 3 of 19 PALLARÉS et al. Together with genomics and morphometrics, physiological characterisation of populations could be also useful in shedding light on unresolved species complexes, but has seldom been incorporatedintointegrativetaxonomicstudies(e.g.Chen&Hare,2008; Degerlund et al., 2012; Muangmai et al., 2015).WhilstA. nevadensis is restricted to the high-mountain lakes of the Sierra Nevada (>2500 m),A. bipustulatus occupies a wider latitudinal and altitudinalrange(fromsealeveltoover3500 m a.s.l., Table 1)acrossthe WesternPalearcticandoccursinallthemainIberianmountainsystems and a wide diversity of freshwater habitats, including alpine lakes. Therefore, some degree of divergence in the environmental nichemightbeexpectedamongstthesetaxa(e.g.differingthermal tolerance,acriticalaspectofaspeciesfundamentalniche;Arribas et al., 2012; Calosi et al., 2010). The aim of this study was to use A. nevadensis and A. bipustulatus as a study system to analyse the role of Pleistocene climatic changes, local adaptation and phenotypic plasticity along elevation gradients onpatternsofgenomicandphenotypicvariationinalpinetaxa.To thisend,wefirstusedsinglenucleotidepolymorphism(SNP)data from populations covering the entire altitudinal distribution range of A. nevadensisandmultipleIberianandtrans-Palearcticpopulations of A. bipustulatusto(i)investigatespatialpatternsofgenetic structureanddelineatelineageswithinthecomplex,(ii)infertheir timingofdiversificationandpastdemographichistory,and(iii)detect signatures of ongoing or historical hybridisation and genetic introgressionamongidentifiedlineages.Second,weusedgeometric morphometricsandphysiologicalexperiments(thermaltolerance)to (iv)assesswhetherpatternsofphenotypicvariationarecongruent withgenomic-basedinferencesandtheextenttowhichsuchvariation is associated with phenotypic plasticity and/or local adaptation along altitudinal gradients. 2 | MATERIALS AND METHODS 2.1 | Study area and sampling OurstudyareacoverstheSierraNevadamountainrangeinsoutheastern Iberia, to which the endemic A. nevadensis is restricted, and severalpopulationsofthewidespreadWesternPalearcticA. bipustulatus(Table 1; Figure S1). We sampled 26 populations covering thefullaltitudinalrangeofeachtaxonintheIberianPeninsula,from waterbodies located at sea level to alpine lakes in the major mountain ranges. Additionally, five populations of A. bipustulatus from otherEuropeanregionsandtheMiddleEastwereincluded(Table 1; Figure S1)andAgabus bigutattus(Olivier,1795)wasusedasanoutgroupinphylogenomicanalyses.Weusedanaquatichandnetto collecteightto12specimensperlocality.Specimenswerestored in96%ethanolandpreservedat−20°Cforgenomicanalyses.Ina subset of localities where beetles were more abundant, additional specimens were collected for either morphometric and/or physiological analyses. The specific populations and sample sizes used for each analysis are presented in Table 1. 2.2 | Genomic library preparation and processing We extracted and purified DNA from each specimen using NucleoSpinTissuekits(Macherey-Nagel,Düren,Germany).WeprocessedDNAofA. nevadensis, A. bipustulatus and A. bigutattus into differentgenomiclibrariesusingthedouble-digestion restriction- fragment-based procedure (ddRAD-seq) described in Peterson etal.(2012).Inbrief,wedigestedDNAwiththerestrictionenzymes MseI and EcoRI (New EnglandBiolabs,Ipswich,MA,USA)andligatedIlluminaadaptorsincludingunique7-bpbarcodestothedigestedfragmentsofeachindividual.Wepooledligationproducts, size-selected them between 350 and 450 bp with a Pippin Prep machine(SageScience,Beverly,MA,USA)andamplifiedthefragmentsbyPCRwith12 cyclesusingtheiProofTMHigh-FidelityDNA Polymerase(BIO-RAD,Veenendaal,TheNetherlands).Single-read 201-bp sequencing was performed on an Illumina NovaSeq6000 platform.Weusedthedifferentprogramsdistributedaspartofthe stacksv.1.35pipeline(Catchenetal.,2013)tofilterandassemble our sequences into de novo loci, call genotypes, calculate genetic diversitystatistics,andexportinputfilesforalldownstreamanalyses.Unlessotherwiseindicated,weexportedonlythefirstSNPper RADlocus,andretainedlociwithaminimumstackdepth ≥5(m = 5), aminimumminorallelefrequency(MAF) ≥ 0.01(min_maf = 0.01)and thatwererepresentedinatleast80%ofthepopulations(p = 25)and 50%oftheindividualswithineachpopulation(r = 0.5).Formoredetailsongenomicdatafilteringandassembling,seeAppendixS1. 2.3 | Analyses of genetic structure and admixture We first performed a comprehensive suite of analyses to infer patterns of genetic structure, differentiation, admixture and hybridisation amongst studied lineages and populations. These included(i)geneticclusteringanalysesinstructurev.2.3.3(Pritchard et al., 2000), (ii) principal component analyses (PCA) of genetic variation (Jombart,2008),(iii)estimatesofgeneticdifferentiation (FST) between populations, (iv) reconstructions of phylogenomic relationships amongstlineages/populations in svdquartets(Chifman &Kubatko,2014)and(v)identificationofhybridcategories using NewHybridsv.1.1(Anderson&Thompson,2002).Next,weanalysed whether the probability of assignment of populations to the two geneticlineagescoexistingwithintheSierraNevadamountainrange (see section 3.2) is best explained by their geographical location and/or environmental factors. 2.3.1 | Geneticclusteringanalyses Weranstructure analyses assuming correlated allele frequencies andadmixtureandwithoutusingpriorpopulationinformation.We conducted 15 independent runs for each value of K(fromK = 1 to K = 8) toestimatethe mostlikelynumberofgeneticclusters with200,000MCMCcycles,followingaburn-instepof100,000 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 19 | PALLARÉS et al. TABLE 1 Localitiessampledandthenumberofindividuals(N)usedforgenomic,geometricmorphometricandphysiologicalanalyses. Taxon Locality N No Code Name Country Lat Lon Altitude (ma.s.l.) Genomics Geometric morphometry Physiology Agabus bipustulatus 1IRAN StreamnearGhachsar,AlborzMts. Iran 36.181 51.319 3500 8 5♀, 3♂ A. bipustulatus 2SARD RíoPisciaroni,Sardinia Italy 40.858 9.157 1030 8 A. bipustulatus 3ALPS Poolsnr.Chandolin,Alps Switzerland 46.254 7.634 2400 10 A. bipustulatus 4PENN PoolsatHighCupNick,Cumbria, Pennines United Kingdom 54.643 −2.415 680 10 A. bipustulatus 5SOME DitchatChiltonPolden,Somerset United Kingdom 51.177 −2.881 2 4 15♀, 15♂ A. bipustulatus 6AZUL IbónAzulSuperior,Pyrenees Spain 42.789 −0.246 2407 815♀,15♂76 A. bipustulatus 7ARME IbóndeArmeña,Pyrenees Spain 42.516 0.353 1850 10 A. bipustulatus 8LLOR Lagos de Lloroza, Picos de Europa Spain 43.164 −4.812 1870 8 A. bipustulatus 9MOLI Pleta de Molières, Pyrenees Spain 42.627 0.718 2000 10 A. bipustulatus 10 MONE Lago Moñetas, Picos de Europa Spain 43.192 −4.789 1710 8 A. bipustulatus 11 NEIL Poolnr.LagunaLarga,SierradeNeila Spain 42.045 −3.061 1895 6 A. bipustulatus 12 URBI Zona encharcada en Laguna Helada, Picos de Urbión Spain 41.995 −2.861 2000 813♀, 10♂ A. bipustulatus 13 GRAN LagunaGrandedeGredos,Sierrade Gredos Spain 40.254 −5.275 1943 823♀, 15♂70 A. bipustulatus 14 POZA PradodelasPozas,SierradeGredos Spain 40.270 −5.246 1923 8 A. bipustulatus 15 PENA Poolnr.LagunaGrandedePeñalara, SierradeGuadarrama Spain 40.837 −3.951 1940 8 A. bipustulatus 16 CLAV Pools nr. Laguna de los Claveles, SierradeGuadarrama Spain 40.850 −3.948 2116 815♀,15♂66 A. bipustulatus 17 PAJA LagunadelosPájaros,Sierrade Guadarrama Spain 40.861 −3.948 2170 8 A. bipustulatus 18 MURT RíoMúrtigas,LaNava,Huelva Spain 37.957 −6.745 419 815♀, 15♂22 A. bipustulatus 19 FSAL FuenteSalobre,AlbaidadelAljarafe, Sevilla Spain 37.426 −6.165 163 8 3♀, 4♂ A. bipustulatus 20 ESPU FuenteBlanca,SierraEspuña Spain 37.886 −1.565 1142 924♀, 17♂60 A. nevadensis 21 SJUA LagunillodeSanJuan,SierraNevada Spain 37.088 −3.372 2520 8 A. nevadensis 22 LAVR Laguna de los Lavaderos de la Reina, SierraNevada Spain 37.124 −3.273 2635 10 A. nevadensis 23 JUNT LagunadeJuntillas,SierraNevada Spain 37.110 −3.264 2930 8 A. nevadensis 24 HOND LagunaHondera,SierraNevada Spain 37.048 −3.294 2897 9 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 5 of 19 PALLARÉS et al. iterations.Weretainedthe10runshavingthehighestlikelihood for each value of K and determined the number of genetic clusters that best describes our data according to log probabilities of the data(LnPr(X|K);Pritchardetal.,2000)andtheΔKmethod(Evanno et al., 2005),asimplementedinstructure Harvesterv.0.7(Earl& vonHoldt, 2012).Weusedclumppv.1.1.2andtheGreedyalgorithm to align multiple runs of structure for the same Kvalue(Jakobsson &Rosenberg,2007)anddistructv.1.1(Rosenberg,2004)tovisualise the individuals' probabilities of population membership in bar plots. 2.3.2 | Principalcomponentanalysesofgenetic variation WeranaPCAofgeneticvariationasimplementedinther v. 4.2.3 (R Core Team, 2021) package adegenetv.2.1.10(Jombart,2008). Before running the PCA, we replaced any missing data with the mean allele frequency of the corresponding locus estimated across allsamples(Jombart,2008). 2.3.3 | Geneticdifferentiationbetweenpopulations WecalculatedgeneticdifferentiationbetweeneachpairofpopulationsusingtheWeirandCockerhamweightedfixationindex(FST; Weir&Cockerham,1984)asimplementedinarlequiNv.3.5(Excoffier &Lischer,2010). These analyseswere performedfor populations with a sample size of n ≥ 5afterexcludingthoseindividualsidentified by structureasbeinghybrid/admixed(i.e.q < 0.99forK = 3;seesection 3.2).WedeterminedstatisticalsignificancewithFisher'sexact tests after 10,000 permutations, applying a false discovery rate adjustment(5%,q < 0.05)tocontrolformultipletests. 2.3.4 | Phylogenomicanalyses We ran svdquartets, as implemented in PAUP* v. 4.0a169 (Swofford,2002),toestimatetherelationshipsamongstpopulations and lineages identified by structure(i.e.apopulation/speciestree). To reduce the confounding effects of contemporary hybridisation on phylogenetic reconstructions, we excluded from the dataset hybrid/admixedindividualsidentifiedbystructure(i.e. q < 0.99for K = 3;seesection3.2; e.g. Maier et al., 2019).WeusedA. bigutattus as an outgroup, evaluated 100,000 random quartets from the data set and quantified uncertainty in relationships using 100 bootstrapping replicates. 2.3.5 | Identificationofhybridcategories WeperformedaBayesianassignmenttestofsamplesintodiscrete hybrid/parental categories using NewHybrids, which computes the Taxon Locality N No Code Name Country Lat Lon Altitude (ma.s.l.) Genomics Geometric morphometry Physiology A. nevadensis 25 MOSC LagunadelaMosca,SierraNevada Spain 37.060 −3.315 2895 8 A. nevadensis 26 CALD LagunadeLaCaldera,SierraNevada Spain 37.055 −3.329 3030 918♀, 10♂67 A. nevadensis 27 AVER LagunadeAguasVerdes,Sierra Nevada Spain 37.049 −3.368 3055 814♀, 15♂66 A. nevadensis 28 VIRG LagunillosdelaVirgen,Sierra Nevada Spain 37.053 −3.379 2945 812♀, 4♂ A. nevadensis 29 MEDE LagunilloMediodelaErmita,Sierra Nevada Spain 37.050 −3.385 2870 8 A. nevadensis 30 LLAN LagunadeLanjarón,SierraNevada Spain 37.038 −3.400 2975 8 A. nevadensis 31 CUAD LagunaCuadrada,SierraNevada Spain 37.027 −3.419 2910 814♀, 13♂ Note:AmapofthestudyareaisshowninFigure S1. TABLE 1 (Continued) 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 19 | PALLARÉS et al. posteriorprobability(PP)distributionthateachsamplefallsintoone ofsixgenotypicclasses:parentalclasses(P1andP2),first(F1)and second-generation (F2)hybrids,andbackcrosses to both parental classes(BC1andBC2).NewHybrids analyses were performed for two datasets,oneincludingthetrans-PalearcticandIberianlineagesof A. bipustulatus and another including the Iberian lineage of A. bipustulatus and A. nevadensis(seesection3.2).Toovercomecomputational limitations of NewHybrids, we used the gl.nhybrids function in the r package dartRv.2.9.7(Gruberetal.,2018)toselectthesubset of 200 loci with the highest discriminatory power between parental classes.Weconsideredasreferenceparentalgenotypesthoseindividualswithahighprobabilityofassignment(q > 0.99)totheirrespective genetic clusters, as inferred by structure analyses for K = 3 (seesection3.2).WeranNewHybrids with default parameters and 50,000MCMCstepsafter10,000burn-instepsoneachofthetwo datasets. 2.3.6 | Driversofgeneticadmixturewithinthe SierraNevada SomeputativepopulationsofA. nevadensisfromtheSierraNevada exhibited different degrees of admixed ancestry with the Iberian lineage of A. bipustulatus and, in some cases, even included purebred specimens of this lineage, that is, samples with a high probabilityofassignment(structure q-value>0.99)tothisgeneticcluster (seesection3.2).Forthisreason,weusedsimplelinearregressions toexploretherelationshipbetweenthepopulation-averageprobability of assignment to the Iberian lineage of A. bipustulatus and (i)climaticconditions,(ii)altitudeand(iii)the‘accessibility’toeach population from the lowlands, measured as the distance between thefocalpopulationandthe2500 mcontour,aproxyofthedistributional range limit of A. nevadensis. Climatic conditions were estimated usingthe 19 present-day bioclimatic variables downloaded fromWorldClimv.2.1(Fick&Hijmans,2017)at30arc-sresolution (ca.1 kmattheEquator)fortheSierraNevadaanditssurroundings. WeperformedaPCAonbioclimaticvariablesandobtained,foreach population,scoresofthefirstprincipalcomponent(PC),whichexplained 85% of the climatic variance and was mainly negatively correlated with mean annual temperature and the mean temperatures of the wettest, driest and coldest quarters and positively correlated with annual precipitation and precipitation in the driest and warmest quarters(seefactorloadingsinTable S1). 2.4 | Testing alternative models of lineage divergence We used the coalescent-based approach implemented in fastsimcoal2 (Excoffier et al., 2013) to test alternative models of divergence amongst A. nevadensis and the two lineages of A. bipustulatus. Specifically,wetestedamodelofdivergenceinstrictisolation(SI) andmodelsofisolation-with-migrationconsideringeithersymmetric (IMS)orasymmetric(IMA)geneflowamongstlineages(Figure S2). For fastsimcoal2 analyses we considered the three lineages inferred by structure for K = 3 and the topology yielded by phylogenomic analyses in svdquartets(seesection3.2).Theseanalysesaimedatinvestigating historical process of population divergence and genetic introgression amongst the three main lineages. For this reason, and in order to remove the confounding effects of contemporary hybridisationand recentadmixture,weexcludedallhybrid/admixed individualsfromthedataset(i.e.structure q < 0.99forK = 3,asfor phylogenomic reconstructions; see Results section; e.g. Bertola et al., 2024; Momigliano et al., 2021;Nogueralesetal.,2024).Note alsothatincludinghybridindividuals(e.g.F1andF2)intheseanalyses would require arbitrary decisions about how to assign them to each of the three discrete parental populations. Divergence times were estimated assuming two generations per year, although voltinism may decrease under unfavourable climatic conditions (Čiamporová-Zaovičová&Čiampor,2011;Galewski&Tranda,1978; Nilsson&Holmen,1995).Fordetailsonfastsimcoal2 analyses and modelselection,seeAppendixS2. 2.5 | Genetic diversity and past demographic history First, we calculated different estimates of genetic diversity for eachstudiedpopulation(Table 1)usingtheprogrampopulations from stacks(Catchenetal.,2013)andusedone-wayanalysesof variance(ANOVAs)totestforsignificantdifferencesingeneticdiversity between the three lineages inferred by structure analyses (seesection3.2).Second,wereconstructedthepastdemographic history from each lineage using the program stairway plot v. 2.1, which implements a flexible multi-epoch demographic model basedonthesite-frequency-spectrum(SFS)thatdoesnotrequire whole-genomesequencedataorreferencegenomeinformation (Liu&Fu,2020).WecomputedtheSFSforeachlineageasdescribed for fastsimcoal2analyses(AppendixS2)andranstairway plot considering two generations per year, assuming a mutation rateof2.8 × 10−9persitepergeneration(Keightleyetal.,2014), and performing 200 bootstrap replicates to estimate 95% confidence intervals. 2.6 | Geometric morphometric analyses We used landmark-based geometric morphometric analyses to examine shape and size variation amongst (i) the two currently recognised taxa, (ii) the lineages inferred by genetic clustering analyses for K = 3 (section 3.2) and (iii) all sampled populations (Table 1). We excluded populations with hybrid/admixed individuals identified by structure(i.e.q < 0.99forK = 3)fromthese analyses. Unfortunately, the number of genotyped specimens per population(n = 8–10)wasinsufficienttoperformrobustmorphometric analyses comparing purebred specimens with individuals 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 7 of 19 PALLARÉS et al. exhibiting different degrees of admixed ancestry (i.e. different earlygenerationhybridclasses).Wetookdigitalimagesoftheright elytronandcaptured11landmarks(2Dconfiguration;Figure S3). Weexaminedelytralshapeasaproxyforbodyshape,whichisone of the characters often use to distinguish between Agabus species(Millánetal.,2014).Thecoordinatesofthelandmarkswere mapped onto images using tpsdigv.2.32(Rohlf,2015).Weperformed generalised Procrustes analyses to remove the effects of location, size, and rotation of the relative positions of landmarks amongst specimens. Centroid size was calculated as the square root of the sum of the square distances from the landmarks to the centroidthattheydefined(Zelditchetal.,2004)andwasusedas aproxyforspecimensize.Wetestedforsizedifferencesamongst thecurrentlyrecognisedtaxa,inferredlineagesandpopulations, usingANOVAswithcentroidsizeasthedependentvariable,followed by post hoc pairwise comparisons. We checked that our results on shape variation were not affected by the low sample sizes of some populations (Table 1) by using data from those populations with sample sizes >25 and estimatingProcrustesdistancesbetweenpopulationsusing(i)thefull sample size and (ii) four randomly chosen specimens of each sex (n = 100 runs).Resultsshowed no significantdifferencesbetween observedandsubsampleddistances(p > .05).WeusedProcrustes ANOVA(Collyeretal.,2015;Goodall,1991)toassessshapedifferencesbetweenthecurrenttaxa,theinferredlineagesandthesampledpopulations.Weusedtheresidualrandomisationpermutation procedure(RRP)toestimateeffectsizesandthesignificanceofthe terms(Anderson&TerBraak,2003).Wethenperformedposthoc pairwise comparisons of Procrustes distance between lineages and populations.Additionally,weperformedcanonicalvariateanalyses (CVA), which provides axes that maximise discrimination among groups(Zelditchetal.,2004),tovisualiseshapevariationamongst inferred lineages and populations. Finally, to assess the degree to which morphological variation is driven by local adaptation to altitudinal and climatic gradients, we exploredtherelationshipbetweenelytrumshape(scoresofthefirst CV axis) and both altitude and climatic conditions across Iberian populationsofthecomplex.Climaticconditionswereestimatedwith aPCAusingbioclimaticvariables,asdescribedinsection2.3.6, but for all Iberian populations. The scores from the first and second PCs, which together accounted for 77% of the climatic variance, were obtained for each population. PC1 was mainly negatively correlated with the maximum temperature of the warmest month and positively with annual precipitation and PC2 was negatively correlated with temperature seasonality and positively with minimum temperatureofthecoldestmonth(seefactorloadingsinTable S1). For size and shape analyses, models were performed first includingsexanditsinteractionwithtaxa,lineageorpopulationaspredictors.Then,assignificanteffectsofsexwerefound(seesection3.5), eachsexwasanalysedseparately. Morphometric analyses were performed in the r package geomorphv.4.0.5(Adams&Otarola-Castillo,2013)andthesoftware morpHoJv.1.07a(Klingenberg,2011). 2.7 | Thermal limits experiments Wecomparedthethermaltolerance(upperandlowerthermallimits andtheirplasticityestimatedthroughanexperimentalapproach)between A. nevadensis and the Iberian lineage of A. bipustulatus.Weused thermal tolerance data of several populations of A. bipustulatus from a previousstudy(Pallarésetal.,2024)andreplicatedtheexperimental procedure to obtain thermal limits of two populations of A. nevadensis. Thepopulationsusedfortheseexperiments(Table 1)onlyincluded purebred individuals of each corresponding lineage. Specimens of A. nevadensis were collected alive in summer 2022 and transported within24 htothelaboratoryin500 mLcontainerswithmoistenedfilterpaper,placedinaportablerefrigeratorat10°C.Uponarrival,they wereallowedtohabituatetolaboratoryconditionsfor3 dayspriorto experimentsat10°Canda12:12 L:Dphotoperiodinaclimaticchamber(SANYOMLR-351).Then,groupsofindividualswereacclimatedat 10,15or20°Cfor7 daysinclimaticchambers.Maintenanceconditions inthelaboratoryaredescribedinPallarésetal.(2024).Afteracclimation,setsofindividualswererandomlydividedinsub-groupsof10–15 beetles to estimate upper and lower thermal limits. Heat tolerance was assessed by estimating the heat coma temperature(HCT)astheupperthermallimit.HCT,definedasthetemperature at which individuals experience paralysis prior to death, precededbyspasmodicmovementsoflegsandantennae(Chown &Terblanche,2006),wasestimated in air(i.e. ondryspecimens), employing a dynamic method in which temperature is increased and the time to reach a specific physiological response is recorded (Lutterschmidt&Hutchison,1997).Weusedaheatingrateof1°C/ min. Body surface temperature at the moment of paralysis was measured with infrared thermography. Coldtolerancewasestimatedusingthesupercoolingpoint(SCP) asalowerthermallimit.SCPisthetemperatureatwhichthebody fluidsoftheorganismbegintofreezewhenspecimensareexposed tocooling.SCPwasestimatedasthelowertemperaturereachedbefore the release of the latent heat of crystallisation, employing also adynamicmethodwithacoolingrateof−1°C/min,andalsousing infraredthermography.Fulldetailsofeachexperimentareshown inAppendixS3andPallarésetal.(2024).Allspecimensweresexed afterexperimentsandstoredin96%ethanolforuseinmorphometric analyses. DifferencesinHCTandSCPamongsttaxaandpopulations,and the effect of prior acclimation temperature, were determined using generalisedlinearmodels(GLMs)withanormalerrorstructureand identitylinkfunction.Sexwasalsoincludedasafixedfactor. 3 | RESULTS 3.1 | Genomic dataset The average number of reads retained per individual after the differentqualityfilteringstepswas2,724,121(range = 154,963–7,169,390 reads).On average, thisrepresented81%(range = 49–90%)ofthe 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 19 | PALLARÉS et al. totalnumberofreadsrecoveredforeachindividual.Afterfiltering loci(seeAppendixS1),thefinaldatasetincludingallgenotypedpopulationscontained1940unlinkedSNPs,withameancoveragedepth of 32×(mode = 34×;range = 6–58×)andanaverageproportionof missingdataof28%(mode = 19%;range = 14–70%). 3.2 | Analyses of genetic structure and admixture structure analyses showed that ΔK peaked at K = 2(ΔK = 4680) and K = 3(ΔK = 1257),followedbyasharpdecline(ΔK < 180)athigherK- values(Figure S4).However,LnPr(X|K)increasedfromK = 2toK = 7, reaching a plateau at K = 8(Figure S4).ForK = 2,onegeneticcluster grouped the putative populations from A. nevadensis with the great majority of Iberian populations of A. bipustulatus, whereas the second genetic cluster included the populations of A. bipustulatus sampled across the rest of the Palearctic (hereafter, BIP-PAL, for simplicity; Figure 1a). All individuals from two populations from the Pyrenees (ARMEandAZUL)werefullyassigned(q > 0.99)tothegeneticcluster BIP-PALmostlyrepresentedinextra-Iberianpopulations.Remarkably, individualswithahighprobabilityofassignment(q > 0.90)toeachof the two genetic clusters were syntopic in two populations from the CantabrianMountains(LLORandMONE)inNorthIberia(Figure 1a). Severalpopulations(PENN,SOME,SARD,LLORandMOLI)presented individualswithdifferentdegreesofadmixedancestrybetweenthe two lineages of A. bipustulatus, suggesting ongoing or historical hybridisationandintrogression(Figure 1a).ForK = 3,mostputativepopulations of A. nevadensis(hereafter,NEV)splitfromIberianpopulations of A. bipustulatus (hereafter, BIP-IBE). However, several individuals sampled in putative populations of A. nevadensis(SJUA,LAVRand HOND)wereassignedwithahighprobabilityofmembership(q > 0.99) tothegeneticclusterBIP-IBEmostlyrepresentedinIberianpopulations of A. bipustulatus; in these populations, individuals assigned with a high probability of membership to both genetic clusters were syntopic(Figure 1b,e).SeveralpopulationsfromSierraNevadaalsocontainedindividualswithdifferentdegreesofadmixedancestrybetween these two clusters, indicating ongoing hybridisation and introgression (Figure 1b,e).structure analyses for higher K-values(K = 4–7)revealed furthergeneticstructure(Figure S5). ThePCAyieldedresultsinlinewiththoseobtainedforstructure, separating three genetic clusters corresponding with the lineages BIP-PAL, BIP-IBEand NEV(Figure 1f).PC1separatedthelineage BIP-PALfromthelineagesBIP-IBEandNEV,PC2separatedBIP-IBE from NEV,and somesampleswithintermediatePC scores correspondingtoadmixed/hybridindividuals(Figure 1f). ExceptforthreecomparisonsinvolvingonepopulationofBIP- IBE(MOLI)andthreepopulationsofNEV(MOSC,AVERandVIRG), all pairwise FST values involving populations assigned to different lineages were significantly different from zero (FST range: 0.025– 0.606; Table S2).AllpairwiseFSTvaluesbetweenpopulationsofBIP- PALweresignificantlydifferentfromzero(FST range: 0.084–0.569; Table S2).Exceptforafewcomparisonsinvolvingthenearbypopulations AVER, VIRG and LLAN, all pairwise FST values between populationsofthelineageNEVwerealsosignificantlydifferentfrom zero(FST range: 0.000–0.268; Table S2).Incontrast,nopairwiseFST valuesbetweenpopulationsofBIP-IBEweresignificantlydifferent fromzero(FST range: 0.000–0.016; Table S2). Phylogenomic analyses in svdquartets supported the results from structure and revealed that A. nevadensis is nested within A. bipustulatus,whichisaparaphyletictaxon(Figure S6).Thetrans-Palearctic lineage of A. bipustulatus(BIP-PAL)issistertoacladeincludingthe Iberian lineage of A. bipustulatus(BIP-IBE)andA. nevadensis(NEV), whicharesisterlineages(Figure S6).However,thephylogeneticrelationships amongst lineages and populations were not well resolved in mostcases(bootstrapsupport<95%)andonepopulationofA. bipustulatus sympatric with A. nevadensis was included within the clade of A. nevadensis with a basal relationship with the rest of the populations. NewHybridsanalysesfortheBIP-PALandBIP-IBEdatasetunambiguously assigned (PP > 0.95) one individual from the population MOLItoabackcross(BC)withBIP-IBE,66individualstoBIP-PAL, and96individualstoBIP-IBE(Figure 1c).NewHybrids analyses for the BIP-IBE and NEV dataset unambiguously assigned (PP > 0.95) fourindividualstosecondgenerationhybrids(F2),83individualsto theBIP-IBElineage,and66individualstotheNEVlineage.Theremaining 18 samples could not be unambiguously assigned to a single genotypicclass(PP < 0.95;Figure 1d).Theseresultssuggestthatthe admixtureidentifiedbystructure in several individuals, especially thosecollectedoutsidetheSierraNevada(e.g.SOME,PENN,and LLOR),isprobablyduetosharedancestralalleles(i.e.retainedancestry)ratherthanaconsequenceofhybridisation. IntheSierraNevadapopulations,themeanprobabilityofassignment to the genetic cluster corresponding to the BIP-IBE lineage showedsignificantnegativecorrelationswith(i)thefirstPCsummarisingclimaticconditions(slope ± S.D. = −0.055 ± 0.016,p = .006, R2 = .533, n = 11), which mainly summarises annual and seasonal temperatureandprecipitationvariables(Table S1),(ii)thedistance to the 2500 m isohyet (slope ± S.D. = −0.0003 ± 0.0001, p = .017, R2 = .429, n = 11) and (iii) altitude (slope ± S.D. = −0.002 ± 0.0002, p < .001,R2 = .821,n = 11;Figure S7). 3.3 | Testing alternative models of lineage divergence themodelbestexplainingtheformationofthethreelineagesofA. nevadensis and A. bipustulatusisascenarioofisolation-with-migrationand asymmetricgeneflow(IMA; Table 2; Figure 2);othertestedmodelsreceivingmuchlowerstatisticalsupport(ΔAIC>99; Table 2).Considering twogenerationsperyear,thesplitoftheBIP-PALfromthetwoother lineages was estimated to have taken place during the last glacial period (ca.57 ka),whereastheIberianlineagesBIP-IBEandNEVdivergedat theendofthelastglacialperiod(ca.14 ka;Table 3; Figure 2).Effective migration rates per generation between demes were asymmetric and significantly different (i.e. 95% confidence intervals do not overlap; Table 3). Remarkably, gene flow from BIP-IBE to NEV was five-fold higherthanintheoppositedirection(Table 3; Figure 2). 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 9 of 19 PALLARÉS et al. 3.4 | Genetic diversity and past demographic history GeneticdiversitystatisticsarepresentedinTable S3.Geneticdiversity differed amongst populations assigned to the three lineages (one-wayANOVAs; HO: F2,20 = 6.05, p = .009; HE: F2,20 = 7.21, p = .004;π: F2,20 = 7.95,p = .003;FIS: F2,20 = 8.33,p = .002).Posthoc Tukey's tests revealed that these differences were due to the higher levels of genetic diversity in the Iberian lineage of A. bipustulatus thaninthetrans-PalearcticlineageofA. bipustulatus(HO: p = .007; HE: p = .004;π: p = .003;FIS: p = .006)andA. nevadensis(FIS: p = .011; for all other statistics, p-values>.05).stairway plot analyses showed thatthethreelineageshaveexperienceddemographicexpansions justbefore(trans-PalearcticlineageofA. bipustulatus)orjustafter (IberianlineageofA. bipustulatus and A. nevadensis)thelastglacial maximum(LGM),followedbydemographicstabilitysincetheonset FIGURE 1 Resultsofgeneticstructureandadmixtureanalyses.(a,b)Geneticassignmentsbasedonstructurefor(a)K = 2and(b)K = 3; species names, as traditionally assigned; each individual is represented by a vertical bar, which is partitioned into K coloured segments showingtheindividual'sprobabilityofbelongingtotheclusterwiththatcolour.(c,d)GeneticassignmentsbasedonNewHybrids for comparisonsinvolving(c)thetrans-Palearctic(BIP-PAL)andIberian(BIP-IBE)lineagesofA. bipustulatusand(d)BIP-IBEandA. nevadensis (NEV).Eachindividualisrepresentedbyaverticalbar,whichispartitionedintoK coloured segments showing the individual's probability of belongingtoeachofthesixgenotypicclassesinferred:P1,P2,F1,F2,BC1andBC2(fordetails,seesection2.3.5).(e)Geneticassignment ofindividualsfrompopulationsintheSierraNevada,asinferredfromstructure for K = 3.Theblacklinerepresentsthe2500 mcontour. Insect images show A. bipustulatus(left)andA. nevadensis(right)(author:JACarbonell).(f)principalcomponentanalysis(PCA)ofgenetic variation.ShapesandcoloursinthePCAcorrespondtothegenotypicclassesassignedinNewHybrids analyses. Dots indicate individuals unambiguouslyassigned(PP > 0.95)tooneoftheparentalgenotypes(P1orP2).Yellowandreddiamondsindicateindividualsthatwere mostly,butnotunambiguously(0.95 > PP > 0.70),assignedtoBIP-IBEandNEV,respectively.GreydiamondscorrespondtoF2individuals involvinghybridisationbetweenBIP-IBEandNEV(see(c))andthelightreddiamondcorrespondstoabackcrossresultedfromhybridisation betweenBIP-IBEandBIP-PAL(see(d)).WiththeexceptionofpopulationsALPSandAZULfromBIP-PAL(ellipses),otherpopulationswithin eachlineagelargelyoverlapinthePCAandarenotoutlinedforthesakeofclarity.PopulationcodesasdescribedinTable 1. 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 16 of 19 | PALLARÉS et al. Alves,P.C.,Melo-Ferreira,J.,Frieyas,H.,&Boursot,P.(2008).Theubiquitous mountain hare mitochondria: Multiple introgressive hybridization in hares, genus Lepus. Philosophical Transactions of the Royal Society B: Biological Sciences, 363, 2831–2839. Alves,V.M.,Hernandez,M.I.,&Lobo,J.M.(2018).Elytraabsorbultraviolet radiation but transmit infrared radiation in Neotropical Canthon species (Coleoptera, Scarabaeinae). Photochemistry and Photobiology, 94, 532–539. Anderson,E.C.,&Thompson,E.A.(2002).Amodel-basedmethodfor identifying species hybrids using multilocus genetic data. Genetics, 160, 1217–1229. Anderson,M.J.,&TerBraak,C.J.F.(2003).Permutationtestsformulti- factorial analysis of variance. Journal of Statistical Computation and Simulation, 73, 85–113. Angus,R.B.,Clery,M.J.,Carter,J.C.,&Wenczek,D.E.(2013).Karyotypes of some medium-sized Dytiscidae (Agabinae and Colymbetinae) (Coleoptera).Comparative Cytogenetics, 7, 171–190. Antonelli,A.,Kissling,W.D.,Flantua,S.G.,Bermúdez,M.A.,Mulch,A., Muellner-Riehl,A.N.,Kreft,H.,Linder,H.P.,Badgley,C.,Fjeldså, J.,Fritz,S.A.,Rahbek,C.,Herman,F.,Hooghiemstra,H.,&Hoorn, C.(2018).Geologicalandclimaticinfluencesonmountainbiodiversity. Nature Geoscience, 11, 718–725. Arribas,P.,Velasco,J.,Abellán,P.,Sánchez-Fernández,D.,Andújar,C., Calosi,P.,Millán,A.,Ribera,I.,&Bilton,D.T.(2012).Dispersalability rather than ecological tolerance drives differences in range size betweenlenticandloticwaterbeetles(Coleoptera:Hydrophilidae). Journal of Biogeography, 39, 984–994. Arroyo,J.,Abellán,P.,Arista,M.,Ariza,M.J.,deCastro,A.,Escudero, M., Lorite, J., Martínez-Borda, E., Mejías, J. A., Molina-Venegas, R., Pleguezuelos, J. M., Simón-Porcar, V., & Viruel, J. (2022). Sierra Nevada, a Mediterranean biodiversity super hotspot. In R.Zamora&M.Oliva(Eds.),The landscape of the Sierra Nevada: A unique Laboratory of Global Processes in Spain(pp.11–30).Springer International Publishing. Avise,J.C.(2000).Phylogeography: The history and formation of species. Harvard University Press. Baker,A.J.(2008).Islandsinthesky:TheimpactofPleistoceneclimate cycles on biodiversity. Journal of Biology, 7, 32. Balfour-Browne,W.A.F.(1950).British water beetles(Vol.2).RaySociety. Behm,J.E.,Ives,A.R.,&Boughman,J.W.(2010).Breakdowninpostmating isolation and the collapse of a species pair through hybridization. American Naturalist, 175, 11–26. Bennett, K. D., & Provan, J. (2008). What do we mean by ‘refugia’? Quaternary Science Reviews, 27, 2449–2455. Bergsten,J.,Bilton,D.T.,Fujisawa,T.,Elliott,M.,Monaghan,M.T.,Balke, M.,Hendrich,L.,Geijer,J.,Herrmann,J.,Foster,G.N.,Ribera,I., Nilsson,A.N.,Barraclough,T.G.,&Vogler,A.P.(2012).Theeffect of geographical scale of sampling on DNA barcoding. Systematic Biology, 61, 851–869. Bertola,L.D.,Quinn,L.,Hanghøj,K.,Garcia-Erill,G.,Rasmussen,M.S., Balboa,R.F.,Meisner,J.,Bøggild,T.,Wang,X.,Lin,L.,Nursyifa,C., Liu,X.,Li,Z.,Chege,M.,Moodley,Y.,Brüniche-Olsen,A.,Kuja,J., Schubert,M.,Agaba,M.,…Heller,R.(2024).Giraffelineagesare shaped by major ancient admixture events. Current Biology, 34, 1576–1586. Butlin, R. (1987). Speciation by reinforcement. Trends in Ecology & Evolution, 2, 8–13. Calosi, P., Bilton, D. T., Spicer, J. I., Votier, S. C., & Atfield, A. (2010). Whatdeterminesaspecies'geographicalrange?Thermalbiology and latitudinal range size relationships in European diving beetles (Coleoptera:Dytiscidae).Journal of Animal Ecology, 79, 194–204. Carbonell,J.A.,Pallarés,S.,Velasco,J.,Millán,A.,&Abellán,P.(2024). Thermaltolerancedoesnotexplainthealtitudinalsegregationof lowland and alpine aquatic insects. Journal of Thermal Biology, 121, 103862. Catchen, J., Hohenlohe, P. A., Bassham, S., Amores, A., & Cresko, W. A. (2013). stacks: An analysis tool set for population genomics. Molecular Ecology, 22(11),3124–3140. Chen, G., & Hare, M. (2008). Cryptic ecological diversification of a planktonic estuarine copepod, Acartia tonsa. Molecular Ecology, 17, 1451–1468. Chifman,J.,&Kubatko,L.(2014).QuartetinferencefromSNPdataunder the coalescent model. Bioinformatics, 30(23),3317–3324. Chown,S.L.,&Terblanche,J.S.(2006).Physiologicaldiversityininsects: Ecologicalandevolutionarycontexts.Advances in Insect Physiology, 33, 50–152. Čiamporová-Zaovičová,Z.,&Čiampor,F.(2011).Aquaticbeetlesofthe alpinelakes:Diversity,ecologyandsmall-scalepopulationgenetics. Knowledge and Management of Aquatic Ecosystems, 402, 10. Coates,D.J.,Byrne,M.,&Moritz,C.(2018).Geneticdiversityandconservationunits:Dealingwiththespecies-populationcontinuumin the age of genomics. Frontiers in Ecology and Evolution, 6, 165. Collyer,M.L.,Sekora,D.J.,&Adams,D.C.(2015).Amethodforanalysis of phenotypic change for phenotypes described by high- dimensional data. Heredity, 115, 357–365. Degerlund, M., Huseby, S., Zingone, A., Sarno, D., & Landfald, B. (2012).FunctionaldiversityincrypticspeciesofChaetoceros socialisLauder(Bacillariophyceae).Journal of Plankton Research, 34, 416–431. Drotz,M.K.,Brodin,T.,&Nilsson,A.N.(2010).Multipleoriginsofelytral reticulation modifications in the west Palearctic Agabus bipustulatuscomplex(Coleoptera,Dytiscidae).PLoS One, 5, e9034. Drotz,M.K.,Brodin,T.,Saura,A.,&Giles,B.E.(2012).Ecotypedifferentiation in the face of gene flow within the diving beetle Agabus bipustulatus(Linnaeus,1767)innorthernScandinavia.PLoS One, 7, e31381. Drotz,M.K.,Saura,A.,&Nilsson,A.N.(2001).Thespeciesdelimitation problem applied to the Agabus bipustulatuscomplex(Coleoptera, Dytiscidae)innorthScandinavia.Biological Journal of the Linnean Society, 73, 11–22. Dynesius,M.,&Jansson,R.(2000).Evolutionaryconsequencesofchanges in species' geographical distributions driven by Milankovitch climate oscillations. Proceedings of the National Academy of Sciences United States of America, 97, 9115–9120. Dynesius, M., & Jansson, R. (2014). Persistence of within-species lineages: A neglected control of speciation rates. Evolution, 68, 923–934. Earl,D.A.,&vonHoldt,B.M.(2012).structure Harvester:Awebsiteand program for visualizing structure output and implementing the Evanno method. Conservation Genetics Resources, 4(2),359–361. Evanno,G.,Regnaut,S.,&Goudet,J.(2005).Detectingthenumberof clusters of individuals using the software v: A simulation study. Molecular Ecology, 14(8),2611–2620. Excoffier,L.,Dupanloup,I.,Huerta-Sanchez,E.,Sousa,V.C.,&Foll,M. (2013).RobustdemographicinferencefromgenomicandSNPdata. PLoS Genetics, 9(10),e1003905. Excoffier,L.,&Lischer,H.E.(2010).arlequiNsuitever3.5:Anewseriesof programstoperformpopulationgeneticsanalysesunderLinuxand Windows.Molecular Ecology Resources, 10(3),564–567. Fick,S.E.,&Hijmans,R.J.(2017).WorldClim2:New1kmspatialresolution climate surfaces for global land areas. International Journal of Climatology, 37, 4302–4315. Flantua,S.G.,Payne,D.,Borregaard,M.K.,Beierkuhnlein,C.,Steinbauer, M.J.,Dullinger,S.,Essl,F.,Irl,S.D.H.,Kienle,D.,Kreft,H.,Lenzner, B.,Norder,S.J.,Rijsdijk,K.F.,Rumpf,S.B.,Weigelt,P.,&Field,R. (2020).Snapshotisolationandisolationhistorychallengetheanalogy between mountains and islands used to understand endemism. Global Ecology and Biogeography, 29, 1651–1673. Galewski, K., & Tranda, E. (1978). Fauna Słodkowodna Polski, Chrz szcze (Coleoptera), Rodziny Pływakowate (Dytiscidae), Fliskowate 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 17 of 19 PALLARÉS et al. (Haliplidae), Mokrzelicowate (Hygrobiidae), Kretakowate (Gyrinidae)(p. 396).PWN. García-Vázquez, D., Bilton, D. T., Foster, G. N., & Ribera, I. (2017). Pleistocene range shifts, refugia and the origin of widespread species in western Palaearctic water beetles. Molecular Phylogenetics and Evolution, 114, 122–136. Garrick,R.C.,Benavides,E.,Russello,M.A.,Hyseni,C.,Edwards,D.L., Gibbs,J.P.,Tapia,W.,Ciofi,C.,&Caccone,A.(2014).Lineagefusion inGalápagosgianttortoises.Molecular Ecology, 23, 5276–5290. Gonzalez,V.H.,Oyen,K.,Vitale,N.,&Ospina,R.(2022).Neotropical stingless bees display a strong response in cold tolerance with changes in elevation. Conservation Physiology, 10, coac073. Gómez, A., & Lunt, D. H. (2007). Refugia within refugia: Patterns of phylogeographicconcordanceintheIberianPeninsula.InS.Weiss &N.Ferrand(Eds.),Phylogeography of southern European Refugia: Evolutionary perspectives on the origins and conservation of European biodiversity(pp.155–188).Springer. Gómez-Ortiz,A.,Oliva,M.,Salvà-Catarineu,M.,&Salvador-Franch,F. (2013). The environmental protection of landscapes in the high semiarid Mediterranean mountain of Sierra Nevada National Park(Spain):Historicalevolutionandfutureperspectives.Applied Geography, 42, 227–239. Goodall,C.R.(1991).Procrustesmethodsinthestatisticalanalysisof shape. Journal of the Royal Statistical Society Series B, 53, 285–339. Gruber,B.,Unmack,P.J.,Berry,O.F.,&Georges,A.(2018).dartR:Anr packagetofacilitateanalysisofSNPdatageneratedfromreduced representation genome sequencing. Molecular Ecology Resources, 18(3),691–699. Hedin, M., Carlson, D., & Coyle, F. (2015). Sky Island diversification meets the multispecies coalescent–divergence in the spruce-fir mossspider(Microhexura montivaga,Araneae,Mygalomorphae)on the highestpeaks of southern Appalachia.Molecular Ecology, 24, 3467–3484. Hedrick,P.W.(2013).Adaptiveintrogressioninanimals:Examplesand comparison to new mutation and standing variation as sources of adaptive variation. Molecular Ecology, 22, 4606–4618. Hewitt,G.(2000).Thegeneticlegacyofthequaternaryiceages.Nature, 405, 907–913. Hewitt, G. M. (1999). Post-glacial re-colonization of European biota. Biological Journal of the Linnean Society, 68(1–2),87–112. Jakobsson,M.,&Rosenberg,N.A.(2007).clumpp:Aclustermatchingand permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics, 23(14), 1801–1806. Jombart,T.(2008).Adegenet:Ar package for the multivariate analysis of genetic markers. Bioinformatics, 24(11),1403–1405. Kearns, A. M., Restani, M., Szabo, I., Schrøder-Nielsen, A., Kim, J. A., Richardson,H.M.,Marzluff,J.M.,Fleischer,R.C.,Johnsen,A.,& Omland,K.E.(2018).Genomicevidenceofspeciationreversalin ravens. Nature Communications, 9, 906. Keightley,P.D.,Ness,R.W.,Halligan,D.L.,&Haddrill,P.R.(2014). Estimation of the spontaneous mutation rate per nucleotide site in a Drosophila melanogasterfull-sibfamily.Genetics, 196(1), 313–320. Keller, I., Alexander, J. M., Holderegger, R., & Edwards, P. J. (2013). Widespreadphenotypicandgeneticdivergencealongaltitudinal gradients in animals. Journal of Evolutionary Biology, 26, 2527–2543. Kleindorfer,S.,O'Connor,J.A.,Dudaniec,R.Y.,Myers,S.A.,Robertson, J., & Sulloway, F. J. (2014). Species collapse via hybridization in Darwin's tree finches. The American Naturalist, 183, 325–341. Klingenberg, C. P. (2011). MorphoJ: An integrated software package for geometric morphometrics. Molecular Ecology Resources, 11, 353–357. Körner,C.(2007).Theuseof‘altitude’inecologicalresearch.Trends in Ecology and Evolution, 22, 569–574. Liu,X.M.,&Fu,Y.X.(2020).StairwayPlot2: Demographic history inferencewithfoldedSNPfrequencyspectra.Genome Biology, 21(1), 280. Love,S.J.,Schweitzer,J.A.,Woolbright,S.A.,&Bailey,J.K.(2023).Sky islands are a global tool for predicting the ecological and evolutionary consequences of climate change. Annual Review of Ecology, Evolution, and Systematics, 54, 219–236. Lukicheva,S.,&Mardulyn,P.(2021).Whole-genomesequencingreveals asymmetric introgression between two sister species of cold- resistant leaf beetles. Molecular Eecology, 30, 4077–4089. Lutterschmidt, W. I., & Hutchison, V. H. (1997). The critical thermal maximum: History and critique. Canadian Journal of Zoology, 75, 1561–1574. Maier,P.A.,Vandergast,A.G.,Ostoja,S.M.,Aguilar,A.,&Bohonak,A. J. (2019). Pleistocene glacial cycles drove lineage diversification andfusionintheYosemitetoad(Anaxyrus canorus).Evolution, 73, 2476–2496. Mavárez,J.,&Linares,M.(2008).Homoploidhybridspeciationinanimals. Molecular Ecology, 17, 4181–4185. McCulloch,G.A.,Foster,B.J.,Dutoit,L.,Ingram,T.,Hay,E.,Veale,A.J., Dearden,P.K.,&Waters,J.M.(2019).Ecologicalgradientsdrive insect wing loss and speciation: The role of the alpine treeline. Molecular Ecology, 28, 3141–3150. Médail,F.,&Quézel,P.(1997).Hot-spots analysisforconservationof plant biodiversity in the Mediterranean Basin. Annals of the Missouri Botanical Garden, 84, 112–127. Médail,F.,&Quézel,P.(1999).BiodiversityhotspotsintheMediterranean Basin:Settingglobalconservationpriorities.Conservation Biology, 13, 1510–1513. Millán, A., Picazo, F., Sánchez-Fernández, D., Abellán, P., & Ribera, I. (2013). Los Coleópteros acuáticos amenazados (Coleoptera). In F. Ruano, M. Tierno de Figueroa, & A. Tinaut (Eds.), Los Insectos de Sierra Nevada. 200 años de historia (pp. 443–456). Asociación Española de Entomología. Millán,A.,Sánchez-Fernández,D.,Abellán,P.,Picazo,F.,Carbonell,J.A., Lobo,J.M.,&Ribera,I.(2014).Atlas de los coleópteros acuáticos de España peninsular.MinisteriodeAgricultura,AlimentaciónyMedio Ambiente. Miller,K.B.,&Bergsten,J.(2016).Diving beetles of the world: Systematics and biology of the Dytiscidae.JHUPress. Momigliano,P.,Florin,A.B.,&Merilä,J.(2021).Biasesindemographic modeling affect our understanding of recent divergence. Molecular Biology and Evolution, 38(7),2967–2985. Muangmai,N.,Preuss,M.,&Zuccarello,G.C.(2015).Comparativephysiological studies on the growth of cryptic species of Bostrychia intricata(Rhodomelaceae,Rhodophyta)invarioussalinityandtemperature conditions. Phycological Research, 63, 300–306. Múrria, C., Sáinz-Bariáin, M., Vogler, A. P., Viza, A., González, M., & Zamora-Muñoz, C. (2020). Vulnerability to climate change for two endemic high-elevation, low-dispersive Annitella species (Trichoptera)inSierraNevada,thesouthernmosthighmountainin Europe. Insect Conservation and Diversity, 13, 283–295. Nilsson, A. N., & Hájek, J. (2021). A World Catalogue of the Family Dytiscidae, or the Diving Beetles (Coleoptera, Adephaga). Version 1.I.2021. http:// www. water beetl es. eu Nilsson,A.N.,&Holmen,M.(1995).TheaquaticAdephaga(Coleoptera) of Fennoscandia and Denmark. II. Dytiscidae. Fauna Entomologica Scandinavica, 32, 1–192. Nilsson,A.N.,&Persson,S.(1990).Dimorphismofthemetasternalwing in Agabus raffrayi and A. labiatus (Coleoptera: Dytiscidae) questioned. Aquatic Insects, 12, 135–144. Noguerales, V., Arjona, Y., García-Olivares, V., Machado, A., López, H.,Patiño,J.,&Emerson,B.C.(2024).Geneticlegaciesofmega- landslides: Cycles of isolation and contact across flank collapses in an oceanic Island. Molecular Ecology, 33(9),e17341. 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 18 of 19 | PALLARÉS et al. Noguerales, V., & Ortego, J. (2022). Genomic evidence of speciation by fusion in a recent radiation of grasshoppers. Evolution, 76, 2618–2633. Ortego,J.,Gutiérrez-Rodríguez,J.,&Noguerales,V.(2021).Demographic consequences of dispersal-related trait shift in two recently divergedtaxaofmontanegrasshoppers.Evolution, 75, 1998–2013. Ortego,J.,&Knowles,L.L.(2022).Geographicalisolationversusdispersal: Relictual alpine grasshoppers support a model of interglacial diversification with limited hybridization. Molecular Ecology, 31, 296–312. Pallarés,S.,Carbonell,J.A.,Picazo,F.,Bilton,D.T.,Millán,A.,&Abellán, P.(2024).Intraspecificvariationofthermaltoleranceinfreshwater insects along elevational gradients: The case of a widespread diving beetle. bioRxiv. https:// doi. org/ 10. 1101/ 2024. 03. 04. 583263 Pallarés, S., Millán, A., Mirón, J. M., Velasco, J., Sánchez-Fernández, D.,Botella-Cruz,M.,&Abellán,P.(2020).Assessingthecapacity of endemic alpine water beetles to face climate change. Insect Conservation and Diversity, 13, 271–282. Peterson,B.K.,Weber,J.N.,Kay,E.H.,Fisher,H.S.,&Hoekstra,H.E. (2012).DoubledigestRADseq:Aninexpensivemethodfordenovo SNPdiscoveryandgenotypinginmodelandnon-modelspecies. PLoS One, 7(5),e37135. Pritchard,J.K.,Stephens,M.,&Donnelly,P.(2000).Inferenceofpopulation structure using multilocus genotype data. Genetics, 155(2), 945–959. RCoreTeam.(2021).R: A language and environment for statistical computing.RFoundationforStatisticalComputing.h t t p s : // w w w . R - p r o j e  ct. org/ Ribera,I.,Castro,A.,Díaz,J.A.,Garrido,J.,Izquierdo,A.,Jäch,M.A., &Valladares,L.F.(2011).Thegeographyofspeciationinnarrow- rangeendemicsofthe‘Haenydra’lineage(Coleoptera,Hydraenidae, Hydraena).Journal of Biogeography, 38, 502–516. Ribera,I.,Foster,G.N.,&Holt,W.V.(1997).Functionaltypesofdivingbeetle(Coleoptera:HygrobiidaeandDytiscidae),asidentified by comparative swimming behaviour. Biological Journal of Linnaean Socity, 61, 537–558. Ribera,I.,Hernando,C.,&Aguilera,P.(1998).Anannotatedchecklistof theIberianwaterbeetles(Coleoptera).Zapateri, 8, 43–111. Ribera, I., & Nilsson, A. (1995). Morphometric patterns among diving beetles (Coleoptera: Noteridae, Hygrobiidae, and Dytiscidae). Canadian Journal of Zoology, 73, 2343–2360. Ribera,I.,&Vogler,A.P.(2004).SpeciationofIberiandivingbeetlesin Pleistocenerefugia(Coleoptera,Dytiscidae).Molecular Ecology, 13, 179–193. Rohlf,F.J.(2015).Thetps series of software. HystrixItalian Journal of Mammalogy, 26, 9–12. Rosenberg,N.A.(2004).Distruct:Aprogramforthegraphicaldisplayof population structure. Molecular Ecology Notes, 4(1),137–138. Ruano,F.,TiernodeFigueroa,J.,&Tinaut,A.(2013).Los insectos de Sierra Nevada: 2000 años de historia.AsociaciónEspañoladeEntomología. Rubalcaba,J.G.,Verberk,W.C.,Hendriks,A.J.,Saris,B.,&Woods,H. A.(2020).Oxygenlimitationmayaffectthetemperatureandsize dependence of metabolism in aquatic ectotherms. Proceedings of the National Academy of Sciences, 117(50),31963–31968. Schmitt,T.(2007).MolecularbiogeographyofEurope:Pleistocenecycles and postglacial trends. Frontiers in Zoology, 4, 11. Schoville,S.D.,Roderick,G.K.,&Kavanaugh,D.H.(2012).Testingthe ‘Pleistocene species pump'in alpine habitats: Lineage diversification of flightless ground beetles (Coleoptera: Carabidae: Nebria) in relation to altitudinal zonation. Biological Journal of the Linnean Society, 107, 95–111. Seehausen,O.(2006).Conservation:Losingbiodiversitybyreversespeciation. Current Biology, 16, R334–R337. Seehausen,O.,Takimoto,G.,Roy,D.,&Jokela,J.(2008).Speciationreversal and biodiversity dynamics with hybridization in changing environments. Molecular Ecology, 17, 30–44. Shah, A. A., Gill, B. A., Encalada, A. C., Flecker, A. S., Funk, W. C., Guayasamin,J.M.,Kondratieff,B.C.,Poff,N.L.R.,Thomas,S.A., Zamudio,K.R.,&Ghalambor,C.K.(2017).Climatevariabilitypredicts thermal limits of aquatic insects across elevation and latitude. Functional Ecology, 31(11),2118–2127. Sharp,D.(1882).OnaquaticcarnivorousColeopteraorDytiscidae.The Scientific transactions of the Royal Dublin Society, Series 2, 2, 17–1003. Slatyer,R.A.,&Schoville,S.D.(2016).Physiologicallimitsalonganelevational gradient in a radiation of montane ground beetles. PLoS One, 11. e0151959. Sommaruga,R.(2001).TheroleofsolarUVradiationintheecologyof alpine lakes. Journal of Photochemistry and Photobiology B: Biology, 62(1–2),35–42. Stanbrook,R.,Wheater,C.P.,Harris,W.E.,&Jones,M.(2021).Habitat type and altitude work in tandem to drive the community structure of dung beetles in Afromontane forest. Journal of Insect Conservation, 25, 159–173. Stanton,D.W.,Frandsen,P.,Waples,R.K.,Heller,R.,Russo,I.R.M., Orozco-terWengel,P.A.,TingskovPedersen,C.-E.,Siegismund,H. R.,&Bruford,M.W.(2019).Moregristforthemill?Speciesdelimitation in the genomic era and its implications for conservation. Conservation Genetics, 20, 101–113. Steinbauer,M.J.,Irl,S.D.H.,&Beierkuhnlein,C.(2013).Elevationdriven ecologicalisolationpromotes-diversificationonMediterraneanislands. Acta Oecologica, 47, 52–56. Stevens,G.C.(1989).Thelatitudinalgradientingeographicalrange:How somanyspeciescoexistinthetropics.The American Naturalist, 133, 240–256. Stewart,J.R.,Lister,A.M.,Barnes,I.,&Dalen,L.(2010).Refugiarevisited: Individualistic responses in space and time. Proceedings of the Royal Society B: Biological Sciences, 277, 661–671. Swofford,D.L.(2002).PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4.SinauerAssociates. Taylor,E.B.,Boughman,J.W.,Groenenboom,M.,Sniatynski,M.,Schluter, D., & Gow, J. L. (2006). Speciation in reverse: Morphological and genetic evidence of the collapse of a three-spined stickleback (Gasterosteus aculeatus) species pair. Molecular Ecology, 15, 343–355. Tonzo,V.,&Ortego,J.(2021).Glacialconnectivityandcurrentpopulationfragmentationinskyislandsexplainthecontemporarydistributionofgenomicvariationintwonarrow-endemicmontanegrasshoppers from a biodiversity hotspot. Diversity and Distributions, 27, 1619–1633. Tonzo,V.,Papadopoulou,A.,&Ortego,J.(2019).Genomicdatareveal deepgeneticstructurebutnosupportforcurrenttaxonomicdesignationinagrasshopperspeciescomplex.Molecular Ecology, 28, 3869–3886. Tribsch,A.(2004).Areasofendemismofvascularplantsintheeastern AlpsinrelationtoPleistoceneglaciation.Journal of Biogeography, 31, 747–760. Tsuchiya, Y.,Takami,Y.,Okuzaki,Y.,&Sota, T. (2012).Geneticdifferencesandphenotypicplasticityinbodysizebetweenhigh-andlow- altitude populations of the ground beetle Carabus tosanus. Journal of Evolutionary Biology, 25, 1835–1842. Vonlanthen, P., Bittner, D., Hudson, A. G., Young, K. A., Müller, R., Lundsgaard-Hansen,B., Roy,D.,DiPiazza,S.,Largiader,C.R.,& Seehausen,O.(2012).Eutrophicationcausesspeciationreversalin whitefish adaptive radiations. Nature, 482, 357–362. Webb,W.C.,Marzluff,J.M.,&Omland,K.E.(2011).Randominterbreeding between cryptic lineages of the common raven: Evidence for speciation in reverse. Molecular Ecology, 20, 2390–2402. Weir,B. S.,&Cockerham,C.C. (1984).EstimatingF-statisticsforthe analysis of population structure. Evolution, 38, 1358–1370. Weir,J.T.,&Schluter,D.(2004).Icesheetspromotespeciationinboreal birds. Proceedings of the Royal Society B: Biological Sciences, 271, 1881–1887. 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 19 of 19 PALLARÉS et al. Zamora,R.,&Oliva,M.(Eds.).(2022).The landscape of the Sierra Nevada: A unique Laboratory of Global Processes in Spain.SpringerNature. Zelditch, M. L., Swiderski, D. L., Sheets, H. D., & Fink, W. L. (2004). Geometric morphometrics for biologists: A primer.ElsevierAcademic Press. SUPPORTING INFORMATION Additional supporting information can be found online in the SupportingInformationsectionattheendofthisarticle. How to cite this article: Pallarés,S.,Ortego,J.,Carbonell,J.A., Franco-Fuentes,E.,Bilton,D.T.,Millán,A.,&Abellán,P.(2024). Genomic,morphologicalandphysiologicaldatasupportfast ecotypic differentiation and incipient speciation in an alpine diving beetle. Molecular Ecology, 33, e17487. https://doi. org/10.1111/mec.17487 1365294x, 2024, 17, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/mec.17487 by Readcube (Labtiva Inc.), Wiley Online Library on [24/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License