Posted on Authorea 1 Oct 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160157511.10593343 | This a preprint and has not been peer reviewed. Data may be preliminary. sesn pce onaiscnb atclrycalnigfrtx htaentesl morphologically of easily comparisons quantitative not context, are this that In taxa preferences. for habitat challenging similar Wiens particularly superficially 1997; have be or Mayden can morphological, maydistinguished 1963; on boundaries which emphases 1957, species of their 1942, each Assessing in (Mayr species, differ Freudenstein assessments 1998; that distinguishing Queiroz concepts species and de species contradictory divergences 2004; of Loera resolving or plethora 2009; for inconsistent Wilkins a genetic, 2007; information to produce historical, Queiroz led a ecological diverse de has from or by 2004; This suffer affected Wiens genetic, identification are 2003; 2012). Marshall and speciation al. & of delimitation (Sites et processes species factors the in MacDonald stochastic because employed and 2013; part ecological, procedures Magurran in the 2004; limitations, 2019), (Mace of IPBES initiatives number conservation 2017; modern al. of amidst et (Coates consequences units planet political operational our and of the losses economic, comprise biodiversity social, and habitat biological, rapid greater the increasingly has delimitation Species Introduction Speyeria and ecological combines that delimitation Keywords: species hesperis integrated S. to concepts. of approach delimitation species an related species of S. current reconciles utility of the and the that data populations suggest to demonstrate suggest genomic southern analyses We modelling and and integrity. these northern niche revised, genetic of between be applied their Results flow should then reinforcing gene lineages. We and contemporary genomic to hindering recovered combination contributing expansion. is the a hesperis, range conditions to among ecological attributed flow post-glacial to be gene and may adaptation to which zerene, that barriers hesperis, S. and substantial S. divergence species, recovered within ecological also another we assess but with SNPs, atlantis, introgression S. genomic and past apply Using muddled We hesperis of its S. for discordance. delimitation. between notorious mito-nuclear only species is not and integrated which divergence, complex, variation in species morphological aid multidisciplinary Nymphalidae) to delimitations, new, approach (Lepidoptera: data an present atlantis-hesperis species present sequence Speyeria and we the next-generation Here, dynamics, to with otherwise. approach genomics population classify models this both to on niche difficult in focused been ecological advances complex, have research combining that Recent often taxa for to in taxa. and boundaries boon many heterogenous species for a clarifying is challenge for been opportunities speciation a have remained of modelling has process changes ecological boundaries the rapid and species However, amidst of particularly characterization biodiversity, landscapes. robust planet’s natural and our and categorizing conditions and environmental understanding to to essential is delimitation Species Abstract 2020 1, October 3 2 1 Campbell Erin a butterflies using of delimitation group confusing species ecological and genomic Reconciling ffiito o available not Affiliation Entomology of Museum Texas Alberta of University clgclseito,hbttsiaiiy pce eiiain ouaingnmc,introgression genomics, population delimitation, species suitability, habitat speciation, ecological , 1 ahr MacDonald Zachary , tal. et 2016). 1 dGage Ed , 1 2 ad Gage Randy , tal. et 08 Stanton 2018; 3 n ei Sperling Felix and , tal. et 09.Wiespecies While 2019). 3 Posted on Authorea 1 Oct 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160157511.10593343 | This a preprint and has not been peer reviewed. Data may be preliminary. tts eefe eerdt ste“otenlnae) n hs curn hogottesouthwestern the United throughout the occurring in those Dakota and South lineage”), and “northern Montana the and as Canada to in referred Saskatchewan hereafter and States; Alberta, Columbia, British between as SNPs well in as divergence, genetic (Scott al. (Campbell alone and morphology populations morphological using identify sympatric of reliably between assessments hybridization on Campbell of 2017; based lack species, apparent distinct an Hammond as 1981; Brown taxa and these Miller 1951; to (Grey Mountains 2009). similarity considered Rocky variation Dunford morphological have as the 1975; treatments well Moeck along taxonomic as 1947; south Some areas Grey and some & Columbia in Passos British species (dos each contact between to species species similarity within Manitoba two morphological The from substantial 2019). areas exhibiting (Pelham Colorado, forest Manitoba mixed southeastern in and in recognized Dakota other South taxonomically to is east variation and More America Newfoundland. to Mountains hesperis Rocky S. the in from subspecies America North five include These 2019). Graham 2003; Marshall Jezkova & 2008, (Sites evolutionary Stockman data diverged & genetic recently of Bond among basis 2006, conservatism the Wiens and on divergence delineated potential Elith often niche and 2000; lineages, assess Zimmermann associations, to & habitat Guisan framework niches, 2001; comparative Peterson ecological 1985; quantify (Austin to species al. single used Manel of be 2009; distributions may geographic 2001, (Schluter factors, species environmental of and processes reinforcement inferring to and Balkenhol approach diversification integrative 2003; ecological an facilitated to has contribute (ENMs) these that models of niche ecological combination of due development some either approaches, or al. other introgression, using et historical characterize ambiguity, to (Hohenlohe difficult morphological factors historically (Andrews diversification, been organisms have recent non-model that to in taxa divergences in greatly limits genomic (SNPs), species fine-scale polymorphisms recent, nucleotide al. single detect of et to (Baird thousands capacity RADseq of our as genotyping phylogenetic such extending enabled techniques strengthen have sequencing methods and DNA related high-throughput speciation so. of of done development convincingly modes The not on mutation- have approaches or inferences traditional valuable selection when without provide inferences ecologically- speciation thereby that (e.g., can niche suggest while implicated frameworks speciation), would are ecological processes species (i.e., speciation, order Graham speciation other sister underlying that 2003; between process suggests (Sperling principal divergence has conservatism integrity a niche selection is genomic selection significant divergent species’ divergent events, ecologically-based example, based a whether speciation For of into ongoing reinforcement insight and 2004). and permits historical speciation information to of of (Carstens contributed resolution sources species two sister the these parapatric in of or aid allopatric can for divergences particularly ecological and genomic oc 95 ufr 09.Attlo 6seisaecretyrcgie in recognized evolutionary currently understood Scott are poorly 1947; with species peris Grey complexes 16 & species 1947; Passos of several (dos Grey and total relationships & subspecies A Passos variable (dos morphologically 2009). lineages 110 Dunford component among 1975; relationships Moeck evolutionary ambiguous and variability genus butterfly The 06 01 Phillips 2011; 2006, 21)hv eetyson ae nalmtdnme fseies usata eoi divergence genomic substantial specimens, of number limited a on based shown, recently have (2019) Ewrs 84 and 1864) (Edwards, 06.Sc eoi aahv rvnvlal o lrfigpplto yaisadre-assessing and dynamics population clarifying for valuable proven have data genomic Such 2016). 08 Hinojosa 2018; hc a 1sbpce curn ndirmaosadoe oet hogotwsenNorth western throughout forests open and meadows drier in occurring subspecies 21 has which , tal. et tal. et sensu .hesperis S. tal. et Speyeria 09 Riva 2019; 06.EM,gnrlyprmtrzdb eaigseis curne ogeographic to occurrences species’ relating by parameterized generally ENMs, 2016). oi 02.Icroaino clgclifrainit eei pce delimitation species genetic into information ecological of Incorporation 2012). Nosil tal. et tal et .atlantis S. 03 Wagner 2013; cde,17 Lpdpea ypaia)i elkonfrisphenotypic its for known well is Nymphalidae) (Lepidoptera: 1872 Scudder, ouain curn ot n ato h ok onan (throughout Mountains Rocky the of east and north occurring populations 06 imrane l 00.Hwvr Nsmyas eue na in used be also may ENMs However, 2010). al. et Zimmermann 2006; . 09 Hundsdoerfer 2019; tal. et Ewrs 82 omalrecmlxcnann 6sbpce (Pelham subspecies 26 containing complex large a form 1862) (Edwards, 09 Thompson 2019; .atlantis S. tal. et tal. et .hesperis S. tal. et tal. et 03 Escudero 2013; 98 ufr 09 ehm21) mn these, Among 2019). Pelham 2009; Dunford 1998; htaebodydsrbtdi oie odad across woodlands conifer in distributed broadly are that tal. et 09 Loera 2009, 98 pe arn20) diinly Campbell Additionally, 2005). Warren & Opler 1998; 2 tal. et ob useisof subspecies a be to 09.Aogiegnmcavne,continued advances, genomic Alongside 2019). 09.Hwvr h aarmi icl to difficult remain taxa the However, 2019). tal. et tal. et tal. et 02 Newton 2012; 03.Frhroe h integration the Furthermore, 2013). 04 Vargas 2014; tal. et 03.Rcn okrecognizes work Recent 2013). tal. et .atlantis S. Speyeria tal. et tal. et 07 eMoya de 2017; tal. et 07 Abdelkrim 2017; 2020). swl sover as well as , ae noverall on based tal. et 04 oa & Kozak 2004, 08 and 2008) .hes- S. tal. et tal. et tal. et et et Posted on Authorea 1 Oct 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160157511.10593343 | This a preprint and has not been peer reviewed. Data may be preliminary. hlgntcaaye o ohthe both for analyses maximum Phylogenetic a to analyses locus genetic per population data and missing Phylogenetic global the reduce further (Danecek to 0.1.14 and 20%. 3% of vcftools of in frequency allele the filtering minor in minimum SNP processing additional final Paris conducted of We during principle output “r80” was (the population locus single any of 80% in in construction catalog the during following: locus the for except settings novo Campbell follows (Poland mitochondrial extraction, DNA processing and of generation data subspecies Molecular 14 in specimens of 113 specimens of outgroup comprised was of dataset subspecies Campbell among total by relationships The analysis in . 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SNPs informative. 1026 phylogenetically The contained 65.6). dataset depth: SNP locus our filtering, After analyses phylogenetic and construction Dataset southern and northern the Results of that reinforcement suggest would to distance contributes Euclidean out likely hesperis inte- partialling selection genomic after divergent the distance reinforcing lineage ecologically-based mechanism and principal distance from a resistance southern resulting as between and flow out northern gene ruled the reduced be of that cannot grity for suggest (vicariance) would controlling dispersal distance after to Euclidean barriers out between lineage partialling li- exist same after different dispersal distance the to of southern lineage barriers of individuals and significant between individuals whether northern evaluates distances between effectively the resistance those analysis This whether than distance. evaluate greater Euclidean different to significantly to test were or mantel 0) neages partial = Euclidean a (value matrix of used lineage pairwise matrix We same third pairwise the A a 2005). to 1). Bivand generated = belonged & (value also individuals (Pebesma lineages We sp whether 2007), package 1996). indicated in R Beier distance”) al. the individuals) & (“lineage using et sequenced (McRae in- individuals (Chandra of distances all them surface) localities between circuit organized (i.e., distances resistance to and nodes (i.e., analogous 2017) between graph are time Etten a distances commute (van random-walk resistance gdistance expected package These measuring matrix. R pairwise the a using to individuals all between distances .h dorothea h. S. lineages. .h brico h. S. , .hesperis S. .hesperis S. Ewrs 84 rmAioa hlgntccmaio fteSPand SNP the of comparison Phylogenetic Arizona. from 1874) (Edwards, .h dennisi h. S. K Bidvl 89 rmClfri,btddntehbtcnitn egahcsbcutrn.The sub-clustering. geographic consistent exhibit not did but California, from 1869) (Boisduval, rmNwMxc n rzn,adbodysprtdasuhr e eia ouainof population Mexican New southern a separated broadly and Arizona, and Mexico New from Fg 1,wihgopdabodynorthern broadly a grouped which S1), (Fig. 2 = K Ewrs 89 ape nsuhatr Utah, southeastern in sampled 1879) (Edwards, tal. et .h tetonia h. S. .Ti resolved This 6. = oc,14 ape nteSni onan fNwMxc,wihcutrdwith clustered which Mexico, New of Mountains Sandia the in sampled 1947 Moeck, oln,18 ape nteScaet onan rmtemr otenpopulation northern more the from Mountains Sacramento the in sampled 1988 Holland, ieg a itrto sister was lineage lse seiesfo th dh,suhr otn,suhetr ooao New Colorado, southwestern Montana, southern Idaho, Utah, from (specimens cluster (2019). oda ct pmr 98and 1998 Spomer, & Scott Kondla, o ass&Ge,14 rmAlberta, from 1945 Grey, & Passos dos COI .zerene S. .hesperis S. .hesperis S. .h viola h. S. aae osse f68sts(7 nain n 1vrat,5 fwihwere which of 58 variant), 71 and invariant (577 sites 648 of consisted dataset o ass&Ge,14 ape nnrhr thadsuhr Montana, southern and Utah northern in sampled 1945 Grey, & Passos dos .cybele S. .hesperis S. .zrn,S atlantis S. zerene, S. .atlantis, S. Fg a.LnPr( 2a). (Fig. o ass&Ge 95fo dh.Amnpyei ld contained clade monophyletic A Idaho. from 1945 Grey & Passos dos notregorpial-endcutr:1 cnrl (specimens “central” 1. clusters: geographically-defined three into , iegs infiatcreainbtenrssac itneand distance resistance between correlation significant A lineages. .zerene S. .atlantis S. .h ratonensis h. S. iegs lentvl,teasneo infiatcorrelation significant a of absence the Alternatively, lineages. n otie subspecies contained and n tefhdtomjrgorpi ruig.Alarge A groupings. geographic major two had itself and , 7 and , K n northern and , .zerene S. supported ) .h beani h. S. ct,18 rmsuhatr ooao and Colorado, southeastern from 1981 Scott, .h hutchinsi h. S. K .hesperis S. .h electa h. S. .hesperis S. niaigsbtutr ntedata. the in substructure indicating , smnpyei lds(i.1a) (Fig. clades monophyletic as rmBiihColumbia British from COI K .hesperis S. and 6 = .h beani h. S. lse seiesfo British from (specimens cluster re(i.1) agl consistent largely 1b), (Fig. tree iegs h otenlineage northern The lineages. o ass&Ge,14 from 1947 Grey, & Passos dos Ewrs 88 ape in sampled 1878) (Edwards, COI sdsic clusters distinct as Δ Bre Benjamin, & (Barnes K diinlyhda had additionally aaesrecovered datasets .atlantis, S. .h lurana h. S. , n a and , , .h. S. and Δ but S. K Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. otenppltos(i.2) usrcueaayi of analysis Substructure 2b). (Fig. Idaho populations the northern and populations, Utah Colorado southern southwestern Mexico the New Similarly, northern the of Two nausicaa Arizona. and Mexico zerene re oee h eodms omntplg 71 ftesmldtes hw nprl)depicted MCC purple) the in in shown presented trees, topology the sampled the of the as 87.4% of same for the (7.1% accounted was topology which panel), common topology, right most recovered 3a, zerene second major Fig. the The in however blue tree, tree. in between the (shown relationship on trees the sampled species in discordance indicated three indicated additionally remaining results tree the BFD* MCC the of The visualization DensiTree panel). left 3a, northern (Fig. and 0.87 of southern probability the that posterior a 1) with of supported probability (posterior clade northern the LnPr( of both analysis substructuring; Substructure geographic Alberta. and Montana eaieBysFco B) niaigta hsmdlwstebs upre ytedt Tbe1.The 1). (Table data the northern by the supported for support best strong the indicated was tree model (MCC) this credibility that clade indicating maximum ( (BF), model Factor species” Bayes “4 negative the southern BFD*, in and tested , models species five the Californian Of the of analyses rest introgression the and with delimitation consistent Species haplotype a had and California, from instead 2a). hesperis (Fig. S. cluster one single hybrids: a likely supporting into further the S1), populations of Utah (Fig. that southern to and score central DIC the lumped it that given supported and TESS locality, sampling to corresponded that data estimate the in sub-structure meaningful any eaawsietclt h major with the admixed to identical was Nevada third the Interestingly, the from distinct haplogroups. of most consisted the was group containing and group One Utah and Montana, haplogroups. Idaho, distinct Alberta, three with network, Comparatively, “ location. major sampling the in one from observed Manitoba; different not and minimally from distinct Alberta were marginally in Manitoba were sampled Colorado 1940) from each Chermock, specimens for 1998 group haplotype Spomer, & major the and specimens canadensis “distinct” the For between differences species. nucleotide either two within or variation one haplotype distinct little depicted very network was there haplotype spanning minimum The hesperis with admixed was sbslt h te he species three other the to basal as rmClfri htwr o hrdb any by shared not were that California from population. 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S. .zerene S. .hesperis S. 5 = . hshpoyevrainlreycreae osmln oain-eastern - location sampling to correlated largely variation haplotype this K hsaayi a agl ogun ihthe with congruent largely was analysis This . nteSrcueo ESaaye fSP;tesnl likely single the SNPs; of analyses TESS or Structure the in a h ihs agnllklho siae(L)adtems strongly most the and (MLE) estimate likelihood marginal highest the had ) ESrsls u aldt d n ennflgorpi substructuring geographic meaningful any add to failed but results, TESS 5 = n one and , n lotalteseiessmldhdietclhpoye eadesof regardless haplotypes identical had sampled specimens the all almost and K ape rmsuhr thsae netywt h otenNwMexico New northern the with ancestry shared Utah southern from sampled sotmlfrti nlss ohTS n tutr niae few a indicated Structure and TESS Both analysis. this for optimal as 5 = K , rmClfri a nemdaebtenthe between intermediate was California from .zerene S. u h ld containing clade the but .hesperis S. samr ennflresult. meaningful more a is 1 = .zerene S. K .zerene S. .h electa h. S. . ld iegdsihl ale hntecaecontaining clade the than earlier slightly diverged clade and ) hr oooyacutn o h eann .%o h sampled the of 5.4% remaining the for accounting topology third A .h viola h. S. a uhmr altp iest ntemnmmspanning minimum the in diversity haplotype more much had .hesperis S. altp htwsfudin found was that haplotype Δ altp Fg c,btteeidvdasddntappear not did individuals these but 2c), (Fig. haplotype K rmClfri hrdacsr ihtesuhr Utah southern the with ancestry shared California from niae noptimal an indicated .zerene S. pcmn eeitreit ewe h eta and central the between intermediate were specimens 8 pcmn eeitreit ewe h eta and central the between intermediate were specimens priori a hollandi .h dorothea h. S. .zerene S. ape rmClfri (putatively California from sampled .alni hollandi atlantis S. pcmn ape rmNvd,Ua,Idaho, Utah, Nevada, from sampled specimens .hesperis S. .zerene S. .atlantis S. pce Fg c.I l ae hr eeonly were there cases all In 2c). (Fig. species altp.Ti egahcptenwas pattern geographic This haplotype. ” .zrn,S atlantis, S. zerene, S. .hesperis S. niae eei ieecsin differences genetic indicated K K pcmn n l h Arizona the all and specimens .zerene S. n southern and lse i o niaeadditional indicate not did cluster tutr eut,ecp that except results, Structure 6 = .alni hollandi atlantis S. and f2 oee hsddntproduce not did this however 2, of .zrn gunderi zerene S. and .hesperis S. rmAbraadoefrom one and Alberta from .hesperis S. .alni sorocko atlantis S. .hesperis S. .atlantis S. pcmn ape from sampled specimens K .zerene S. .hesperis S. .zerene S. northern a similar a had 6 = lses however clusters, and and n second a and , Cemc & (Chermock ape from sampled .zerene S. .h irene h. S. Δ yrdwas hybrid specimens. .hesperis S. .atlantis S. .atlantis S. .atlantis S. .atlantis S. K .zerene S. a less was Kondla cannot .h. S. and S. S. ) Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. niae osgicn euto ntepwro ahlnaesnl Nswe rdcigterandom “within- the the predicting of when coefficient ENMs the null Specifically, lineage’s each scores each lineages. AUC to of other resulting confined power the addressing localities the for models generated in generated effects randomly reduction localities mixed with significant Linear built no polygon. ENMs indicated convex null minimum using buffered niches. repeated lineage’s ecological were and associations analyses habitat These their in divergent significantly “within- are the comparison: lineages of pairwise southern coefficient and each northern The for ENMs and lineage’s negative lineages. each two significantly of other was power the northern variable the of predictor in each reduction binary of significant lineage” Contributions localities a was the ENMs. there predicting to the indicated when of contribution models each effects greatest For mixed the lineages. 2. Linear had Table among in days considerably reported degree varied of are convex variables growing permuted, minimum other density and as randomly buffered of cover measured was highest their variable ENMs, land each the to to lineages, after variables adjacent that three scores predictor and AUC environmental and are in within and drop lineages niches, found geographic the genomic of generally ecological contributions three is and Relative the lineage polygon. associations each that for suggests habitat habitat landscape respective suitable study their entire in the divergent across surfaces as suitability polygon, southern habitat convex and minimum 0.063), = buffered lineage’s s.e. scores: each AUC within mean by suitability indicated habitat predicted sufficiently ENMs divergence niche Ecological northern and Mexico/Arizona central the into ( population from admixture 61 the Of 0.01, = weight (migration 0.09, atlantis = weight (migration between events migration significant, statistically still the but than rather 0.07, substantial, branch = less the weight two of (migration Pritchard indicated interior & (Pickrell the also populations along model between unsampled plotted from This edge admixture migration or of a 2012). admixture historical 0); addition indicate = continued may containing p that terminus branch 0.41, suggesting the = similar, weight of The highly migration interior fit. models were improve the for greatly events from not plateau migration did event to point five seemed this 1. this and past but events Fig. four, S2), migration in phylogeny three, (Fig. events SNP for improved migration the indicated plots of with generally addition consistent model migration the largely each with was likelihood for fit maximum residuals S2) population unrooted Fig. pairwise The the 3b, containing species. Heatmaps (Fig. these TreeMix between in introgression for produced test phylogeny to TreeMix used we analyses, between relationship discordant the Given depicted The orange) in (shown trees ouainand in population populations: some between weights models Though 0.017). .atlantis S. mgainwih 0.02, = weight (migration .hesperis S. .zerene S. .zerene S. f et o nrgeso htw optd nytrewr infiat(al 2;oeindicated one S2); (Table significant were three only computed, we that introgression for tests 3 p ) n nte from another and 0), = /northern .atlantis S. .zerene S. .hesperis S. n h ld otiigteNwMxc/rzn n central and Mexico/Arizona New the containing clade the and n h ld otiigtecnrladNwMexico/Arizona New and central the containing clade the and ( β -0.292; = m p .hesperis S. and 4 p a o-infiat(irto egt 0.02, weight: (migration non-significant was .hesperis S. p n h otenUa ouainof population Utah southern the and .4)wr non-significant. were 0.345) = .hesperis S. .3) n between and 0.037), = ouain( population .5) ewe h e Mexico/Arizona New the between 0.058), = .hesperis S. .atlantis S. m .zerene S. p p a iia eiul Fg 2,te niae o-infiatmigration non-significant indicated they S2), (Fig. residuals similar had 5 0.028), = .9) n ewe h eta n northern and central the between and 0.092), = itrrltosi a ossetyrcvrdi ahsmldtopology. sampled each in recovered consistently was relationship sister m ma .0,se .8)(i.4.Vsa npcino predicted of inspection Visual 4). (Fig. 0.085) = s.e. 0.803, = (mean ( β .zerene S. h irto debtenteNwMexico/Arizona New the between edge migration the 4 -0.333; = ma .0,se .8) northern 0.084), = s.e. 0.809, = (mean .zerene S. p ouain notecentral the into populations stesse ao othe to taxon sister the as .0) h hr etsgetdgn o rmteNew the from flow gene suggested test third The 0.003). = .atlantis S. .zerene S. n h e eioAioapplto of population Mexico/Arizona New the and p and .zerene S. .2) oehr hs eut niaeta l three all that indicate results these Together, 0.023). = 9 .alni/.hesperis atlantis/S. S. n southern and otecentral the to and m .atlantis S. oe niae usata migration substantial a indicated model 3 .hesperis S. .atlantis/ S. .hesperis S. .hesperis S. p .hesperis S. .6) n in and 0.169), = .hesperis S. mgainwih 0.07, = weight (migration norpyoeei n BFD* and phylogenetic our in notecentral the into northern .hesperis S. ouain( population ( β .hesperis S. .hesperis S. .hesperis S. -0.6811; = ouain(i.3b; (Fig. population vs. ouain and populations m .hesperis S. between-lineage” -;teresidual the 3-5; ma 0.810, = (mean .atlantis S. m vs. p h edges the 5 populations populations populations .hesperis S. .hesperis S. .hesperis S. 0.035). = p between- < clade. 0.00), p and S. = Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. u N hlgn Fg a.Hwvr h eainhpof on relationship work the phylogenetic However, between molecular recent 1a). as between interesting, (Fig. relationship phylogeny sister SNP a our and between species, history distinct evolutionary as shared complex, zerene a S. suggest analyses genomic Our between introgression historical of contexts for conservation group Evidence in meaningful species approach and confusing This stable more notoriously science. concepts. and be citizen a species quantitatively ultimately ecological and uses should incorporate and that study genetic delimitations to of Our species integration phenotypic difficult supports analytical accounts. only the been an history – demonstrate are have characteristics natural to genes – butterflies ecological to environment by But relegated informed their usually delimitations more available. with are Species increasingly individuals become as species. of have renaissance a interactions a methods makes undergone what analytical have of and data part genetic technology by sequencing informed of sophisticated are present separation that might delimitations geographic that Species habitat the suitable to of attributed arrangements to best less Discussion not is are and lineage this parapatry) dispersal. each lineage, to (i.e., of barriers lineages other individuals significant the the dispersing of of although ranges that, individuals the suggests contact related result to significantly This not likely was distance. lineage) different Euclidean vs. and (same ( distance distance lineage resistance that lineages. to indicated hesperis three test S. the mantel among in partial A habitat flow available gene in to differences barriers from of arising Assessment biases to attributed be cannot niches 0.015; atlantis for S. non-significant was variable predictor binary hl h eut forSPbsdaaye n h ako eunevraini h iohnra haplotype mitochondrial the in variation sequence of lack the between and analyses shared SNP-based our of results and the between While admixture introgression SNP-based probable by indicated to explicitly due more also is but analyses, phylogenetic our southern al. et neto i nrgeso ffr luil n etbehptei htcudepanteosre haplotype observed the explain could that hypothesis testable and plausible a offers introgression throughout via infection haplotypes mitochondrial particular (Werren(McHugh variation of haplotype spread reducing Ahmed the is species, facilitate process and can populations a that such bacteria (Sonsthagen facilitating endosymbiotic haplotypes recently for has mitochondrial event variable bottleneck candidate severe other a A of to leading loss sweep the selective strong of caused a range that the suggesting variability, across genetic SNPs in nuclear mtDNA, to contrast In in and haplotype TreeMix this them; of pervasiveness between the flow gene of entire zerene, the direction throughout the occurs clarify sufficiently from introgression not indicated do they species, p 09.I slkl httercvrdplpyyof polyphyly recovered the that likely is It 2019). ugssta toiiae in originated it that suggests tal. et .zerene S. .1) hs eut niaeta bevddffrne nhbttascain n ecological and associations habitat in differences observed that indicate results These 0.810). = .hesperis S. tal. et F*seisdlmtto upre eaaino otenadsouthern and northern of separation supported delimitation species BFD* . n southern and 2015). 03,btntytwti the within yet not but 2013), .zerene S. and Wolbachia and .hesperis S. r -0.111; = .hesperis S. .zerene S. and .zerene S. .hesperis S. .hesperis S. netoshv enrpre nseveral in reported been have infections / p rsne eei atydet h msino other of omission the to due partly is here presented .atlantis S. .8)atrpriligotterltosi ewe ieg distance lineage between relationship the out partialling after 0.987) = ( .hesperis S. into .zerene S. β 0.020; = ape ag,btol nteNvdnpr fterneof range the of part Nevadan the in only but range, sampled .hesperis S. upr yohsso nrgeso ewe hs non-sister these between introgression of hypothesis a support .hesperis S. d Moya (de .atlantis-hesperis S. .atlantis S. Wolbachia n ovldt t origin. its validate to and p xaddsmln fbt pce snee oclarify to needed is species both of sampling Expanded . .5) n otenadsouthern and northern and 0.751), = 10 .atlantis S. oee u neec htthe that inference our however , Speyeria and .hesperis S. tal. et COI n northern and etg&Wlah 94 maternally-inherited, 1924, Wolbach, & Hertig .hesperis S. .zerene S. .zerene S. altp nlss(i.2 i.3 al S2). Table 3, Fig. 2, (Fig. analyses haplotype 07 Campbell 2017; eeal niae o-itrrelationship non-sister a indicates generally tal. et n northern and ope (Hamm complex ontso h aeovosreduction obvious same the show not do Speyeria 08 Kodandaramaiah 2008; n h itrrltosi between relationship sister the and oteohrcae aid hsis This varied. clades other the to tal. et .hesperis S. .hesperis S. 07 us ign 2005). Jiggins & Hurst 2017; tal. et .hesperis S. .alni,S hesperis, S. atlantis, S. pce,including species, tal. et ( 07 09 Thompson 2019; 2017, β .hesperis S. and -0.012; = .hesperis S. Speyeria 2014). .hesperis S. ossetwith consistent , .zerene, S. tal. et p Wolbachia haplotype pce in species 0.822), = .zerene S. f lineages tests 3 which 2013; ( β and S. = Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. eetv fti eetrneepnin swl sdet e arest ipra n eeflwi the in flow gene and dispersal to barriers few to due northern as of southern well habitat of and as forest habitat and retreat, expansion, prairie/open northern other glacial in range homogenous each structure last recent relatively with population the this genes recovered of exchange following of lack to reflective the habitat continued then available initially accurate, newly is have hypothesis may into lineages region these geographic that this northern from in southern poleward substructure and any northern recover sampled to southern failed of TESS population and northern Structure between Both flow southern 3a). and hesperis northern (Fig. splitting S. for species support clear distinct indicated into SNPs using delimitation species BFD* maintained be Our from should regions. distinct lineage sympatric subspecies evolving the in between independently sorocko, even differentiation species, SNP a. between and S. flow mitochondrial hollandi, little gene relatively limit indicate to also enough results significant likely are Riva associations surveys genetic 2009; other Dunford with 2001; consistent are results these between Canada); Alberta, in (mostly (Thompson ranges their of southern portions the between within admixture clusters of genetic northern any the to that than show consistently results hesperis Our S. in limits species of 4). in divergence Assessment (Fig. geographically-structured lineages clear the demonstrate between landscape associations study southern entire habitat and genomic the to northern across lineages’ barriers between habitat parapatry the with suitable present-day of associated that speciation reinforcement suggest hesperis therefore mutation-order S. and We or diversification vicariance). selection (i.e., to without dispersal dis- contributing speciation selection to divergent process than barriers ecologically-based rather significant principal that integrity, hypothesis any the the detect between to been not support habitat did has further available southern analyses conferring localities in them, resistance-based and between random differences our northern persal from distributions, to although their built Furthermore, attributed in ENMs be allopatric divergence). null cannot niche separation, divergences (spurious geographic niche lineages of observed degree that to indicate between correspond estimated approximately divergences to genomic indica- of hesperis an that S. as these to of also corresponds Comparison but ing divergence, events. between niche speciation greatest was hesperis of ecological divergence strength reflect niche that likely relative suggest ern divergences the associations models genomic of habitat across not estimates measure their coefficient or in a whether differ as of “within- significantly only tor the data, not of genomic magnitude interpreted relative of be The basis niches. the ecological on and delineated the lineages, that tionary indicated ENMs taxa. of these between Comparison relationship historical divergence the niche clarify of further Evidence may which specimens, sampled the in Nevadan between sharing .hesperis S. hc eeapoiaeyeuvln ntersrnt fpiws ih iegne hsorder- This divergence. niche pairwise of strength their in equivalent approximately were which , .atlantis S. sacneune ahrta as,o clgclybsddvretslcin spoetosof projections as selection, divergent ecologically-based of cause, than rather consequence, a is Fg ,Fg ,Fg 3) Fig. 2, Fig. 1, (Fig. tal. et ept ra egahcsmln,adTeMxand TreeMix and sampling, geographic broad despite olwdb northern by followed , 09 Campbell 2019; .hesperis S. and . .hesperis S. .hesperis, S. .atlantis S. and .hesperis S. .zrn gunderi zerene S. tal. et .a canadensis a. S. ntesuhr ok onan.Tehpoyesaigbetween sharing haplotype The Mountains. Rocky southern the in .hesperis S. 09,adterslso u Nsidct htdvrecsi habitat in divergences that indicate ENMs our of results the and 2019), tal. et Fg ,TbeS) hsi loteapoiaebudr ewe the between boundary approximate the also is This S2). Table 2, (Fig. n northern and southern nrgosweete oocr(Bird co-occur they where regions in . .hesperis S. lhuhdge fdvrecsbtenpiso iegsappears lineages of pairs between divergences of degree Although 09.Ps okhsntddffrne nmcohbttpreference micro-habitat in differences noted has work Past 2019). .atlantis, S. and and iegs oi spsil htnorthern that possible is it so lineages, .hesperis S. .hesperis, S. eaiet h te aai hsstudy this in taxa other the to relative .hesperis S. .atlantis S. .hesperis S. .hesperis, S. .hesperis S. s southern vs. 11 northern n eonto fteseissau of status species the of recognition and , and nteasneo otmoaynceradmixture nuclear contemporary of absence the in ieg smr eeial iia to similar genetically more is lineage ieg,btw i o eoe n evidence any recover not did we but lineage, vs. .hesperis S. .zerene, S. ept apigbt pce nsympatric in species both sampling despite .hesperis S. .hesperis S. ewe-ieg”cecetetmt may estimate coefficient between-lineage” .atlantis S. f nlssidctdpoal gene probable indicated analyses 3 atclryi rna h central the near or in particularly n southern and , and tal. et oprdt h oedisjunct more the to compared n otenadsouthern and northern and .atlantis S. .hesperis S. 95 up Shepard & Guppy 1995; . .hesperis S. Thus, .hesperis S. .atlantis S. .zerene S. .hesperis S. s northern vs. .atlantis S. a epartly be may r largely are .hesperis S. .atlantis S. .atlantis S. .atlantis S. s south- vs. expanded .zerene S. fthis If . evolu- san is S. Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. he,M . ruoJr .V,Wlh .J n aaaa .Y 21)Wlahai utrisand butterflies in Wolbachia (2015) Y. 1-9. A. 12, Kawahara, Zoology, and in J. Frontiers frequency. J. infection Welch, in N. V., structure Puillandre, E. geographic Araujo-Jnr, moths: and Z., P, integrative M. Zaharias, an Ahmed, in Y., 4591-4611. sequencing Kantor, 27, RAD Ecology, using A., Conoidea) Molecular (Turridae, Fedosov, framework. gastropods taxonomy B., marine of Buge, species Delimiting L, (2018) Aznar-Cormando, J., Abdelkrim, well support. and as computational Alberta for support, of References (www.westgrid.ca) sequencing University Westgrid MaxEnt. for the and Universit´e Laval in (www.computecanada.ca) at at Canada modelling Unit Syst`emes (IBIS) Compute niche Services des as to Int´egrative We Biology et related Bean Biologie Molecular IDs. guidance the de the specimen their acknowledge with Institut and for help to collection, Majchrzak to wish Yasmine specimens specimen additionally and museum We with their Peers to help Michael access Lee, thank for James for also Valgardson University Fisher, Young and Kirby Mark Brigham Ferguson, E.O.C. and Simon at to Dupuis, Biodiversity Museum Sperling, Julian in Thomas Grants Campbell, (ACA) Barb Nazari, thank Association Vazrick Conservation to wish Alberta authors Doctoral by to The – and grant Scholarship Z.G.M. Discovery Z.G.M, Graduate (NSERC) to Canada Council D) Bell Research – Graham Engineering (CGS Alexander and NSERC Sciences an Natural (RGPIN-2018-04920), taxa. a F.A.H.S by of funded range was broad work a This in diversification conservation, lineage for of units patterns Acknowledgements biodiversity influence meaningful that delimit factors taxonomies, delimitation extrinsic stable informing species inte- characterize to of distinctiveness, readily contribute and method ecological to species be multidisciplinary to positioned This could for thus divergence and management. useful is genomic biodiversity science, particularly and links citizen habitat be that in of framework should prioritization visibility coalescent-based conservation analyses high delimitations a multispecies ecological or into species and and impacts grated phylogeographic genetic conservation genetic have for in of that priors subsequent integration modelling delimitations in Bayesian an approach ecological inform this Such of on reciprocally selection. build incorporation modelling. to divergent to the used opportunity ecologically-based great particular, be - maintenance is mechanism may In the there likely that attribute a suggest work. to to we us systematic results, lineages enabled our these have to of surfaces addition each In resistance in landscape integrity and genomic ENMs of using analyses between and divergences genomic , species-level substantiate results Our several of status Conclusions conservation between the introgression to detected due also We pursued be where persist. Basin, may Great Pleistocene the and the and California before in diverged particularly assessment, that genomic lineages and sampling study, further this requires that as in genomic to Following sampled referred their name species. be maintain distinct to level, subspecies continue as nonetheless southern should recognized lineage that oldest be northern environment the should the lineages 2019), the two (Pelham and priority the taxonomic that and species recommend northern other therefore We between with integrity. divergences interactions ecological by and shaped evolutionary of recent lineages support southern unambiguously results Our a the likely in to even is revision lineages, Taxonomic selection southern divergent and dispersal. northern ecologically-based to the barriers that of of suggest reinforcement absence and similarly divergence ENMs underlying process that complex. principal emphasizes this and in expansion, boundaries range this to southern prior and northern and .hsei/.nausicaa hesperis/S. S. .nausicaa S. orpeetteetr otenlnae hstxnmcrvso ean hypothesis a remains revision taxonomic This lineage. southern entire the represent to , .hesperis S. .hesperis S. .hesperis S. htmyb oreo ula eete-pce redsodne hsshould This discordance. tree tree-species gene nuclear of source a be may that u loidct htteelnae urnl xs sdsic entities distinct as exist currently lineages these that indicate also but , ute ugssta nrgeso ewe hs pce occurred species these between introgression that suggests further COI 12 .zerene S. sol atal nomtv o lrfigspecies clarifying for informative partially only is .h nausicaa h. S. subspecies. .atlantis S. hudb lvtdt species to elevated be should , , .hesperis S. and , .hesperis S. .nausicaa S. .zerene S. and , Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. 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Kondla, 16(7), its A., evolution, and J. & speciation Scott, ecology ecological in for Trends Evidence species. (2009) of D. origin Schluter, the and Ecology (2001) D. Schluter, 11231. 8(1), reports, Scientific agassizii). < www.butterfliesofamerica.com 18 > Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. O n G odce aaaaye n rt h nta aucitdat l uhr otiue to contributed authors all draft; Figures manuscript revisions. and draft initial Tables manuscript the and wrote collection, and specimen analyses design, data study conducted ZGM and EOC Campbell citation: Data contributions acceptance. Author manuscript New pending respectively, (2010) SRA, C. NCBI J. and Svenning, & B., COI P. Pearman, H., 985-989. Accessibility Press. C. 33(6), Ecography, California Data Graham, of modelling. C., University distribution (CA): T. 914–923. species Berkeley 163, Jr, in idea. Nat. Edwards trends the E., Am. of N. it? history study A Zimmermann, we Species: should (2009) how S. and J. biology. speciation Wilkins, invertebrate is of What manipulators (2004) master J. Wolbachia: J. (2008) Wiens, E. 741-751. M. 6, Clark, Microbiology, and Reviews L., Nature Baldo, H., J. Werren, eesqec n a dA/w-nyeGSsqecn lswl emd vial nGenBank on available made be will files sequencing GBS ddRAD/two-enzyme raw and sequence gene 19 tal. et (2020). Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. eta ufc sdpce nteright. image: the right correspond on Top depicted colours (A). is haplotype in surface and results ventral sequences, subclustering between and image: differences clustering left nucleotide Structure of the number geographic to the and genetic indicate between (C) correspondence and in show northern to (B) between hesperis in S. divergence map the genetic on The charts major structure. pie as a plotted indicate are analysis consistently (A) results southern TESS and Structure 2: Figure indicate nodes on circles Grey (A). in membership group their to according values coloured bootstrap are (B) in Specimens 1: Figure .hesperis S. aiu ieiodpyoeisbsdo A ula Nsad()mitochondrial (B) and SNPs nuclear (A) on based phylogenies likelihood Maximum egahcassmn fpplto eei lseigof clustering genetic population of assessment Geographic n altp hrn between sharing haplotype and .hsei beani hesperis S. COI > 75%. altp ewr o h aeseies()dpcsalc fgorpi tutr in structure geographic of lack a depicts (C) specimens same the for network haplotype iegs ouain of Populations lineages. h oslwn ufc ssono h etsd fec pcmn n the and specimen, each of side left the on shown is surface wing dorsal The . .hesperis S. .hesperis S. 20 n Nevadan and and .atlantis S. .zerene S. .atlantis S. dnie nthe in identified ace ln h branches the along Hatches . and .alni hollandi atlantis S. .hesperis S. K COI 6Structure =6 sn SNPs. using sequence. lower ; Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. infiatmgaineet hwn uaieitorsinbtenppltosof populations between introgression output putative (left) and showing phylogeny events likelihood the migration maximum in recovered the significant populations the depicts the for (B) using TreeMix support topology. by branch that and depicting poor tree, distribution of MCC posterior source the the on as nodes (= zerene SNPs theta both between of at discordance estimates red zerene genealogical the in the southern indicate indicated visualizes and branches are (A) northern in the values (overlaid supporting along probability tree colour), values (MCC) Posterior (in grey DensiTree credibility by clade species. maximum output distinct a cloud” both as “tree as a visualized as model and black), species” “4 BFD* the depicts 3: Figure .atlantis S. and /southern .alni/.hesperis. atlantis/S. S. F*seisdlmtto n rei nrgeso eut sn Ns h etpnli (A) in panel left The SNPs. using results introgression TreeMix and delimitation species BFD* h oe eiul o hsaayi r niae ytehampo h ih n(B). in right the on heatmap the by indicated are analysis this for residuals model The . .hesperis S. ld nteMCte,adidctstreatraerltosisbetween relationships alternate three indicates and tree, MCC the on clade ecnae bv ahDnirerpeettepooto ftesi the in trees of proportion the represent DensiTree each above Percentages K 21 tutr nlss(i.2,wt he statistically three with 2), (Fig. analysis Structure 6 = N e o ahlnae h ih ae in panel right The lineage. each for ) .zrn,S hesperis, S. zerene, S. .hesperis S. S. S. Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. otnnaiy0005 .600 6.05 7.16 0 9.61 34.62 0.41 26.7 0 0 3.97 4.46 5.34 0 s.e. 0 6.22 41.59 0.18 39.52 1.26 0.38 0 0 4.06 mean 20.12 3.98 0 0.62 0 12.79 0.56 0.17 0 10.5 0 42.71 s.e. 3.02 0.43 0.28 0 35.04 5.1 0 18.74 2.61 0.19 1.9 13.97 0.44 5.44 19.54 0 15.13 1.89 mean 0 54.78 1.47 0.84 4.73 10.07 BF 3.1 s.e. precipitation Aug) MLE to 0 (Jun summer 15.87 6.58 snow as precipitation mean temperature minimum extreme days degree growing days 9.99 degree chilling 0 month warmest continentality the of month temperature coldest mean the of temperature mean cover land load heat ruggedness terrain variable Predictor therefore was model species” “4 the support; model strongest indicate optimal. values considered (BF) Factor Bayes negative 1: Table ” species” ”6 ” species” ”4 ”north species” ”3 species” ”2 tested priori a groups received a Species cell grid suitability. 1-km higher Each model indicating values BFD* area. higher study five with entire the 0–1, of the from average across ( ranging The suitability score points habitat. habitat suitability available predict Background habitat sample to to used evaluation. polygon were convex model minimum models for buffered lineage’s used each localities within occurrence of 20% hesperis 4: Figure n C southern (C) and , F*mdlslcinrsls h ihs agnllklho siain(L)admost and (MLE) estimation likelihood marginal highest The results. selection model BFD* rdce aia utblt ufcsbsdo Nsfr(A) for ENMs on based surfaces suitability habitat Predicted S atlantis” ”S. ” .zerene S. atlantis S. .hesperis S. .hesperis S. ”, ,” ”, .hesperis S. north + ” , S atlantis ”S. .zerene” S. .atlantis S. + .hesperis S. .atlantis S. o ahlnae v Nswr ul,ec ihodn different a withholding each built, were ENMs five lineage, each For . ,”north ”, ”north , ,” ”, .alni .atlantis S. atlantis S. .zerene S. ,”south ”, ”south ”, .hesperis” S. .hesperis S. 22 -13863.6 ” .hesperis S. hesperis S. ,”eta” suhU” N Z 267117587 -22657.1 AZ” & ”NM UT”, ”south ”central”, ”, ”south , ,” ”, + northern .hesperis” S. .zerene S. .zerene S. pyraatlantis Speyeria n .hesperis S. 13751067.8 -14397.5 ” 1848-97.6 -13814.8 ” 000 eegenerated were 10,000) = B northern (B) , northern

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S. southern .hesperis S. southern .hesperis S. Posted on Authorea 1 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160157511.10593343 — This a preprint and has not been peer reviewed. Data may be preliminary. sdfrmdleauto.Tema n tnaddvain(.. frltv otiuin o ahpredictor each for localities reported. contributions occurrence relative are of of ENMs 20% (s.e.) different permuted. five deviation a randomly the standard withholding was and across each variable mean variable built, The each were evaluation. precipitation. after ENMs model Aug) values five for to AUC lineages, used three in (Jun the drop summer of the and warmest each as For snow, and measured as coldest was precipitation the contribution temperature, Relative of minimum temperatures mean extreme cover mean month, days), land 0 between coldest gree and below difference the aspect), degree-days the of and (continentality), month, temperature months slope warmest mean terrain the included on of variables (based temperature predictor load Environmental heat categories). ruggedness, (12 terrain included variables dictor atlantis 2: Table northern , h eaiecnrbto % fgorpi n niomna rdco aibe to variables predictor environmental and geographic of (%) contribution relative The .hesperis S. n southern and , ° .hesperis S. ciln eredy) eredy bv 5 above degree-days days), degree (chilling C 23 clgclncemdl EM) egahcpre- Geographic (ENMs). models niche ecological ° goigde- (growing C Speyeria