Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. atta,frmn pce,i eosrtdol eylmtditaseisvrain(..gnrlybelow generally the (i.e. to variation due intra-species standard limited the gold very of barcode only 5’-region DNA demonstrated the the it , become For species, quickly delimitation. many has species for gene for that, (COI) extensively also fact been I between but since subunit identification has gap oxidase and specimen 2003), “morphological” method cytochrome for DeWaard, identification & a To only cheap Ball, 2016). and not of Cywinska, Eberhard, fast, generational Hebert, used absence & reliable, 2002; Frey, or Djurakic, a an & (Karanovic, as sexual Fleming, diversity to introduced by (Brunner, cryptic was conversely lead of barcoding levels or DNA will within-species substantial morphology, taxa, complement in which minimize between result to of variation may morphological way and Both of However, species a lack characters. species. in variable a within of traits delimited by sets polymorphisms were morphological challenged in often They in variation variation is 2010). between-species Vences, examining maximise & by to taxonomists, Riva, described and for la 2019; variation were S´anchez-Bayo Wyckhuys, task De & 2017; species urgent Traditionally, al., Miralles, et an 2019). (Padial, (Hallmann is al., insects the hypotheses et of species Seibold group 2014, hyperdiverse Pievani, testable the 2011; providing for al., especially et and (Barnosky diversity observed currently cryptic losses uncovering biodiversity of levels unprecedented the Given Introduction 1. species. fast bee a single as a within UCEs paraphyly of mitochondrial evidence suitability conclusive and first the species, the delimitation for distinct provided species study among evidence this explicit introgression Furthermore, strong mitochondrial provided lineages. provide six diverged both and results recently using for loci in These even assessed nuclear delimitation was homologous complexes. species for robust 1860 species method method and and investigated genomic paraphyly. 686 quick all mitochondrial This between in or a recovered introgression, hypotheses as delimitation. mitochondrial UCEs diversity, (UCEs) of cryptic species To elements harbour sequencing of DNA. to genetic The cases suspected high-quality nuclear complexes problematic require ultraconserved species with such often bee of addressed different and address instance use be repeatable, to for only the approach poorly can barcodes, examined single intensive, genomic barcoding share we labour one DNA can limitations, expensive, mitochondrial as species these within-species are of more regarded overcome deep which limitations or to is markers, intrinsic two or what diversity These genomic contrast, cryptic nuclear instance, In to introgression. For either mitochondrial isolation. point to can reproductive history. which due no barcodes, species cases with DNA numerous the distinct divergences nevertheless two reflect mitochondrial are with COI there necessarily associated Although taxa, be not can delimitations. most do species for species barcodes identifications morphological assisting mitochondrial complement in where to successful Today, proven biodiversity. barcoding largely managing DNA and has mitochondrial surveying barcoding measuring, on for rely essential often are hypotheses taxonomists delimitation species testable and Accurate Abstract 2020 5, May 2 1 bees Gueuning European Morgan Central in enigma UCEs systematic long-standing delimitation: resolve species for informative yet Ultraconserved gocp tnotWadenswil Biologie Standort de Agroscope Institut Neuchatel de Universite 1 ur Frey Juerg , 2 n hitpePraz Christophe and , 1 1 Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. ighwUE a vroetemi rwak o pce eiiainuigDAbroigdeveloped complexes: investiga- barcoding species include species, DNA We European using per bees. delimitation following “barcodes” European species the for Central multiple ; on drawbacks in of main focused delimitation the We and species overcome above. introgression can for mitochondrial UCEs UCEs how putative of an ting use both remains the of species examine examples diverged we conserved. study, recently highly this among are However, In divergence markers. they nuclear definition capture for by requirements to since mentioned variation above question, the enough open (Faircloth of harbor timescales” many evolutionary fulfil essentially they that multiple to and can whether spanning so appear quick datasets markers UCEs a that repeatable, 2012). genetic as extent al., of introduced the (iv) et “thousands were to obtain (UCEs) species; universal elements to (v) diverged way ultraconserved available; numerousuniversal 2012, recently is cover In material to other. capture more markers each nuclear once to complement complemented for variable scale be reflect genomic sufficiently can to (ii) datasets markers, (iii) genes; mitochondrial mitochondrial loci; and and nuclear nuclear independent (i) last both on: of This based flow datasets. repeatability. be gene earlier of should complementing essentially lack species-delimitation continuously datasets work molecular data the taxonomic Ideally, importantly, new since to with delimitation, More due hypotheses, species joinable required. for previous drawback hardly upon DNA severe builds are a is high- high-quality is taxa RAD-seq provide RAD-sequencing of and/or of can microsatellites, amount limitation studies and for different and/or powerful as from very workload However, obtained are RAD- dynamics. cost, slightly methods as population by are such These on techniques that hampered 2019). primers information genomic-reduction approaches al., specific include Alternative intraspecific et clade resolution information. resolution, (Lemopoulos require higher genomic sequencing therefore or available ddRAD and equal on or clade-specific provide base are and designed loci (McKendrick universal that more be delimitation is to to species such limitation impediment have markers for major genetic major that resolution one population a ample 2017), used is provide al., have which et microsatellites studies ITS), Although some insects, (e.g. microsatellites. resolution, in genes increased and as 2018), For ribosomal internal Flot, workflow. multi-copy & sequencing (i.e. the (Dellicour these the resolution 2018; of markers of phylogenetic usefulness al., variation of DNA the lack et within-genome However, the ribosomal Martinet by by 2019). limited multicopy Thaochan, 2009; often & Wahlberg, or is Lelej, markers & 28S) Williams, nuclear well-suited, Chichvarkhin, been 2017; or (Leneveu, similarly Praz, have & explored [EF-1a] B´enon, nuclear markers for Alvarez, Soltani, were single-copy alpha nuclear quest instance, [ITS]) of For 1 the spacer drawbacks. types factor marker, serious transcribed Several with elongation species unsuccessful. associated (i.e. a all far are genes as so candidates was COI current markers but of explored, nuclear suitability robust uncommon genes 2012). and use the Sperling, universal should & to delimitation Roe, contrast species (Dupuis, markers, In origin molecular nuclear al., for and information et and of mitochondrial sources 2013) Hinojosa both multiple Satler, 2015; on of & rely Ruedi, should can Reid, & delimitation they Pelletier, species 2012), Naciri, Consequently, (Carstens, Hoffman, 2016). (Andriollo, & al., estimates Cant, et Jamie, Mutanen biodiversity & Jordan, 2019; or Kropf, Nichols, Klopfstein, phylogenies 2014; see Praz, skew (but & rare considerably Guex, considered Baur, generally Neumeyer, are events 2016; Wolff, these Baur, White, Although introgression, with acting 2012)]. 2017; forces mitochondrial Brelsford, thought, evolutionary Zouros, [e.g. & to (Toews initially level & subject organelle than be (Ladoukakis the can recombination complicated at genomes solely mitochondrial or more Furthermore, heteroplasmy 2008). is Gemmell, body leakage, & inheritance growing paternal Pierson, a mitochondrial of example, that For of cases emerged. number reporting rare recently A have is signal. results erroneous literature barcoding an of problematic to such leads for COI-barcoding reasons & where Genoud, possible examples M¨uller, Meyer & numerous Praz, 2003; nevertheless, Boev´e, 2019; are, al., & 2015). There Notton, et Hebert, No¨el, powerful Sonet, & Hebert (Pauly, a Morini`ere, Haszprunar, bees be 2002; Schmid-Egger, to Schmidt, in al., shown 2019; delimitation was et species COI-barcoding Brunner identification, for morphological (e.g., tool with species combination between In nrn abra/ieai;A ost/rpnu;A aatnc/rmeaarse aigosmal- Lasioglossum carantonica/trimmerana/rosae; A. dorsata/propinqua; A. barbareae/cineraria; Andrena 2 Wolbachia nrn amieti/allosa/bicolor/montana Andrena neto rsxbae asymmetries; sex-biased or infection Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. 04.Nvrhls,bt aaehbtdffrne ntrso opooy olncletn eaiu and, behaviour collecting pollen morphology, of Westrich, terms 2008; in Peeters, differences & Achterberg, exhibit Van taxa Groenenberg, Reemer, both 2016; Nevertheless, (Falk, 2014). species deli- same the species differentiation. of controversial morphological nerations with unclear and enigma generations long-standing divergent a First, morphologically is to due complex hypotheses species mitation this in 2015). delimitation carantonica/trimmerana/rosae al., Taxonomical Andrena et (Schmidt 3: although barcodes complex separated identical Species as share 2.1.3. taxa considered both generally because are challenged recently species was both view differences, this phenological and biogeographical for generations two 2010). with al. phenologies, et (Amiet genders both in than characters of combination a by cases barbareae barbareae/cineraria A. Andrena 2: as complex to Species referred undescribed 2.1.2. is an species representing this probably complex; and species Greece this in (2019). in collected included specimens were two species dataset, the complement To within clades sympatric species two distinct the the addition, in which In from group. unit this paraphyletic in a species valid for among 2.4% distances and to 3.7% approximately with species, high, for for cryptic clades clades undescribed sympatric then sympatric two until two revealed new, both 2010) also a of Neumeyer, validity analyses of M¨uller, taxon & presence These Hermann, the the revealed confirmed Amiet, (LW-rhodopsin) also (F gene but status nuclear a debated and with (COI) taxa two with where co-occur region alpine “ the (hereafter in group this within delimitation allosa/amieti/bicolor/montana Species All Andrena (2015). 1: complex al. Species et 2.1.1. complexes. Schmidt of species of exception six the the study on with comprehensive information included, provides the were section the Switzerland on in in based identifications introgression mainly nitida/limita mitochondrial COI-based of selected, and cases were morphological reported fauna between discrepancies bee showing Swiss complexes species Six complexes Species 2.1. Methods and species. these Material of status 2. the on morphology conclusions of definite characteristics enable combined not the does on barcodes the based DNA evidence documented COI-based suggested current and been were that low-divergence so have delimitations, below); differentiation morphological details morphological see with 2015; ti/allosa/bicolor/montana associated al., et not (Schmidt divergences cases controversial these the of for four for suggested goodeniana/succincta Nomada pigenum/bavaricum/cupromicans; .bicolor A. .barbareae A. nrn rosae Andrena and or and .bicolor A. .amieti A. .cineraria A. oltshederae/succinctus Colletes hc smil etitdt h ls ohseisaepllci u xii different exhibit but polylectic are species Both Alps. the to restricted mainly is which , and .carantonica A. .stragulata A. .amieti A. namely , ersn diinlcytcseiswti hsgop ean unclear. remains group, this within species cryptic additional represent ld Pa ta.21) oto hs ae r locnrvrilwt epc to respect with controversial also are cases these of Most 2019). al. et (Praz clade r iln pce,mrhlgclyvr ls,atog dnial nmost in identifiable although close, very morphologically species, sibling are .allosa A. eei itne ewe hs ypti ldswr comparatively were clades sympatric these between distances Genetic . / .barbareae A. trimmerana r osdrdb otatost ersn h umradsrn ge- spring and summer the represent to authors most by considered are .bicolor A. and o hc o nuhmtra a vial.Tefollowing The available. was material enough not which for , bicolor ope Shite l 05;wiede within-species deep while 2015); al. et (Schmidt complex .montana A. n n for one and .allosa A. 3 .bicolor A. and gop)hsln eandcnrvril especially controversial, remained long has -group”) .amieti A. rs.Wehrtemtcodilcae found clades mitochondrial the Whether arose. .cineraria A. hlgntcaaye namitochondrial a on analyses Phylogenetic . as eotdi cmd ta. 05 and 2015) al., et Schmidt in reported (also iohnra nrgesoshv been have introgressions Mitochondrial . epciey n ec comparable hence and respectively, , .cineraria A. eas ftermorphological, their of Because . Andrena .amieti A. .allosa A. a ie distribution wider a has p,a nPa tal. et Praz in as sp3, Pa ta. 2019). al., et (Praz and .amieti A. nrn amie- Andrena .montana A. Andrena formed , Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. pcmnue nti td a enulae noteBL platform. every BOLD of the sequence onto and COI uploaded metadata the been as addition, has well in study as (S1-S2); this sampling, dry. information in the Supplementary preserved used on as and specimen information provided Further pinned are identification. acetate, maps for for ethyl sampling 4 Praz); morphology in at (C. to us EtOH killed addition sampled of 70% were in one additionally in surveys by were preserved identified list sympatry morphologically were red were in 2018 the in species/clades within collected several the between For collected Samples or project Greece. Bees populations bee” and 2018. Swiss large Italy of France, in harbour List in to “Red collected the known were of specimens sites frame Additional the 2019. in and Switzerland 2008 across sampled were specimens Most that suggested study previous 2008). Sampling Lunau, a 2.2. & where (Diestelhorst Germany, barcode in described COI least As share 2019). at not al., misidentification, did et to species (Creedy due both England in be found potentially found was the could (2015) result in similar al. possibly A for et and cluster. above Schmidt European phenology southern 1997). hosts, a their (Kuhlmann, and in secretions for differ therefore glandular clusters is to divergent mandibular and two appear patterns of both colour composition on although relying chemical errors, mainly identification is species to two prone these between separation morphological goodeniana/succincta The Nomada 6: complex Species 2.1.6. mor- tentative. genital however remain male taxon is in of this females male differences of no clear Identification since on 1970). However, based Edmer, 2001; accepted and Neumeyer, generally challenging, M¨uller, & is Herrmann, complex (Amiet, this phology in delimitation alpigenum/bavaricum/cupromicans Species Lasioglossum level, 5: pre- complex genetic were Species the sharing species 2.1.5. barcode At observed both scale. the however, underlie European recovered, could the were errors 2015). identification at taxa al., that two et especially suggesting (Schmidt the COI, errors, share to to to separation corresponding found the subject viously mostly Morphologically, and clades 1997). complicated Scheuchl, distinct is & two Schmid-Egger taxa 2002; both Schwarz, of & Gusenleitner 2010; al., author, the on dorsata/propinqua Depending Andrena 4: complex Species 2.1.4. Germany. or Switzerland in reported of those but from area, differentiated be cannot generation carantonica for of summer two generation and the first May) of to the 2014). those of Westrich, mandible, males carantonica 2006; the while Neve, A. of and & morphology females; Peeters, the separate Reemer, in to Meer, allows der character van logical 2010; of al., differentiation morphological et Second, (Amiet sites nesting possibly, .carantonica A. .dorsata/propinqua A. r nw C rzadR atn nulse aa.Bt aaoelpi hi distribution their in overlap taxa Both data). unpublished Paxton, R. and Praz (C. known are ohtx xii itntpeooiswt nyoegnrto for generation one only with phenologies distinct exhibit taxa Both . .bavaricum L. .trimmerana A. smc oeaudn than abundant more much is .succincta N. .dorsata A. .bavaricum L. and h eotdCIbroesaigbetween sharing barcode COI reported the , .cupromicans L. MrhArl ueJl) lhuhioae aesme pcmn of specimens late-summer isolated although June-July), (March-April, and otenErpa lse otiigseiesof specimens containing cluster European northern a : .carantonica A. a ensqecd dnicto ftesqecdseiesfor specimens sequenced the of identification sequenced, been had .propinqua A. eercnl ugse osaetesm O barcode. COI same the share to suggested recently were 4 .trimmerana, A. r osdrdsprt rcnpcfi aa(me et (Amiet taxa conspecific or separate considered are and .carantonica/trimmerana A. .trimmerana A. ° oesr odDApeevto.Albees All preservation. DNA good ensure to C o hc nyafwocrecswere occurrences few a only which for scalnig ocermorpho- clear No challenging. is .goodeniana N. .carantonica A. hnlg a used was phenology , .trimmerana A. .bicolor A. and .goodeniana N. .succincta N. complex, (April differ A. Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. ope eecmue ihMG- Kmr tce,L,Kyz aua 08 sn h Tajima-Nei the using 2018) Tamura, & Knyaz, Li, Stecher, “ (Kumar, the as MEGA-x model. for parameters with produced same computed were were the trees complex using separate two v8.2.11 for comprehensiveness, RAxML For one with trees. (i.e. complex RAxML species COI Maximum each the analyses. for phylogenetic for for produced used were were amplicons trees UCE likelihood the of alignments concatenated remaining The with analyses were specimens alignments UCE remove to Remaining 2.4. applied out. was filtered step were filtering data. locus additional missing a An 90% using format. than sharing 30-50 than complex fasta less specimens (< more by in species related of saved shared close by number and Loci relatively aligned maximum 2016). concatenated are (Faircloth, the and complex internal-trimmed of extracted each not within 75% and were edge-trimmed species reads reference were Since with UCE matching alignments 2013). coverage MYA), corresponding assembled Standley, and a the & and were 2007) against (“–careful”) (Katoh reads (Harris, mapped option MAFFT were Clean carful 2016), Lastz contigs mainly (“–sc”), (Faircloth, 2014). using Obtained flag processed Usadel, (“–cov-cutoff”). illumprocessor single-cell file and five the & with of using merged Lohse, value 2013) cleaned were cutoff al., (Bolger, were runs et trimmomatic (Nurk all data around v3.12.0 from Raw SPAdes wrapped data 2016). tool and (Faircloth, Miseq a bp; tools the 300 PHYLUCE on x using demultiplexed (2 were kits reads data Fastq v3 UCE Illumina of five processing using Bioinformatic Miseq 2.3. protocol a enrichment on The sequenced ng. 65 were 500 at Switzerland). h Pools using 24 location, individuals cycles. of Illumina, 12 14 step of hybridization using with a pool enriched amplification with UCE Arborbiosci). single recommendations were myBaits, a manufacturer’s Libraries Principal/Full, on the pools). 2.5Kv2 followed row 8 performed each of was (UCE and total enrichment v2 kit for manufacturer’sEach Qubit (i.e. hybridization equimolarly a the v2 using pooled of Hymenopteran quantified were fourth were the plate amplifications products PCR PCR one 96-well PCR 2017). using volumes. Faircloth, a (Roche) recommended of & Ward, the kit 96 Longino, length in prep each Branstetter, fragment performed in containing were Hyper bp described 500 libraries Kapa (as to dual-indexed a volumes sonicated recommended using independent were Two constructed specimen (Diagenode). were per ultrasonicator specimens using DNA quantified Bioruptor ng were 56 50 a Extracts at and protocol. using buffer manufacturer’s Scientific) K the (Thermofisher following proteinase v4 robot a Qubit extraction in 96 overnight Biosprint performed Qiagen were a extractions DNA body Whole preparation library UCE to 2.4. sequences truncated Since were sequences replicates. primers, bp). bootstrap different 265 and 100 (i.e. NGS) and length Sanger, model same (i.e. RAxML the was GAMMA methods with different sequences built GTR using within were the obtained trees codons were using Phylogenetic stop species sequences. 2014) of consensus per absence (Stamatakis, the aligned The v8.2.11 skimming were 2013). were and Standley, sequences translating sequences & Consensus by reverse (Katoh assembler. confirmed and sequences v7.308 Geneious Raw forward MAFFT the 2019). between using using al., sequences complex specimen et consensus each Gueuning and or Hallwachs, in for Sanger v11.0.5 & protocols constructed by Geneious (following Janzen, either into 2013) Burns, sequenced Penton, imported al., was et were (Hebert, specimens (Leray LepF/LepR mlCOIntF/HCO all pairs or of primer 2004) fragment the barcode using (COI) sequencing I NGS-barcode unit oxidase cytochrome The phylogeny and sequencing COI 2.3. .amieti A. n n for one and .bicolor A. .Gntcdsacsbtenseislnae o ahspecies each for species/lineages between distances Genetic ). 5 ° ,floe yaPCR a by followed C, ° n uie using purified and C bicolor -goup” Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. lh n eawr epcieystt n .0.Teaaye a u w ie ihaMM f50,000 of MCMC a with prior, times two tau run the was For period. analyses respectively. burnin The 0.004, 10% 0.002. and and a 3 3 with to to and set set generations respectively were were species. sequences) beta two their and between to alpha sites delimitation assigned different were species of 75% specimens proportion unguided the that after (i.e. so obtained amieti designed model UCE) A. one was A11 to file “ the corresponding population the (each using For The files (Yang nexus ran step. the (BPP) analysis filtering were on model threshold an 2014) analyses phylogeography Rannala, performed BPP and & we Yang (1000 Phylogenetics The Third, analysis; the generations Bayesian 2010). twice 11,000 (III) the to Rannala, as (“thinning”). using set set & generations sequences was was MCMC concatenated 10 “species” The every the complex. possible on sampled (Reid species the of and (bGMYC) the number from burnin) model in maximum results present GMYC generations the Second, species group, the (II) expected version. species of of each single-threshold implementation number For the Bayesian 2012). using a Carstens, run using & was cross-validated analysis were the analyses and trees first 10 ultrametric and The “birthRate”. software. 0 logCombiner for between the the the distribution using using using computational trees uniform tree R For 100 last a into 2018). of Suchard, imported total with & a then model Baele, to were Xie, resampled yule assessed Drummond, were was a convergence (Rambaut, trees Chain followed v1.6 purposes, generations. molecular TRACER 1000 strict Priors every software a sampling the 1.0. with and using generations model of million substitution 250 rate JC69 for ran the fixed MCMC were using a complex 2014) species al., with each et a for clock (Bouckaert Trees performed v2.5.2 we trees. 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(DAPC) verify “ the components first to centering principal two used or of the scaling was analyses using without package discriminant 2011) 2007) clusters, dartR (Wilkinson, genetic Dufour, the ggplot2 & “ using with Dray the using package; equilibrium plotted Datasets performed (ade4 Hardy-Weinberg were data. analyses function missing from component 15% Principal ” conversion, departure 2018). than Georges, After less significant & containing Berry, object. Unmack, for sites genlight (Gruber, variable “ a screened only package into the retain then adegenet files to using converting were filtered the were before R using matrices SNPs into mainly SNPs binary the R, imported extracts in and first sequences conducted were aligned were Sequences tests 2008). distance (Jombart, genetic and analyses Multivariate optim.a.score bicolor -group”, bicolor noe lh n eaprmtr fteivre am itiuino h ht ro (average prior theta the of distribution gamma inverted the of parameters beta and Alpha one. in and .bicolor A. ucin eut fpseirmmesi rbblte o ahseie eeplotted were specimen each for probabilities membership posterior of Results function. ” gop,bsdo h hlgn trees, phylogeny the on based -group”, .bicolor A. splits akg Ead uiaa arcog,20) nevlo pce ubrwsset was number species of Interval 2009). 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Data may be preliminary. h MV Tbe1 eitdsrn eei ieec ie 34% -au ? .01 ewe the between of lineages 0.0001) within both lineages between [?] allosa both obtained p-value between was 43.43%; distance 2) genetic (Table (i.e. index difference fixation admixture. separa- genetic significant of clear strong levels a depicted of considerable lack two suggesting 1) The is (Table ML1. specimens AMOVA to alpine The probability the 100% for a lineages with mitochondrial assigned between were Italy tion south in sampled specimens of lineages both Between S7). information (Supplementary C eut hr ohseiesaefudmrial wyfo h main the from away marginally belong- found of are S5) specimens information Supplementary both 3.8%; where results For (i.e. PCA probability ML2. slight to to a ing 54.36% with and membership (hereafter probability ML1 mixed posterior showed to 45.64% belong with probability, to membership “pp”) mixed revealed France) Southern from ainS) l pcmn eecretyrasge iha10 ebrhppoaiiyfrtretx (i.e. taxa three for infor- probability Supplementary membership variance; 100% a total with the reassigned of allosa correctly 70.3% were A. 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Andrena iegsbtn infiatdffrnebtentetwo the between difference significant no but lineages L.Temxdmmesi rbblt o hs w pcmn r ogun ihthe with congruent are specimens two those for probability membership mixed The ML1. .amieti/bicolor A. sp3, L n L ie .06 n .09,respectively). 0.00299, and 0.00261 (i.e. ML2 and ML1 .amieti A. .montana A. eei lse sinet eemc esspotdadol h two the only and supported less much were assignments cluster genetic , ie ,5,3 p.I oa,svnseiesddntps h 90% the pass not did specimens seven total, In bp). 1,459,230 (i.e. .For ). .bicolor A. .bicolor A. ± .bicolor A. 7 340 cosalseiscmlxs(Supplementary complexes species all across 23,430) L,ol n pcmn(..“GBIFCH00135933” (i.e. specimen one only ML1, ie .03)wssihl ihrta between that higher slightly was 0.00231) (i.e. ± 09.Atrrtiigol oimthn the matching loci only retaining After 5099). L,oeseie GIC0170)also (GBIFCH00117401) specimen one ML2, ± 2 .goodeniana/succincta N. .21 -au .3;Supplemental 0.039; = p-value 0.7221, = 5,1)rasprseie.Teaverage The specimen. per reads 356,015) .amieti A. .bicolor A. .bicolor A. iegs h oet yet lowest, The lineages. Ft=018.Nei’s 0.138). = (Fst L aggregation ML2 ie 2,2 bp) 429,820 (i.e. .amieti A. .bico- A. A. , Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. h P nlsshwvrhgl upre h rsneo he itntseis ihtefloigtree following values. the BIC with closed species, very distinct had three scenarios of clustering presence three the All supported [(( highly topology S10). however information analyses both (Supplementary BPP separate 4 The to = failed K analyses with bGMYC and between GMYC difference (Table and The no species showed S7). both information species between (Supplementary three difference all significant removing with when depicted however PCA Fst The that pairwise view and 1-2). AMOVA the of although supporting between in generations 0.00061), distance clades trees, summer monophyletic Genetic and UCE supported Spring and respectively. strongly mitochondrial 1). as Figure appeared both spinigera BS; species in 90% three intermixed all ([?] were contrast, tree In UCE 1). the Figure 93%; (BS etr wteln n a itrt the to sister was and Switzerland western iohnra hlgne eoee elspotd(S7-0% oohltccae for which clades from monophyletic 72-100%) (BS and well-supported recovered carantonica/trimmerana/rosae phylogenies Andrena Mitochondrial 3: BPP S10, identifications. the complex information morphological with Species (Supplementary the Congruent solution 3.2.2. to 75%. best corresponded at the specimens supported as all was outgroup) of solution (with Clustering 3 this = run 3). Table K first identified the for DAPC In [( possibilities the phylogeny. analyses, the being: different the tree [(( to three on presence 1) tree respect based depicted = one the with tree run (pp only disagreed supported likely first depicted however highly most run The runs the and second The with congruent species. trees, S9). were three species information runs the the Supplementary BPP among 1; Both relationships = phylogenetic all (pp clades. grouped species distinct analyses three three of Both of The S8). presence information level. the species (Supplementary the as results by analyses identified explained S5). morphologically similar the DAPC be information specimens provided by could The variance (Supplementary bGMYC corroborated observed probability total S7). and were 100% the GMYC information tree of with 52.74% (Supplementary phylogenetic specimens that separated UCE revealed AMOVA all the clearly The for from were membership the Results species reassigned in correctly both species 1). which morphological Figure two in values; the the in PCA BS to species corresponding (100% both clades tree between monophyletic separation UCE well-supported clear two no and with tree discordant, COI were phylogenies nuclear barbareae/cineraria and Andrena Mitochondrial analysis. 2: each complex for Species found for clusters 3.2.1. solution of best knowledge number as prior the 4 a summarized = without 3 K performed Table and analyses 3 DAPC respectively. = the ML1+ML2, Finally, K identified dataset. identifications the species in on tree present species one 5 (i.e. (i) having species for delimited depicted: 5 information runs (ii) allosa parallel Supplementary 0.99; Both two A. 1; The [?] of - S9). 0.05). probability - 0.95 information posterior 0 = (Supplementary = [(( (p (pp congruent probabilities probability highly model posterior high were low a very and had with converged scenarios clusters other All 9 S8): same S8). within information the found (Supplementary identified clusters lineages analyses nine mitochondrial identified the amieti specimens to A. all corresponding on clusters computed two analysis GMYC The .rosae A. .trimmerana. A. .als,A amieti A. allosa, A. , osiuetemrhlgclydffrnitdsrn eeainof generation spring differentiated morphologically the constitute and .amieti A. .carantonica A. .carantonica A. u o for not but , .allosa A. nteDP ohseiswr lontsprtdwt n u were but 3 = K and 2 = K with separated not also were species both DAPC the In L+L) l ihapseirpoaiiyo ;(i)adapseirpoaiiyo 1 of probability posterior a and (iii) 1; of probability posterior a with all ML1+ML2), .rosae A. egdtgte,adsxcutr opsdo nyoeseie.TebGMYC The specimen. one only of composed clusters six and together, merged .trimmerana A. , rs.Oecaewscmoe ftoseiesof specimens two of composed was clade One arose. .trimmerana A. L+L) (( ML1+ML2), rmteaaye,bt pce eesprtdo h rttocomponents two first the on separated were species both analyses, the from .barbareae A. .cineraria A. .carantonica A. .carantonica A. hc a opsdo w ldsfrigaprpyei unit paraphyletic a forming clades two of composed was which , ), .bicolor A. .rosae A. , .cineraria) A. noecae For clade. one in 8 )] and ld;spotfrti itrrltosi a high was relationship sister this for support clade; ML1, . nrn sp3 Andrena .barbareae A. .trimmerana A. .bicolor A. , .vaga)], A. .barbareae A. .carantonica A. .amieti A. .bicolor A. , ( , .bicolor A. .rosae A. ML2), .cineraria A. .rosae A. a eaieylw(e’ = D (Nei’s low relatively was orsodn oteexpected the to corresponding .trimmerana A. L+L and ML1+ML2 ohaaye suggested analyses both , nrn sp3 Andrena n lse ihwith with cluster one , n of and ML1, .stragulata A. and and , .trimmerana A. .trimmerana A. .vaga)) A. .bicolor A. .trimmerana A. .carantonica A. .carantonica A. BPP ape in sampled ) iha with ))] .bicolor A. analyses and .The ]. ML2, A. , , Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. u o ihCI hc ugssta acd hrn cusi hs pce ar.Scn,trespecies three Second, pairs. (i.e. species species these biological in single occurs a ( in sharing follows: barcodes pairs , barcode as DNA that mitochondrial species summarized multiple suggests two presented be which complexes in can COI, with introgression study not our mitochondrial but clearly of of and results hypotheses evidence main species strong and robust The provided provide UCEs we delimitation. here, species examined First, species for bee COI wild cases outperformed of investigated complexes all six all in In species delimit successfully elements Ultraconserved 4.1. Discussion 4. S8-S10). information Supplementary 3, (Table species separated two for of hypothesis support the bootstrap supported of The specimens 1). other (Figure with clades monophyletic of defined specimens well two depicted of monophyly trees UCE goodeniana/succincta and postulated Nomada COI previously 6: as complex species Species distinct 3.2.5. three analysis of GMYC hypothesis the Beside All the trees. supported morphology. both barcode. on and in based COI 3) taxa (Table same other congruent the both were to shared sister but clade over-clustered tree monophyletic that single UCE a the in in clustered value) bootstrap for (95% discordance mitochondrial-nuclear revealed trees nuclear and and mitochondrial alpigenum/bavaricum/cupromicans of Lasioglossum Comparison these 5: complex together, Taken Species 3.2.4. S8-S10). analysis DAPC information and Supplementary analyses 3, BPP that (Table the indicate analyses, species results bGMYC specimens both French and the GMYC separated S7); Both successfully information divergent. Supplementary 1, particularly (Figure not clusters were separated as species both recovered of of of Swiss specimen specimens trees, one other but mitochondrial all 1), In (Figure identifications complex. morphological propinqua to species A. corresponding this clusters two within formed observed specimens were discordances dorsata/propinqua mito-nuclear Andrena Strong 4: complex Species 3.2.3. itntvns fteohrseispiso rpesivsiae ee hc ssrnl ofimdb the by confirmed strongly is which each here, within investigated generations triplets two or the pairs particular, species long-standing data. other in solved UCE the delimitation bees; of species European distinctiveness within UCE-based Central observed addition, of clades In taxonomy of delimitation. mitochondrial the species species. two in mitochondrial cryptic controversies the unresolved and distinct that gap two suggest barcode represent a results of our species absence each the Third, within in ( barcodes resulting arose mitochondrial species respectively), two another , which the from while assemblage morphology, paraphyletic a with formed agreement in were which groups .propinqua A. .rosae A. .dorsata A. .cineraria A. .cupromicans L. and .goodeniana N. GIC0134)cletdi otenFac a itrt elspotdcaecontaining clade well-supported a to sister was France southern in collected (GBIFCH00133244) .goodeniana N. rmti rnhst GIC0134) hlgntctesadPA ae nUCEs on based PCAs and trees Phylogenetic (GBIFCH00133243). site French this from , .trimmerana A. , .trimmerana A. aigosmcupromicans Lasioglossum .bavaricum L. .dorsata A. Fgr ) ohtx eewl eiie ihhgl upre oohltcclades monophyletic supported highly with delimited well were taxa Both 1). (Figure .dorsata A. .goodeniana N. olce ot fteAp.I h ula re,teetoseiesclustered specimens two these trees, nuclear the In Alps. the of south collected a o ntemtcodiltesdet h rsneo w lgtydivergent slightly two of presence the to due trees mitochondrial the in low was and epciey hc ontapa orpeetdsic pce;adthe and species; distinct represent to appear not do which respectively, , ;fraltreseisUE eoee togyspotdmonophyletic supported strongly recovered UCEs species three all for ); n of and and .propinqua A. .cupromicans L. ihhg upr aus h pce eiiaintssas highly also tests delimitation species The values. support high with .propinqua A. and .bavaricum L. r ai species. valid are 9 Fgr ) u apigas nlddoespecimen one included also sampling Our 1). (Figure SplmnayifrainS) l te analyses other all S8), information (Supplementary :UE eei gemn ihmorphology with agreement in were UCEs ): .allosa A. , .dorsata A. nrn bicolor Andrena .alpigenum L. nrn barbareae Andrena and nrn amieti Andrena .carantonica A. .bavaricum L. specimens probably Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. ldso oseicseiesi h C re.W pclt htsc ihwti-pce iegne in divergences within-species high such that speculate the We within trees. nested UCE clades where two the trees variations in mitochondrial the in morphological specimens between such specimens conspecific observed divergent study, of not these our clades also addition, In admittedly In punctuation 2019). was thoroughly). or (it question al. colour with specimens et pile divergent associated Praz as the 2019; exclusively deem within such in however almost al. character We observed are et variable not Pauly rejected. bees were highly 2017; be in in al., completely species differentiation et not near-cryptic (McKendrick morphological can since of species unlikely level cryptic strongly some (i.e. additional as of paraphyly scenario presence mitochondrial this the differentiation. of study, morphological cases slight our geographic other their in strong separated with two two the and the with of populations agree In hypothesis Apennine results the and UCE contradicts Alpine Rather, strongly the clades. strongly result of while mitochondrial two separation This clade, both recovered mitochondrial to 1). UCEs corresponding (Figure one The lineages in specimens alpine clades. clustered all mitochondrial all the including both specimens 2019). population; within over Italian Alpine al. clades distributed southern nearest et were supported the the (Praz specimens 1), from populations Alpine (Figure Alpine km the the 600 tree from from some COI specimens divergent Italy, the included slightly Southern We In morphologically in scenario. are this Apennines exclude specimens the to Apennine from for us least enables and At species Alps divergences. this the between these of recover (i.e. variation distribution to paraphyly known uncover low generally entire mitochondrial too more successfully the is of evolution and can cases of study rate they the UCEs our that whether the in argued is question be delimitation important could species trimmerana an It for UCEs, species. of UCEs diverged evolution diversity? of recently of use cryptic rate the of low with levels the to additional regard overlooked With have UCEs unravel to the necessary Could be 4.3. would coverage here. zone geographic documented distribution wider errors colonized a barcoding the with DNA over Therefore, away work of Further fade 2014). cases Petit, and al., Ruedi, the 2012). zone (Currat, et Brelsford, hybridization taxon Pons the & colonized 2011; at Verheyen, (Toews the towards highest & taxon are Hanssens, native levels Backeljau, as Fazalova, with the introgression such Nevado, from events associated 2008; directed contrast, not im- Excoffier, is In which flow often & unidirectional, gene 2012). most are lineage the Brelsford, they is that A incomplete & because plies sorting Toews footprints separate 2003; biogeographical lineage difficult. number to leave Omland, incomplete often low mechanism necessary & hybridization/introgression Indeed, the (Funk underlying be study, pattern would the mechanisms. biogeographical this distribution other on any In entire investigation the the 2016). from the across diverged al., sorting render recently sampling et sequenced in complete (Mutanen occur and exclusive more events sampled mutually these dispari- specimens necessarily often demographic they Most not of introgressions, often are 2012). Brelsford, but and by & species tedious, followed (Toews be hybridization production) offspring can sorting, biased errors lineage barcoding as incomplete these sharing, ties, to behind barcode of linked mechanism or because biological type are divergences species exact (i.e. within-species distinct the error two deep second Identifying recognize by The to triggered fails 2008). pairs barcoding Crandall, are the between DNA & errors observed when Whiting, these Buhay, occurs (Song, error) often, when insertions II occurs Most nuclear error) as I such species. type artefacts to biological for observed (similar single as error barcodes, a first DNA The distinct two errors. with of and associated types is two species biological to one subject is barcoding COI-based errors barcoding DNA 4.2. Wolbachia .trimmerana A. .bicolor A. and netoso e-isdaymtis(..ml-isddsesl aigbhvoro sex- or behaviour mating dispersal, male-biased (i.e. asymmetries sex-biased or infections .propinqua A. lhuhmr pcmn fbt edr n ohgnrtosaenee oadesthis address to needed are generations both and genders both of specimens more although , yeIerr liaeyla oernosdtcino w yohtclseiswithin species hypothetical two of detection erroneous to lead ultimately errors I Type . .amieti A. nrn barbareae/cineraria Andrena nfc ersn diinl vrokdisacso rpi pce,adthat and species, cryptic of instances overlooked additional, represent fact in ) n orsodn oteSuhr tla ouainadteother the and population Italian Southern the to corresponding one , 10 and .trimmerana A. aigosmcupromicans Lasioglossum and .amieti A. .propinqua A. .amieti A. u apigacross sampling our , / barvaricum .amieti A. , investigated ) .propinqua A. . , A. Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. anyfne yteSisFdrlOc o giutr FA) h apigo eswsfne ythe was by funded work was This bees of study. thank sampling this repective to the (FOAG); valuable (FOEN). of like Agriculture Environment there also aspects for for for would statictical Office Office Frey Federal We Federal and Swiss Swiss Beatrice laboratory the study. and by this bioinformatic, Frei funded for to Daniel mainly specimens regards Paxton, provided in Robert Pisanty, who contribution Gideon collaborators Bossert, all Silas to grateful are We Acknowledgements species bee, the bees. cryp- or European two in reconstruction common of species tree most presence of detection the the the range the suggest impact of geographical enable strongly hybrids entire one will analyses these within our UCEs of Lastly, species whether presence investigated. tic addition, the be divergences In of should extend level analyses what markers. the delimitation to conserved determine further and these to delimi- hybrids, with iteratively taxa species of recovered diverged to for recently The be and very methods quantity. can on simultaneously, capture or that focus quality families UCE should DNA of different work advantages Future low three have promising to tation. would to particularly due datasets represent here belonging RAD-seq investigated datasets, specimens assemble species predictions. the processing these for confirm of gather largely information. possibility and to per-locus work impossible to higher early nearly possibility the (2016) this been the and upon repeatability, assessment, al. build its advantage orthology et results of read an Our Harvey because in necessarily systematics ease not delimitation. in the useful was species samples, more RAD-seq low-quality or is use of phylogeny capture phylogeography, typical sequence was information that question concluded of They inherent amount per-locus-information. RAD- the high higher that the when provided and values; that sequence-capture found support while suggested They branch information also and phylogenies. overall hypotheses shallow more enable phylogenetic for sequence provided RAD-Seq of similar to utility and in seq the study, insects resulted compared our techniques comprehensively in in (2016) both as scale that al. UCEs time et using Harvey Faircloth, specifically shallow Glenn, 2018). methods, Smith, at al., capture (Harvey, et variation vertebrates Zarza in sufficient 2016; gathered delimitation Brumfield, provide evidence & previous species can to adding for UCEs discrimination, UCEs that species confirm of results use Our the data. on UCE species remarks on overall Concluding applied the when 4.5. long caution inflated harboring with specimens ultimately interpreted split be which analyses should GMYC S8) analyses the values, GMYC information cases, Dincˇa, missing & Therefore, (Talavera, most of (Supplementary BPP, In variation levels number. high to species intraspecific trees. yield the high Compared DNA singleton in input 2013; of branches branches low-quality presence al., species. with longer the specimens to et of case, led in (Carstens number particular which especially our rates the In 2018), delimitation inflate Zhu, 2013). species Vila, & to underestimations Ho, tendency or Ling, species the overestimation Luo, morphological had with can morphological congruent cases current analyses less with several were GMYC agreement (b)GMYC in in both and most by results results hypotheses, contrast, the By provided BPP. that was delimitation hypotheses species methods of delineation method The species different 2015). of al., Comparison et Schmidt 4.4. 2019; continental- al., with et barcoding (Hinojosa once achieved expected be previously will than sampling common more scales prove will barcodes mitochondrial .bicolor A. ilb eesr ofrhrutnl hsrmral aeo cryptic of case remarkable this untangle further to necessary be will 11 nrn bicolor Andrena nagn u aae othe to dataset our Enlarging . Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. arlt,B . comc,J . rwod .G,Hre,M . rmed .T,&Gen .C. T. Glenn, & T., R. Brumfield, G., times- evolutionary M. multiple Harvey, spanning G., markers genetic N. of Crawford, cales. thousands anchor E., loci. elements J. genomic Ultraconserved conserved McCormack, (2012). of C., analysis B. the Faircloth, for package software a is PHYLUCE formatics (2016). C. statistics. B. threshold Faircloth, by limits species’ Splits: (2009). G. Gro T. im Version Barraclough, & S.L. T., LATR. Fujisawa, T., Halictus Ezard, Genus related des closely Bienen II. in Die Teil delimitation Apidae) (1970). species W. Multi-locus A. (2012). Edmer, H. 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(12), M Evolutionary BMC Biometrics Entomologia Proceedings Proceedings Biological , Alpine 0030 doi: . , Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. pvle .01 r ihihe nbl olwda seik *.Sgicnewsasse through species. assessed phylogenetically twice, was run closed tests Significance was the analyses significant only the with Statistical (*). species, once two asterisks packages. and than an more species R of followed all pegas composed with bold and complexes once in species dartR For highlighted complex. the permutations. are using species 10,000 dataset 0.0001) per UCEs < performed (AMOVA) the (p-values variance on molecular performed of were Analysis 1: Table Tables C.P. M.G., by written was Manuscript M.G. 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Rannala, & Z., Yang, oeua ilg n Evolution and Biology Molecular .CIsqecsaeacsil nBL hog h olwn DOIs: following the through BOLD on accessible are sequences COI ). oa 5024009100% 27.03% 100% 0.009 47.26% 0.003 100% 56.57% 0.003 0.214 0.002 0.069 100% 0.002 98.25% 0.001 0.037 0.021 25 23 0.002 0.087 variance % 0.002 0.061 11 Total 10 Error 0.049 MSD lineages 0.047 54 Between 53 Total Error lineages Between SSD 27 Total 26 Error lineages Between Total df Error lineages Between source Variance PeerJ , 6 64.di 10.7717/peerj.6045 doi: e6045. , , .4 0.072 0.016 0.145 1.75% 0.026 0.016 2 0.002 0.026 1 0.002 1 1 31 1) 1533.di 10.1093/molbev/msu279 doi: 3125–3135. (12), 16 urnl uaigfie for files curating Currently urnl uaigfiles curating Currently AMOVAs 72.97%* 52.74%* 43.43%* Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. ML1 color bi- A. ML2 eti ami- A. ML1 eti ami- A. allosa A. Fst \ D Tajima- MEGA-x. package; Nei’s using dartR the files using fasta R concatenated Tajima- of in the matrixes (ML) on SNP computed lineages the were mitochondrial on distances asterisks computed genetic both by were complex. net depicted between tests Nei’s are species Fst and computed bold). per bootstraps Fst 10,000 in distance the through (highlighted assessed genetic Only were Fst net pairwise of (*). Tajima-Nei’s 0.05) and [?] Fst (p significance Pairwise 2: Table succincta / goodeniana N. cupromicans / bavaricum L. cupromicans / bavaricum / alpigenum L. propinqua / dorsata A. trimmerana / carantonica A. complex Species .4*033 .1*030 .9*--002100210050005000350.01395 0.01395 0.00510 0.00510 0.00231 0.00231 - 0.01430 0.01430 0.00962 - 0.00962 0.390* 0.00961 0.01661 0.00961 0.390* 0.01661 0.00923 0.313* 0.01179 0.00923 0.01179 0.313* 0.00027 0.01117 0.00027 0.340* 0.01117 0.01080 0.01080 - 0.00299 0.203* 0.00299 0.00261 0.00261 - 0.196* - allosa A. oa 4005004100% 33.87% 100% 61.73% 0.004 0.002 100% 22.97% 0.009 0.006 0.055 0.025 100% 0.017 60.67% 0.005 0.085 0.057 100% 14 72.97% 13 0.003 0.302 0.002 0.081 10 variance 9 % 0.002 Total 0.002 Error 0.039 lineages 0.026 18 Between 16 Total MSD Error 0.038 lineages 0.031 Between 18 Total 17 Error SSD lineages 18 Between 17 Total Error lineages Between df Total Error lineages Between source Variance 0.031 - ML1 amieti A. 0.031 - ML1 amieti A. .3 0.030 0.028 0.030 0.110 0.028 1 0.012 0.221 1 0.008 0.012 2 0.008 1 1 .14 .14 .18 .18 .14 .14 .13 0.01633 0.01633 0.01145 0.01145 0.01085 0.01085 0.01048 0.01048 - - ML2 amieti A. ML2 amieti A. 17 ML1 bicolor A. ML1 bicolor A. .amieti A. ML2 bicolor A. a o significant not was ML2 bicolor A. Statistical 66.13%* 38.28%* 77.03%* 39.33%* 27.03%* A. sp3

A. sp3 A. montana montana A. Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. goodeniana N. nitidulum L. cupromicans L. bavaricum L. alpigenum L. congruens A. propinqua A. dorsata A. rosae A. trimmerana A. carantonica A. vaga A. cineraria A. barbareae A. montana A. sp3 A. ML2 color bi- A. Fst \ D Nei’s Tajima- .08 .08 .08 .08 .18 .18 .18 0.01883 0.01883 0.01883 0.01883 - 0.00381 0.00381 0.00381 0.01114 0.00381 0.01114 - 0.01114 0.00083 0.00083 - - 0.00083 0.057* 0.00083 - 0.057* 0.01134 - 0.01423 0.01134 0.057* 0.01423 - 0.01134 0.01423 0.057* - 0.00418 0.01423 - 0.140* 0.00418 0.01439 0.00418 0.01439 - 0.140* 0.00418 0.01439 - 0.140* - - 0.00406 0.01439 - goodeniana 0.00406 0.00134 0.140* 0.00617 - N. 0.00406 0.00134 0.00617 0.442* - 0.00406 0.00134 - - 0.00617 - 0.442* 0.826* 0.00134 0.00617 0.464* 0.00672 - 0.826* 0.464* 0.00672 - 0.468* - 0.826* 0.00672 - - - 0.468* 0.00672 - 0.826* 0.00061 0.01065 0.795* - 0.00061 - 0.01065 - alpigenum - 0.00061 0.795* - 0.01065 - 0.142* L. 0.312* 0.00061 0.01065 0.795* 0.01022 0.142* 0.312* 0.795* 0.01022 - 0.142* - 0.312* 0.01022 - - - 0.142* 0.01022 0.312* 0.00294 0.355* - 0.00294 - - - 0.01400 dorsata - 0.00294 0.355* - 0.088* 0.01400 A. 0.330* 0.00294 0.355* 0.088* 0.330* - 0.355* - 0.088* - 0.330* - 0.782* - 0.088* 0.01447 0.330* 0.782* 0.01447 0.293* - 0.00549 0.654* - - carantonica - 0.00549 0.293* 0.654* 0.165* A. 0.342* 0.293* 0.629* 0.165* 0.342* 0.293* 0.629* - - 0.165* 0.294* 0.841* 0.165* 0.294* 0.841* 0.637* - - 0.773* barbareae 0.637* 0.138* A. 0.773* 0.401* 0.138* 0.878 0.401* 0.533* 0.533* 0.62* 0.456* 0.456* 0.482* allosa A. goodeniana N. alpigenum L. dorsata A. carantonica A. barbareae A. ML1 amieti A. goodeniana N. 0.002 bavaricum L. dorsata A. carantonica A. barbareae A. ML1 amieti A. dorsata A. carantonica A. barbareae A. ML2 amieti A. goodeniana N. 0.002 - bavaricum L. succincta N. 0.002 - bavaricum L. propinqua A. trimmerana A. cineraria A. ML2 amieti A. 18 succincta N. 0.002 - bavaricum L. propinqua A. trimmerana A. cineraria A. ML1 bicolor A. .11 .11 0.01112 0.01112 0.01112 - - succincta N. - - cupromicans L. propinqua A. trimmerana A. cineraria A. ML1 bicolor A. succincta N. cupromicans L. propinqua A. trimmerana A. cineraria A. ML2 bicolor A. bifasciata N. cupromicans L. congruens A. rosae A. vaga A. ML2 bicolor A. bifasciata N. cupromicans L. congruens A. rosae A. vaga A. A. sp3

bifasciata N. nitidulum L. congruens A. rosae A. vaga A. A. sp3

N. L. A. A. A. bifasciata nitidulum congruens rosae vaga

A. montana nitidulum L. montana A. Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbakt.*ubro lsesfrteotmlKslto ie oetBCvle.Atraieksltoswt iia I austa h pia ouinaepoie nbrackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal the for clusters of *Number brackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal the for clusters of *Number brackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal the for clusters of *Number brackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal the for clusters of *Number brackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal the for clusters of *Number brackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal the for clusters of *Number brackets. in provided are solution K optimal the than values BIC similar with solutions k Alternative value). BIC lowest (i.e. solution K optimal bootstrap Only the for replicates. datasets. bootstrap clusters shown. UCEs 100 of are using and *Number model 70% COI gamma than GTR for higher the using probability obtained brackets. v8.2.11 trees in RAxML provided with phylogenetic built are likelihood were solution Maximum K 1: optimal the Figure than values BIC similar with solutions k Figures Alternative value). BIC lowest (i.e. solution K optimal the for clusters of Number succincta / cupromicans goodeniana / N. bavaricum / alpigenum L. propinqua / rosae dorsata / A. trimmerana / carantonica A. cineraria / barbareae A. mtDNA bicolor A. mtDNA amieti A. in each highlighted For are (S8-S10). identifications information morphological Supplementary than as results methods. or identical RAxML) by providing 1; bold. clusters methods (Figure of groups, Figure number main species as showing either table provides Summary Table3. bifasciata N. succincta N. Fst \ D Nei’s Tajima- .9*040 .9*040 .4*058 .4*058 - 0.02014 0.02014 0.02014 - 0.02014 - - - - 0.548* 0.548* - 0.548* 0.548* 0.490* - 0.490* 0.251* 0.490* 0.251* 0.490* 0.251* 0.251* allosa A. L ML2 / ML1 L ML2 / ML1 ML1 amieti A. ML1 amieti A. ML2 amieti A. ML2 amieti A. 19 ML1 bicolor A. ML1 bicolor A. eut o ahmto are method each for Results ML2 bicolor A. ML2 bicolor A. Trees A. sp3

A. sp3 A. montana montana A. 2 3 2 3 2 1 1 Morphology clusters of Number RAxML 2 2 3 4 1 2 2 COI clusters of Number RAxML 2 3 2 3 2 2 1 dataset UCE clusters of Number -2 DAPC* 2 3 2 (3,4) 2 2 (2) 3 (1,2) 3 dataset UCE clusters of Number (3) (2) GMYC 3 5 3 3 5 2 1 dataset UCE clusters of Number bGMYC 4 3 4 4 5 2 1 dataset UCE clusters of Number 2 3 2 3 2 2 1 BPP dataset UCE clusters of Number Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. 20 Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. 21 Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. 22