Posted on Authorea 6 May 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158879241.12522183 | This a preprint and has not been peer reviewed. Data may be preliminary. iwwe eln ihisc et.Mn uhr aesgetdta oyhgu pce r omdby formed are species polyphagous applied that an suggested from have concern authors a Many the pose pests. highlighting generalists, insect isolation, i.e. with 2012). may sexual ranges, dealing Nosil, hosts of host when 2006; new wide evolution view al., presumptively to the et with Adaptation (Funk byproduct, insects speciation a 2018). Phytophagous in as al., shifts et or plant Orsucci, consequence host 2005; direct of host Dicke, role a development Hence, & and as oviposition plants. Loon, either host location, Van feeding their cause, (Schoonhoven, entails with of associated often host features features and intimacy specific the of source Such with evolution on cope food the 2005). to affect a Dicke, may insects are shifts & allow they plant that Loon, since adaptations Van hosts of (Schoonhoven, their evolution sites the with oviposition interactions and close mating in involved provide often are insects Herbivorous INTRODUCTION 1. natural using strongly control one biological Brazilian for each range. southeastern strategies native from species, reliable its derives design different the from to pest five help of pest cactus will least Rican the population it at Puerto of because source achievement the enemies comprises important the that the pungens an established determine whether is H. also evaluate to This We that to H. and mealybugs. plants. suggested complex, within complex, host the results diversity specific species of Our with species the members associated putative investigate in pest. the to diversification Recent cactus of drove variation Rican one mtDNA America. association each Puerto plant and of South ranges host SNPs from complex. plant species of genome-wide population host a pattern of source establish is to combination the pungens North lato H. a and nor sensu that Caribbean used pest, pungens suggesting the we the populations, in study, local of diversity this among identity cactus differentiation has In threatening the genetic and also substantial about America Rico, to certainty South pointed Puerto from neither studies in pest species is cacti for their polyphagous native There of task a devastating drivers mandatory be pest America. the to a a understand believed to as is was help reported species may pungens been invasive complex Hypogeococcus encompass species mealybug a alternate that The to of complexes adaptation history diversification. of evolutionary species result the the in investigating as Moreover, diversity diverged that management. cryptic species Untangling host-associated of form plants. the in host observed been often have taxa Cryptic Abstract 2020 6, May 6 5 4 3 2 1 Triapitsyn Poveda-Mart´ınezDaniel control mealybug biological cactus for Rican implications Puerto and of pest an source in The use insect: plant herbivorous host by diversification complex Species nttt eEoo´a e´tc vlc´nd unsAires Buenos Ecolog´ıa, Evoluci´on Gen´etica de de y Instituto Blumenau USDA de Administrator, Regional APHIS Universidade the to Advisor Caribbean California of University Agriculture-ARS of Department Invasivas U.S. Especies de Estudio el Fundaci´on para 3 id Diaz-Sotero Hilda , 1 Mar´ıa Aguirre , 4 acl Vitorino Marcelo , 1 ulem Logarzo Guillermo , 1 5 n sea Hasson Esteban and , 1 tpe Hight Stephen , 6 2 Serguei , Posted on Authorea 6 May 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158879241.12522183 | This a preprint and has not been peer reviewed. Data may be preliminary. Agnia aauyadBai)adnnntv Pet ioadsuhr ntdSae)rne by of ranges diet States) the within United diversity of genetic southern i) part investigate: native to and as the variation mtDNA both recognized Rico and comprising SNPs plants (Puerto genome-wide area of host geographic non-native combination large on and a mealybugs to Brazil) effort collecting 2019). Cactaceae, and sampling al., (Poveda-Mart´ınez on the Paraguay et Argentina extended feeding not (Argentina, we mealybugs study, was Rican present pest the the Puerto the with In the of than in population found rather haplotypes source detected latter mitochondrial the haplotype the the that single with of suggesting the ancestor none However, recent matched Argentina. a Argentina from shares in species to new pest closer feeding the appears cactus that pest initially sympatric, cactus suggesting Argentina, Rican trees Puerto in phylogenetic the in Interestingly, collected cactus. populations on ( feeding that Amaranthaceae cies suggests on feeding 2016) species al., mealybug et performance (Aguirre and as based 2019) plants studies identified al., (Poveda-Mart´ınez Recent hosts et compatibility 2010). different reproductive Golubov, assessing 1994; on & (Ben-Dov, and Mandujano, Cactaceae data Cuen, and P´erez molecular Zimmer- Portulacaceae Sandi Zimmermann, on Amaranthaceae, & 2001; of and Neser, Haro, species Australia on & Mathenge, feed in Claps to Klein, cactus reported Hoffmann, was invasive it Paterson, an America) 1981; against 1978, Currently, released Tomley 2011). agent mann, & control (McFadyen as biological Africa reported successful initially South was the cac- pest for (HCM), The threat lybug America. a North represents islands, and smaller Central adjacent pungens Caribbean, 2016; the the Walsh, and in et Rico & diversity Puerto Elfekih Gordon, tus invading McGaughran, in pest Tay, mealybug (e.g., (Anderson, cactus complexes pests The invading species of in 2019). diversity origin al., large the cryptic provide et evaluate trace Ryan methodologies to to These and used 2016). 2018), Hohenlohe, be RADseq al., & as can Luikart, such that Miller, libraries sequencing, datasets Good, by genomic multi-locus Andrews, genotype of 2008; as sequencing al., known high-throughput et genomes, studies on (Baird nuclear genetic of based population reduced-representation methodologies Recently, hybridiza- that a Despr´es, resulting introgressive new 2019). containing known 2017; or potentially of well availability sorting Hefetz, lineage, is lineage from single & mitochondrial it incomplete benefited a Reiner-Brodetzki, However, the to of Seltzer, either to 2017). evaluation due (Eyer, limited al., boundaries the tion information et species on provides of Stouthamer based (Dins- marker misidentification 2010; been (mtDNA) in this Barro, have I cases De 2014; subunit surveys many Feder, oxidase & population & in Buckley, cytochrome most Hood, Riginos, encoding Forbes, far gene Cook, to Powell, So DNA-barcoding 2012; dale, specialized so-called al., 2019). one the et each al., marker, Nosil genetic 2008; et species, Funk, Vidal diverged polyphagous & putative see deeply Nosil, some but (Egan, several that use showed embracing plant studies complexes host of different species number increasing actually, an control are, data, biological insects molecular successful al., of invaders. design et advent of to (Newbold the task effects loss With necessary harmful also biodiversity a reduce but of is or agriculture, causes species prevent to main invasive areas. to only of new the strategies not identification of among invasions territories, proper are in new The species involved to invasive 2015). are spread since species diversity upon cryptic suspected biological problems of of to global guild cases cause a al., in may of relevant et insects members particularly Invasive Bakovic when is variation especially 2018; genetic species, al., interspecific cryptic of et and species Malka intra include between 2017; often Distinguishing Letsch, complexes such & of the since investigate design Gottsberger, insects to the Astrin, herbivorous opportunity for 2019). Friedman, of the crucial diversification (Winter, offer the is origin complexes host in species recent defining species plants specialist in cryptic host complication cryptic addition, of additional and In role an et polyphagous pose Forbes programs. true specialists 2016; control cryptic between Harvey biological of & distinction up Loxdale The made (e.g., complex range. species species specialist A cryptic 2017). or al., populations specialized adapted locally rnr eWlik(eitr:Pedccia) ne h omnnm arsaccu mea- cactus Harrisia name common the under Pseudococcidae), (Hemiptera: Willink de Granara .pnessnulato sensu pungens H. .pnessnulato sensu pungens H. ee culy pce ope opiiga es w pce;one species; two least at comprising complex species a actually, were, , .pnessnustricto sensu pungens H. scniee pce ope,adi t aierne(South range native its in and complex, species a considered is 2 .pnessnulato sensu pungens H. ,adteohra necie spe- undescribed an other the and ), .pnessnustricto sensu pungens H. Hypogeococcus eue a used We . .pungens H. Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. eoe 5 ppie-n ed eesqecdi aeo ie 00(NSuu tUiest of (BBMap, University bbduk at al., et (SNPSaurus using (Jackman 4000 adapters abyss-pe HiSeq Nextera using reference a to done for the us was of trimmed build allowing assembly lane To adults, The then a sequences. as 2017). were in feed females’ (Bushnell, sequences not sequenced in sourceforge.net/projects/bbmap) do paired-end were present males reads Illumina scraps since paired-end Oregon). food reference bp means or clean By 150 a parasitoids S1). (Table obtain genome, bacteria, mealybug to male of able single reads a were (BB- identify from we bbduk DNA procedure using using built this reads was of low-quality genome the and draft trimmed adaptors reference that nextera A remove LLC) sequences. to (SNPsaurus, 2017) (Bushnell, scripts http://sourceforge.net/projects/bbmap/) custom Map, of used University analysis filtering at genotyping (NCBI), data (SNPSaurus The Information genomic 4000 Biotechnology and HiSeq for calling a Centre SNP of National 2.3 lane libraries PRJNA593002. the a number NextRAD at accession in amplified. a available BioProject mode efficiently with are the reads were starting under data single-end primer fragments sequence bp the Raw only 150 of of Oregon). Thus, a GTGTAGAGC. ng sequence in 5 sequence selective PCR sequenced and selective then the were were reaction, the matched DNAs 1/5th adapter Fragmented with that a short samples. DNA sequence at the ligates genomic scaled in 73 also the inhibitors was at of which into reaction cycles presence USA), Nextera 26 fragmented the The CA, for to first due amplified were Diego, fragments. used DNAs the San was Genomic DNA of Inc., genomic ends (2015). (Illumina, the Johnson, reagent to and Flex sequences Etter, DNA Eugene, Waterhouse, LLC, Nextera Russello, (SNPsaurus, with in libraries as genotyping-by-sequencing USA) nextRAD into OR, converted was NY, DNA Island, Genomic Inc., Grand Technologies Technologies, (Nanodrop sequencing (Life spectrophotometer NextRAD Fluorometer ND-1000 2.2 Nanodrop 2.0 a Qubit in USA). using & DE, assessed 2 quantified Wilmington, Blood was adding was DNeasy quality USA), DNA Qiagen and CA, using USA), (Valencia, mealybugs step. instructions female lysis manufacturer’s adult to the of according bodies entire Kit Information using Tissue extraction. and extracted 1 DNA was Figure until DNA in Genomic freezer presented are a species in plants 2012). stored (McFadyen, host 1970s and S1. and the Table coordinates, ethanol geographic in 100% weeds localities, Additionally, cactus sampling in Cactaceae). where concerning against preserved on Cactaceae), agent were control 5 on individuals biological in 5 (n= All a range (n= States United as non-native southeastern Australia United introduced Portulacaceae), was and the southwestern southeastern on Argentina Portulacaceae), from in in 11 samples on and collected n= included Amaranthaceae), and n=4 the samples Amaranthaceae we on and along included on 11 Amaranthaceae Amaranthaceae), also 24 (n= on We Cactaceae; on on States 11 Cactaceae). 93). 18 42 collected n= on (n= n= (n= were Cactaceae; 3 Rico and mealybugs States Puerto on (n= range, Cactaceae United Paraguay 23 native on western continental (n= the 21 in Brazil the In (n= of 25). and Argentina coast n= northwestern Rico Atlantic Portulacaceae, of and diet Puerto and the northern 69; of of in part n= areas as Amaranthaceae, reported invaded of 89; plants range host recently n= the native the on collected the in were in Mealybugs and collected 80) were mealybugs (n= the of extraction Samples DNA and control). collections biological Sample association that (new 2.1 those pest or Puerto the control) the strategies to biological control of species (classical METHODS biological related pest select population 2. closely and the source attack for with search the but host co-evolved to co-evolve whether iv) that able not iii) those be and did complex; will either complex, the we enemies, results, of species natural these members the using on putative Based in the pest. of diversification cactus one Rican the each drove of ranges shifts plant plant host ii) complex, species º ,wt n ftepiesmthn h dpe n xedn nucleotides 9 extending and adapter the matching primers the of one with C, 3 .pnessnulato sensu pungens H. .pnessnulato sensu pungens H. .pnessnulato sensu pungens H. μ fRAeAafter A RNAse of L nSuhAmerica South in (Cactaceae, from Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. netycecet eecluae ysM o ausrnigfo o1,wt 0 eeiin for repetitions 100 with 10, to 1 datasets. from scale ranging values genome uses K accommodate but for of to 2009), sNMF value optimization by Lange, structure each calculated squares & underlying were Novembre, least model coefficients (Alexander, and Ancestry likelihood admixture factorization same and clusters. matrix 2000) the genetic nonnegative Donnelly, to utilizing samples & coefficients assign Stephens, we to Fran¸cois, Fran¸cois, ancestry 2014) (Pritchard, 2015) & & structure, (Frichot individual Bouchard, package population Trouillon, LEA estimates the Mathieu, assess sNMF using (Frichot, To R sNMF in 2019). used mealybugs implemented al., we with as clade (Poveda-Mart´ınez First, et same approaches. the Cactaceae of two part on SNPs employed be after feeding to size shown sample collection Argentina been low have plant to from mealybugs due host Australian pooled and because were also Paraguay location and and sampling filtering, Australia by Argentina, defined from using samples were however, Populations calculated site; (www.r-project.org). were R populations in available between package R estimates with calculated were Fst (Ar) richness allele and (Ho) hierfstat heterozygosity observed datasets, SNPs analyses on phylogeographic MT138931. Based and - MT138921 clustering numbers: genomics, access Population South New under 2.5 (Seoul, analyses. GenBank genetic Inc. in population Finally, deposited and Macrogen phylogenetic were Germany). at in haplotypes employed Hilden, methodology were mtDNA sequences Sanger (Qiagen, All MN013554). using Kit - sequenced (MN013440 Purification were PCR 1% amplicons QIAquick Additional in the Korea). checked 2000). the Ward, were of using & products strands Gadau, purified PCR both (Brady, (2019). and BEN3R al. C1-N-2568 Poveda-Mart´ınez gels et I and following agarose subunit 1994) performed oxidase al., were Cytochrome et reactions the (Simon PCR for Jerry encoding C1-J-2183 gene primers mitochondrial using the of fragment sequencing A test and exact amplification the gene Hardy-Weinberg using 2014). to Mitochondrial tested Fit Gold, was 2.4 SNPs. & populations with outlier Hollenbeck, within SNPs for (Puritz, locus dataset test dDocent each our to in for from Bayescan ensure implemented frequencies excluded used variant et to also We of We and (Danecek indels. (HWE) analyses. (LD) and v1.15 next disequilibrium expectations variants VCFtools linkage the allele minimize two in using employed to than 0.01 SNPs contig more missing than the per 20% lower of SNP than (MAF) independence more one with the selected frequency sites randomly (BBMap allele data, We bbmap minor missing 2011). 10% using gene- al., with than (VCF) 0.95 loci File more Calling with of and Variant individuals The threshold genotypes remove tools). identity to NCBI (BBMap callvariants filtered alignment the using was FuEDEI by an rated as to done available with was blastn calling is genome Genotype using genome draft tools). aligned draft mealybug Reference then reference removed. and were the removed species were bacterial to bp HPun hits 250 Blast than database. shorter nt contigs Assembled 2017). ouain a enderir eecluae sn Ns 6(oa ta. 2017). al., et (Rozas v6 DNAsp using calculated Forster, were (Bandelt, earlier) algorithm defined Joining (as Median populations using ( & Network diversity Stecher, haplotype (Kumar, tide a v7 MEGA build R in to implemented & used fit. W and Clustal best using coefficients. 2016) the aligned Tamura, admixture were with sequences individual value DNA using SNPs). K estimating Mitochondrial performed 1,707 were the and individuals, analyses identify clustering (142 10, prior to dataset in to SNPs (BIC) identified groups 1 the scores between estimates from criterion Fst ranging information pairwise Additional Bayesian values calculated K to then used for We approach Devillard, clusters in (Jombart, second R DAPC of The in components estimation. number implemented principal as ancestry of 2010) analysis of Balloux, discriminant error & a the was evaluate structure population to assess genotypes masked of prediction h,19)ipeetdi oAtv.. Lih&Byn,21) diinly ecluae nucleo- calculated we Additionally, 2015). Bryant, & (Leigh v1.7.1 PopArt in implemented 1999) ¨ ohl, ..aa h CIudracsinnme:JAI00000 lae ed eete apdto mapped then were reads Cleaned JAAOIU000000000. number: accession under NCBI the at 1.1.fa Gue,20) xetdhtrzgst H) lbldffrnito siae Gt n pairwise and (Gst) estimates differentiation global (He), heterozygosity Expected 2005). (Goudet, K π ,hpoyedvriy(d n ubro altps() arieFtetmtsbetween estimates Fst Pairwise (H). haplotypes of number and (Hd) diversity haplotype ), n h optimal the and , COI altpsdtce nPvd-atıe ta. 21)wr eree rmGenBank from retrieved were (2019) Poveda-Mart´ınez al., in et detected haplotypes K au a sesduigtecosetoycieinbsdo the on based criterion cross-entropy the using assessed was value adegenet akg,using package, 4 mmod K and en lseigt dniyteoptimal the identify to clustering means Adegenet Jmat 08 packages 2008) (Jombart, (COI a amplified was ) - Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. uhr,Xe abu,21)t cestepseirpoaiiiso h eutdtopology. resulted the of probabilities posterior trees (Drummond, the the v1.7.5 access TreeAnnotator of using V to 10% complete tree first 2012) The and credibility the Rambaut, U clade removing 2014). & maximum 2014), for generations. al., Xie, a Heled, 1,000 SNAPP et Suchard, generated out & every (Bouckaert we (Rambaut in filter Finally, v2.2.5 2009), sampling v1.6 parameters Densitree to burn-in. generations, Tracer al., in model as MCMC using decided et visualized default checked were 10,000,000 (Li we trees was the for v1.7 of Therefore, data used analysis set bcftools of We the with localities. convergence ran populations SNPs. and sampled we more Stationarity 1,679 might and the or of one sites of one matrix to some more for sites equal new and data or some a allele, no one individuals, in alternative the with of for resulting the of sites number left result or and the a data reference sites of as no monomorphic the reduction missing section) of had the previous of have because to the numbers (see also either Due lowest analyses monomorphic the method. clustering become with the in In could individuals defined of groups selecting demand 2014). the computational individuals, al., of high two et one (Bouckaert include each v2.5.2 only from on Beast data, could Bouckaert, based (Bryant, in we Additionally, SNAPP implemented case, methods, dataset. 2012) coalescent-based each this and RoyChoudhury, the of ML using & tree from estimated Rosenberg, reconstructed was resulted Felsenstein, the tree (PP) of species Probabilities a node Posterior dataset, each and SNPs on values plotted UFboot were mixing respectively, sufficient 2014). haplotypes BI, were and sampling Drummond, stability mtDNA runs generations, and & burn-in, million independent the Xie, as 50 removed Suchard, two and were for runs dataset, run (ESS SNPs the each each of parameters 25% 1,707 for For of first (MCMC) The of Carlo analyses. generations. Monte matrix 1,000 ML Chain every entire Markov in four the implemented with employing fit-model performed same 2012), the Criterion al., Information following Akaike et (UFboot) to (Ronquist GTR+ according bootstrap Haeseler, and ultrafast model v3.2.6 Von dataset 10,000 best-fitted Schmidt, SNPs with the were for 2010) (Nguyen, as approaches Schwartz, (AIC) v1.6.10 GTR & (BI) estimated IQ-TREE Pfeiffer, inference ModelFinder in (Miller, bayesian implemented CIPRES iterations. using was and 2015) datasets (ML) Minh, both likelihood & for maximum ML datasets, performed. mtDNA analyses and coefficients. delimitation regression SNPs species of For and significance the reconstruction assess tree genetic to Species on permutation use 2.7 9,999 package plant the ran host from and correlation and MRM 2007), distance function all Urban, the geographic For using & of regressions influence & matrix 2010). the performing Robert, Fortin, by tested plant packagedivergence & host further Long, We R and (Legendre Jeffrey, Fst significance. the distance pairwise (Smouse, statistical using used geographic test and divergence, for we genetic plant Mantel on controlling host analyses partial plants while second host two a of employed matrix, and conducted influence we matrices distance the divergence then evaluate genetic genetic We to and on 1986) Sokal, geographic use matrices. considering plant distance host first and genetic 1967), families) distance (Mantel, plant geographic tests host of Mantel (the influence categories the three analyze distance with To use. geographic matrix Portulacaceae, plant Cactaceae, binary the host distance a plant collected: of construct were Host constructed patterns To mealybugs we considering S1). SNPs where (Table Thus, nuclear families population distances. Amaranthaceae. on plant each genetic host of and either longitude) considering of based and built level matrix (latitude was population matrix coordinates the at the construct used values we linearized to divergence. matrix, Fst genetic matrices datasets on pairwise distance mtDNA use used of the plant or we analyses distort host analyses, regression would and these multiple and distance In and geographic dispersal tests of mediated correlation importance human of the by combination analyze because areas to a excluded the were among used reached analyses We divergence these locations genetic in analyses. these on populations in plants Non-native collected host range. samples and native the distance divergence from geographic genetic populations of on complex influence distance the geographic analyzed and We plants host of Influence 2.6 > ,0 o NsadESS and SNPs for 1,000 vegan Γ IfrmDAdtst Ircntuto a sesduigMrBayes using assessed was reconstruction BI dataset. mtDNA for +I 244(kae ta. 07 n ,9 emttost assess to permutations 9,999 and 2007) al., et (Oksanen v2.4.4 > 0,frmDA eecekduigTae 16(Rambaut, v1.6 Tracer using checked were mtDNA) for 200, 5 ecodist 201(Goslee v2.0.1 .pungens H. Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. H=001 a he ie rae hnosre eeoyoiy(enH=009,poal sacon- among a greatly as varied probably heterozygosity heterozygosity 0.029), Expected expected Ho= below). mean (mean total (see heterozygosity data, populations observed SNPs among than on native differentiation greater Based the of times 1). across sequence three (Table high was range were differen- non-native diversity 0.061) of the genetic levels (He= in great mitochondrial low but and diversity and nuclear genetic range Overall of levels populations. intermediate among revealed tiation datasets mtDNA The and the SNPs haplotypes. non- in Both MT138931. 27 differentiation - the in and MT138921 distributed in diversity numbers sites, accession 90 Genomic variable under 3.2 (10.16%) amplified and GenBank the 42 of in range and length available (89.83%) The native are conserved sequences generated. the 371 be mtDNA with not in could bp, collected location 413 this was 143 at data sequences, SNPs but included 233 Cactaceae, evaluated we Additionally, of we range. consisted threshold, native strictest dataset species and the the mtDNA structure is Since genetic The dataset population S2. Table in SNPs results in the same detailed the filtering are analyses. essentially at step for delimitation 78X) yielded in coverage filtering that used (average present each thresholds criterium SNPs were in filtering 1,707 removed data other that containing loci loci missing dataset of filtering final 32 of Numbers a After remove 10% with analyses. to loci. up further had 5,383 ended for we we of used individuals, However, total ( the a data of loci. 240.6 recovered 90% missing 14,446 of we least of identified length individuals, numbers total we 141 high a samples, remaining of average with the because 173 an fragments analyses of with 250,856 further contained consisting individual, from genome set per individuals assembled reads a final single-end Considering The million bp. Mb. 1.6 123 of of average, length on read consisted, dataset nextRAD The data mitochondrial generations. and MCMC NextRAD 200,000 (http://species.h-its.org/) using 3.1 server and bPTP values the default in with RESULTS format, performed 3. nexus were was in analyses analysis tree bPTP ultrametric GYMC The the The using Beast. 2012). 1,000 by al., every inferred et tree sampling (Drummond the v1.7.5 generations, TreeAnnotator with 10,000,000 in performed to generated v2.5.2 set was Kapli, Beast tree was in (Zhang, credibility GTR+ tree MCMC (bPTP) ultrametric used an processes We prior. calculated tree first tree Mixed 2014). we Poisson Generalized analysis al., Bayesian GYMC the et and the mtDNA, For (Bouckaert on 2006) 2013). based al., Stamatakis, run et & (BF) were Pavlidis, factor (Pons delimitation Bayes and (GYMC) species the rate of Coalescent 20,000 calculate mutation Yule methods of to same locus used pre-burning single were the a two model using Finally, generations, each calculated 200,000 for MLEs was of The 2 length model burning. statistics MCMC United each as test an the for 10% with and first MLE the steps Rico The discarding 12 Puerto group. considering Brazil, that one above, northeastern difference only as from the as Portulacaceae with appear 1 and States Model Amaranthaceae Brazil. in on southeastern as feeding from groups Amaranthaceae bugs same on Brazil, the feeding northeastern mtDNA: mealybugs from with v) Portulacaceae and considered and States; 2 Amaranthaceae United Model on the Bra- feeding and southeastern mealybugs Rico from iv) i) Puerto Cactaceae Rico; tree: on Puerto phylogenetic feeding and best mealybugs zil estimated iii) Argentina-Paraguay-Australia; the from and Cactaceae mo- DAPC, two sNMF, for stricto with (MLE) su considered obtained estimates 1 likelihood results marginal Model the the to limits. on compared and species based and SNAPP of estimated analysis We the dels 2014) 2014). using Bouckaert, al., performed & et was (Bouckaert Minin, dataset, (Leach´e, Fujita, SNPs (BFD*) the Delimitation Species Factor Bayes ( Hypogeococcus MLE(model1 .pungens H. SPLITS ouain olce nAaataeei retn) i elbg edn on feeding mealybugs ii) Argentina); in Amaranthaceae on collected populations ) akg Ead uiaa arcog,20)i ,uigteultrametric the using R, in 2009) Barraclough, & Fujisawa, (Ezard, package -MLE sacmlxcmrsn orseisacrigt h ao ldsfound clades major the to according species four comprising complex a as ( model2 COI Γ .pungens H. Imdlwt nucreae eae lc n osatsize constant and clock relaxed uncorrelated an with model +I eune ffieidvdasfo aiona ntdSae on States United California, from individuals five of sequences Ks atr,1995). Raftery, & (Kass ) 6 sacmlxo h olwn v pce according species five following the of complex a as .pungens H. ahSampler Path pce complex species .pungen H. akgsicue nBatv2.5.2 Beast in included packages < 0 isn eoye) From genotypes). missing 10% th eeain h maximum The generation. s es stricto sensu .pungen H. COI n mealy- and fragment s sen- Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. ) atlcreaintssbtenhs ln s n eei itnemtie n ewe geographic between and matrices distance genetic and use (Table distance plant geographic host than distance between genetic tests of the predictor correlation in better variation Mantel a genetic was on 2). variation use distance plant genetic geographic host and that in plants showed distance host complex of geographic influence the and of use Analyses plant host Cactaceae- of included Influence H3 3.3 H2. and mealybugs H1 Amaranthaceae-feeding H4 Brazil. from and Paraguay, southeastern differentiated southeastern and from Australia Brazil, in highly Argentina, found southeastern in are only using collected from MT138923) haplogroups mealybugs mealybugs and/or mealybugs (haplotype feeding two other the Amaranthaceae from remaining the by and The on both shared (California), Brazil. is feeding States haplotypes, United MT138924) two (Florida) and (haplotype Rico includes States one Puerto (haplotype plants, which United host haplotype (H2), and as frequent haplogroup Cactaceae Rico Argentina, most second in Puerto collected the The mealybugs Brazil, Amaranthaceae-feeding contained Portulacaceae. by from which shared specimens is (H1), shape, and star-like haplogroup geographic a and first has Cactaceae) one and or The each MT138927) Portulacaceae, data, 2C). and/or mtDNA (Amaranthaceae on (Figure plant based host recognized origin of be type can the substitutions, by two and characterized least hosts at Portulacaceae by separated and/or haplogroups, Amaranthaceae Four As haplotypes on three 0.744). hosts. collected only Cactaceae Hd= samples where on non-native in (mean samples sites, two one non-native and hosts mealybug 0.174), only in in native Cactaceae Hd= lower detected on (mean substantially in was were found 9 found haplotypes were haplotypes and were of 18 hosts number haplotypes range, the Portulacaceae 27 native expected, and/or the of Amaranthaceae in total on between present a collected comparison were 1), the haplotypes (Table All in dataset ranges. 0.945 estimates mtDNA to Fst the BrPRUS-AP differentiation, and Concerning genetic Ar-A S4). strong between (Table comparison revealed Fst Br-A Pairwise the and above BrPRUS-AP structure. in described genetic 0.567 clusters population (BrPRUS-AP). Admixture from five strong (Br-A). (US) ranged and Brazil the States southeastern clusters United considering in between the values Amaranthaceae admixture and on no collected Rico suggested from mealybugs Puerto coefficients species included from (AP) the cluster whereas Portulacaceae populations last (BrPR-C), and/or with The pest Amaranthaceae along cactus on (PR) Brazil feeding Rican northeastern Cactaceae Puerto (Pa), mealybugs of the Paraguay encompassed populations and Argentina, cluster included (Br) from cluster fourth Brazil third native southern The from mealybugs (ArPaAu-C). mealybugs feeding (Au) Amaranthaceae feeding Australia (C) on in Cactaceae feeding collected included mealybugs mealybugs and (Ar) cluster (Figure called values Argentina second previously BIC was included The what and that (A). to cross-entropy 2019) corresponds on cluster al., (Poveda-Mart´ınez based first et The (Ar-A) (K) clusters the respectively). that B, / suggested and populations analyses 2A DAPC ancestral and five sNMF comprises Indeed, complex location. the site in with structuring between associated population sarily 0.969 S3). strong (Table as revealed also high pest) analyses as cactus Clustering or to Rican Amaranthaceae Amaranthaceae) Puerto non-native on on (the feeding the feeding mealybugs PRC in Rican mealybugs and (Puerto (Fst) States PRA PRA (United differentiation (Argentina, between USA 0.078 ARC Genetic and as and 0.827 low Portulacaceae) Cactaceae). Amaranthaceae) as to values on on Portulacaceae) from feeding varied feeding and area mealybugs mealybugs Amaranthaceae feed- Australia (Argentina mealybugs on and (Brazilian ARA feeding BRC Paraguay between mealybugs the between comparison In (Brazilian comparison the BRA 0.908). the in in (Gst= and for than differentiation 0.602 Cactaceae) genetic 1.240) from on the global ranged = ing great considering estimates showed Ar dataset, family, Fst SNPs (mean plant range, the host native range for non-native and detected. native estimated area, the were values the sampling in alleles Fst tions, in 0.003 private pairwise no as higher of and low analysis 20% 1.020), as The was = to richness Ar 0.102) (mean Allele He= areas (mean invaded 0.061). range recently native He= the (mean in area 0.267 from ranging populations 7 .pungens H. pce ope,ntneces- not complex, species .pnessnustricto sensu pungens H. priori a .pungens H. .pungens H. endpopula- defined species species Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. ee.Orgnmcsre loe h dnicto ftesuc ouainfo hc h uroRico Puerto the which the from of population family member plant source host Each the the species. of at five identification specificity of the least degree allowed at high survey of a genomic indicating consisting Our hosts, complex particular level. species with associated a was but complex species, polyphagous that single confirmed study Our S1). (Figure in Amaranthaceae on first DISCUSSION Brazil the 4. on southeastern bPTP, feeding from by Australia mealybugs of and considered in groups Paraguay, consisted were Argentina, one five from second splits that the mealybugs additional group by (bPTP) of Two conformed four fifth and clade four Cactaceae. the (GYMC) the three delimited consider in delimited groups bPTP not methods Both additional and did Portulacaceae. and/or GYMC methods Amaranthaceae on cases, locus (Florida) (GYMC) stricto single six both sensu recovered These pungens In mtDNA, data. H. S1). on SNPs based (Figure with methods groups delimitation identified (bTPT) species 10 locus feeders, single and cactus and Brazil the of bPTP, southeastern clades and from as two other GYMC considered the the be of and may clade BrPR-C). ArPaAu-C) (Br-A) sister (clade (clade the Rico Brazil Australia (BrPRUS- Puerto as and southeastern Portulacaceae Paraguay appeared from Argentina, which and/or latter feeders from clade Amaranthaceae the Amaranthaceae one Moreover, the of on that species. clade feeding separate means the (Florida) three This as States C). well United and as 3A the AP), which (Figure and in data Rico model SNPs Puerto a with supported produced data tree included SNPs species using and method tree netic BFD the on based pungens analysis delimitation Br-A) (clade (clade Species Cactaceae ArBrPRUS-AP. Brazil on of southeastern feeding from clade mealybugs Australia sister feeding and 3B). the Amaranthaceae Paraguay (Figure to as Argentina, close mealy- appears from appeared BrPR-C) feeding that populations (clade ArPaAu-C) cactus included Rico clade including Puerto third clade and The Brazil The southeast ArBrPRUS-AP). from encompassing (clade bugs one Portulacaceae trees, nuclear BI to and/or and respect Amaranthaceae ML with in observed results were contrasting clades showed C). stricto formed major dataset and branch Four mtDNA 3A 3B). independent the (Figure (Figure fourth an with SNPs Br-A) was the obtained (clade clade tree and Brazil phylogenetic fifth southeastern ArPaAu-C) The The in cactus (clade BrPR-C). collected comprised Australia (clade mealybugs clades Rico feeding and Amaranthaceae fourth Puerto Paraguay by and and Argentina, Brazil third from clades, southern encompassing The third second from respectively. first and the the BrPRUS-AP), the first mealybugs: on (clade The related, based feeding (Florida) C). them closely and of States appear all 3A United Portulacaceae, SNAPP, (Figure stricto and/or by clades sensu inferred Amaranthaceae supported pungens tree on well species feeding major the both five and revealed analyses dataset, phylogenetic SNPs BI analysis and delimitation ML species Both and host reconstruction and In tree geographic variation. Species on genetic 2). matrix 3.4 of (Table = distance 47.6% results mtDNA coefficient non-significant explained on geographic yielded based together 0.0001; tests distances distance plant p= regression geographic multiple 0.28497, and and coefficient= plant correlation among plant contrast, differentiation Host (host genetic 0.0001). affected SNPs p= significantly nuclear 0.00709, factors for both localities that showed distance sampling distances geographical geographic for controlling and when plant significant remained matrices distance ( genetic and ( distance significant plant were dataset SNPs nuclear the 0.4; using matrices distance genetic and r 0.6133; = p= ihppltosfo otesenBai,Pet ioadteUie tts(lrd)feigon feeding (Florida) States United the and Rico Puerto Brazil, northeastern from populations with .18 epciey.I diin h ata atlts niae httecreainbtenhost between correlation the that indicated test Mantel partial the addition, In respectively). 0.0128, a ope ffieseis(oe ,TbeS) uprigtepcuedpce ytephyloge- the by depicted picture the supporting S5), Table 1, (Model species five of complex a was .pnessnustricto sensu pungens H. p .01.Lkws,terslso ersigtemti fgntcdsacso ohhost both on distances genetic of matrix the regressing of results the Likewise, 0.0001). = caeA-)ad h eod elbg rmnrhatr rzl uroRc and Rico Puerto Brazil, northeastern from mealybugs second, the and, Ar-A) (clade n h ouainfo otesenBai,Pet ioadteUie States United the and Rico Puerto Brazil, northeastern from population the and .pungens H. A-)adtecaecmrsn elbg rmnrhatr Brazil, northeastern from mealybugs comprising clade the and (Ar-A) omnycle h arsaccu elbg(C) snta not is (HCM), mealybug cactus Harrisia the called commonly , 8 .pnessnustricto sensu pungens H. r 0.5254; = p .pnessensu pungens H. .07and 0.0017 = n the and r= H. H. Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. oto fccu ed.Bt tN n ula Nsrvae eti ereo eei structuring this genetic of of locations degree different at certain biological recorded a which were for revealed clade, haplotypes Australia SNPs mtDNA ArAuPa-C in differentiated nuclear the well introduced and Several within those mtDNA clade. heterogeneity Both and this Paraguay, genetic weeds. within Argentina, of cactus in of degree sampled control certain feeders a cactus 2019). elucidated al., included also et of (Nosil on Doellman results speciation drivers acting 2018; incidental likely Our hosts al., and alternate more et divergence by the the Duque-Gamboa genetic caused may 2012; as produce selection of It divergent to al., appeared that species S1). sufficient et allopatry evidence is (Table on is and populations mealybugs there fragmented fed hosts fact, pest ancestral of alternate clade Rican matter on ArAuPa-C a Puerto selection As the non-native divergence. divergent for that of species argued genera distance Mealybugs putative various be two geographic genera. and these large different Cactaceae, mealybugs, the on to Brazilian feed species besides belong both our that that that by mentioning genera fact cactus indicated the worth as and on is clades populations, fed it these those conspecific of context, and for populations Paraguay, this expected separating Brazil and In be southeastern Argentina analyses. would from mealybugs to which delimitation feeding that native cactus and exceeded mealybugs (ArPaAu-C) (BrPr-C) Argentina feeding from cactus Australia to the introduced al., between et Driscoe divergence little 2018; Genetic Sharanowski, with in Fern´andez, & Bass, plants divergence Zhang, host cryptic 2017; alternate Sousa, al., Bagley, of to et 2011; Forbes 2019). driver adaptation Katakura, 2017; & of important Kahono, Linnen, result (Matsubayashi, & an species the Niemiller, insect as as several diverged use in likely, divergence plant morphological very genetic host that, of species to predictor associated pointed better a results was our priori latter isolation context, matrices the by this distance that than In genetic revealed rather of 2). matrices plants (Table analyses distance host populations regression plant among alternate multiple host divergence and by America and correlation South imposed geographic Mantel in selection on fact, range divergent entire In by the distance. of driven throughout through history differentiation been evolutionary genetic al- mainly the that markers, has nuclear hypothesis of throughout of the insights area number supported new geographic large Argentine results wider and substantially the a for a 2019) to reported of al., results effort use et the the sampling of with our confirmation along of the ranges, lowed extension (Poveda-Mart´ınez non-native The et hosts and pre- 2016). different alternative native asymmetrical al., on with the et divergence, performance associated Aguirre and genetic populations preference 2019; deep mealybug differential al., a the and revealed between Argentina, from that isolation hosts studies reproductive previous postzygotic and the zygotic with agreed gene results of mitochondrial These part analyses the as delimitation considered and species formerly SNPs and populations clustering wide among genomics, genome Population on resources. feeding based as that plants confirmed host results different These hosts. others as and Cactaceae, species or Portulacaceae pungens Amaranthaceae and with Amaranthaceae associated use mealybugs Golubov, indiscriminately the & that between separation Mandujano, clear Cuen, a P´erez Sandi to Zimmermann, pointed Portulacaceae 2001; and Haro, that Apocynaceae de suggesting Cactaceae, & 2010), the Claps to 1994; range host (Ben-Dov, its families extended collections Further 1981). Willink, describe to used specimens The for in strategies pest reliable the design with to associated 4.1 parasitoids, help specialized will as it range. such since native enemies, achievement natural its important using an control is biological This classical derived. was pest cactus yoeccu pungens Hypogeococcus rsmdplpaosiscs rpi iegnehsbe eurnl bevdi h omo host- of form the in observed recurrently been has divergence Cryptic insects. polyphagous presumed Cleistocactus a o oyhgu pce,btaseiscmlxcmoe fcytcseisascae with associated species cryptic of composed complex species a but species, polyphagous a not was and .pungens H. Harrisia .pungens H. pce ope n otpatuse plant host and complex species hra rrCtrvdo pce fohrgnr like genera other of species on thrived BrPr-C whereas , a oyhgu pce.Hwvr u ouaingnmc approach genomics population our However, species. polyphagous a was eeoiial olce na mrnhca ot(rnr de (Granara host Amaranthaceae an on collected originally were .pungens. H. 9 COI .pnessnulato sensu pungens H. Hypogeococcus aaesrvae epgntcdivergence genetic deep revealed datasets elbg (Poveda-Mart´ınezmealybugs neet u present our effect, In . Cereus o native for H. a Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. h reotie ihmDAdt.Hwvr ouvi h neta oti hs lds n oevaluate to and clades, these in in twice host evolved ancestral derived have the to a unveil appeared formed to cactophagy However, species while data. feeding com- SNPs, mtDNA cactus not nuclear with were both on obtained datasets use; based tree was SNPs plant tree the Amaranthaceae and host the on mtDNA in of by feeding clade evolution monophyletic yielded that the results indicated concerning However, mtDNA congruent condition. or more pletely ancestral SNPs of likely nuclear consisted more either it the on since reliable based more trees considered Phylogenetic SNPs. that be distributed dataset, encoding may widely mtDNA gene dataset thousand the mtDNA genomic one the of nuclear of than limitations the pairs to by base due needed depicted hundred is study few history sorting caution present genomes a of lineage the only nuclear word in incomplete A of by conclusions observed. either consisted definitive pattern depicted that the at phylogeny proposed for arriving account authors the before could The cactophagous hybridization with 2019). introgressive related concordant For and/or Hasson, closely (ILS) Despr´es, completely 2019). & of Revale, 2019; not cluster Almeida, al., a is (Hurtado, in et mitogenomes particularly study by Hinojosa mitogenomic data, inferred 2006; a mtDNA Moritz, sorting reported on (2019) lineage & groups only Drosophila al., incomplete Meyer, species based et 2006), (Hickerson, many delimitation Moreyra in Armstrong, taxa species instance, history & diverged obscure evolutionary 3). Ball recently may the (Figure 2004; events in al., trace data Hallwach, hybridization et to mtDNA & Hinojosa past useful with 2017; Janzen, and been al., group Burns, has et same Penton, (Weigand mtDNA the insects (Hebert, the in in though reported collapsed Even been appeared 2019). often have clades inconsistencies these mito-nuclear while Such (Figure SNPs, clades and nuclear supported clade) that to well five (BrPRUS-AP being detect States to difference allowed mtDNAUnited main SNPs with wide Analysis The congruent. genome entirely whereas 3). not clades were major analyses four species delimitation visualized species this and in phylogenetic divergence of genetic Results of patterns affecting be might enemies to complex. associated natural found with only interactions were ecological Paraguay) and stricto Argentina sensu from parasitoids pungens primary the encyrtid of (also members Logarzo all & attack to as- able whereas parasitoid, hyperparasitoids primary complex, and varying widespread parasitoids a impose of as Encyrtidae) may survey also (Hymenoptera: recent competitors could A or factors locations. with enemies environ- geographic other sociated different natural different since to with in adaptation caution interactions pressures by Local with selective induced ecological unexplained. flow interpreted remained and/or gene that be conditions variation limited should mental genetic the of results by distribution followed these the divergence, affect Still, diver- genetic genetic distance. influencing the force by of primary 2) the isolation (Table the of 48% be part least to as at considered seemed influenced mealybugs that factors the two between of gence evidence provided data our 2012). Overall, McFadyen, 2010; Golubov, & Paraguay, Mandujano, and or Cuen, Argentina to Argentina, northwestern authors, central-western South in other inhabiting collected in by samples species (and suggested feeding Australia as cactus in not, the program of but to Australian control debate close Indeed, biological 1984). the genetically Hamon, were the end 1973; bugs to in Williams, 1987; helped used McFadyen, also mealybugs & (Tomley results the Africa) of These of indicative heterogeneity. origin be species the controversial may within for clade the Tucum´an, than this sampled in of rather localities heterogeneity University speciation same (Luc´ıa genetic Claps, incipient the variation that the in suggesting morphological in communication), cacti subtle based personal depicted on revealed Argentina, tree collected Argentina results phylogenetic specimens in the the of survey with Also, morphology genomic concordant 2C). external (Figure sub-clades the isolation internal of evaluation geographic showed of SNPs result nuclear the on as likely clade, widespread p.ihbtn h otencn fSuhAeiaadfudta h vltoayhistory evolutionary the that found and America South of cone southern the inhabiting spp. Hypogeococcus ngrscachamai Anagyrus n h rurCcae(raisne l,21) h nuneo uhdifferential such of influence The 2018). al., et (Triapitsyn clade ArAuPr-C the and elbg nSuhAeiaidentified America South in mealybugs Hypogeococcus raisn oaz gir and Aguirre & Logarzo Triapitsyn, .pnessnustricto sensu pungens H. Hypogeococcus festerianus .pungens H. elbg rmnrhatr rzl uroRc and Rico Puerto Brazil, northeastern from mealybugs 10 .pungens H. Jle rffih,19;Zmemn,P´erez, Zimmermann, 1999; Griffiths, & (Julien pce ope.Hs ln associations plant Host complex. species A-)wr ieetseisaccording species different were (Ar-A) etmsie hypogeococciLeptomastidea ie rle,aohrcactophagous another Trelles, & Lizer COI .lapachosus A. ncnrs,teevolutionary the contrast, In . Hypogeococcus raisn Aguirre Triapitsyn, COI ewr.An network. .pungens H. Triapitsyn mealy- H. Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. o dnicto ftehs lns eapeit h eiino ar ia h aeu important us gave who Vidal fieldwork Mayra for of Permits 17-8130-0757-IA 18-8130-0757-IA. Bill revision Farm Program USDA-APHIS Control the from Biological appreciate obtained USDA-APHIS Font was and We Fabian funding 19-8130-0852-IA, and Partial and assistance, plants. manuscript. technical the host for on the Jimenez suggestions Nadia of and identification Restrepo for Sanchez Fernando Andres thank loss We the two despite vein, diversity cactus this and In ACKNOWLEDGMENTS species native 2020). threatening al., variation. States, et genetic United Koch of southern 2020; and Holway, Clusella-Trullaset, Caribbean Suarez, & the (Tsutsui, the Keegan, populations were of invasive Minnaar, species the individuals Logan, putative negative- of may of novel 2000; potential that in events number evolutionary Case, thriving founder and reduced & of of survival, consequence capable a a fitness, been as that impact have variation ly species genetic suggesting invasive of Many reduction ranges, mtDNA the 1). southern native both (Table despite and environments in process respective Rico diversity colonization Puerto genetic the each of of in in levels involved colonization lower than the showed during populations nuDNA mealybugs events introduced these and cases, founder of both of ranges invasive In Por- occurrence and States. and/or the native United Amaranthaceae the of on in idea diversity feeding fee- the genetic mealybugs of Portulacaceae supported other levels the the and/or and of Puerto Comparisons feeders Amaranthaceae tulacaceae. cactus involving in from the one of collected States, derived United mealybugs mealybugs also for events feeding were invasion two Portulacaceae States United and/or ding continental Amaranthaceae and that focus should Rico showed pest the results against its enemies) Our in mealybugs. indicated natural feeding it for pest the search perspective, cactus the (e.g., practical Brazilian of of a strategies southeastern history control enemies from on but biological evolutionary natural relevance, of the for systematic design and searching untangling the taxonomic the that for Thus, have programs, issue only 2005). control not key southern Kirk, control did biological a the complex & biological classical is and specific In (Hoelmer species more mealybug. Rico area target develop this native Puerto beyond the to from invaded spread of help cactus that has identification may wild and pest correct it pest protecting Mexico) since cactus the at accomplishment (including the California, aimed important America of strategies in an North source pest is throughout the States the hosts of United of cactus al., Identification record (Carrera-Mart´ınez potential et Caribbean. mortality new to cactus plant the the range forests, native to high the dry distribution With 14 to represents of in current 2020). the lead occurring detection its of pest al., often species) half first et this that attacks endangered Triapitsyn the now two deformations of 2015; Since mealybug and tissue presence the endemic high. gall-like 2010), three the is large al., (including Rican diversity causing that et species Puerto cactus (Segarra-Carmona threat cactus the 2005 where Rican the in with Puerto Mexico, Rico of and haplotype Puerto on warning States mtDNA in collected a pest United unique fell samples the is and Moreover, pest in finding cactus same 2). diversity Rican (Figure the This Brazil Puerto Brazil shared pest. of the southeastern California cactus that coast from southern Atlantic found populations we northeastern in with SNPs, and Cactaceae clade genome-wide southeast same In to the the Puerto sampling 3). along into the genetic and and (Argentina), the Paraguay 2 range extended to native (Figure and sampling the clade the in feeding to expanded sampling cactus close pest restricted we clustered cactus Brazilian and Rican pest mtDNA southeastern Puerto cactus only the the Rican using that to study establish similar to previous us population our allowed a mtDNA, and from SNPs derives nuclear popula- on based feeding analyses, cactus Our Brazilian southeastern from derive mealybugs tions cactus delimi- Rican well Puerto other 4.2 continent, the in genus the genus of the and history Ferris of biogeographic the species and cactophagous use ted plant host of evolution the Hypogeococcus .festerianus H. huh nti ae rmnrhatr rzl(iue n ) hs nig suggested findings These 3). and 2 (Figures Brazil northeastern from case, this in though, , .pungens H. hudb nlddi nepne analysis. expanded an in included be should , ope aebe ucsflivdr n otnet pedthroughout spread to continue and invaders successful been have complex .pnessnustricto sensu pungens H. Hypogeococcus .pungens H. 11 pce ope noPet ioadtecontinental the and Rico Puerto into complex species rmSuhAeia uhas such America, South from Pvd-atıe ta. 09.Hwvr when However, 2019). al., (Poveda-Mart´ınez et yoeccu spinosus Hypogeococcus .pungens H. species Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. uhel .(07.BMpsotra lge,adohrbonomtctos eso 37.33. version tools, bioinformatic other and aligner, read short BBMap analysis. coalescent full a (2017). in (https://sourceforge.net/projects/bbmap/) B. trees gene species Bushnell, bypassing Inferring genus (2012). markers: ant A. RoyChoudhury, Evolution genetic & the and biallelic A., Biology of from N. Rosenberg, directly Systematics J., ox- trees Felsenstein, cytochrome R., Bouckaert, gene 1999. D., (2000). January mitochondrial Bryant, in the S. Australia, from Canberra, P. (2014). in data J. Ward, held using A. Conference, analysis & Drummond, In & preliminary I. J., a idase . . Gadau, Formicidae): . D., G., menoptera: Xie, analysis. H., S. evolutionary Bayesian C. 012401. Brady, Wu, for BioRxiv, platform T., software forest. Vaughan, a the D., through 2: Kuhnert, trees BEAST J., Seeing Heled, 2: R., DensiTree economic Bouckaert, and (2014). J. biology Heled, plants, & host R., Coccoidea: Bouckaert, distribution, Homoptera: geographical (Insecta: on world Limited. the data Intercept of with importance. mealybugs the Putoidae) of and catalogue systematic Pseudococcidae A (1994). phylogenies. Y. intraspecific Ben-Dov, inferring for tussock networks Median-joining with Evolution case (1999). and test A. Biology a Rohl, Molecular & identification: P., Forster, pest J., insect H. plant- for Bandelt, Host barcodes DNA (2019). (2006). Lymantriidae). F. J. (Lepidoptera: K. G. moths Armstrong, & Ragland, A. L., & fly, E. S. Ball, Johnson, C., fruit & Stauffer, cherry L., European . . . J. the 4648-4666. A., Feder, in (20), Z. M., differentiation Lewis, Schebeck, genomic markers. L., RAD H., related A. sequenced Shiver, Schuler, using C., use V., mapping M. host Bakovic, Currey, genetic and S., and discovery geography T. SNP Atwood, History, Rapid D., P. (2017). (2008). Etter, R. A., C. N. Baird, Linnen, ( & sawfly pine L., redheaded the power M. in the variation Niemiller, Ecology genetic Harnessing of C., patterns (2016). wide V. A. genome P. Sousa, shape Hohenlohe, K., & R. G., genomics. Luikart, structure Bagley, evolutionary R., Population and M. ecological (2016). Miller, for M., K. RADseq J. T. of Walsh, Good, R., & K. K., pest, Andrews, Gordon, global the A., in McGaughran, unrelated flow T., in gene ancestry W. and Tay, of J., estimation C. model-based Fast Anderson, (2009). K. Rico. Lange, Puerto & in J., individuals. Novembre, Cactaceae H., of D. Logarzo, pest Alexander, & mealybug E., the Hasson, of S., identification Triapitsyn, the of Science A., aid biology Bottero, to the Saracho Argentina on del E., in Studies L. Carrera (2016). Claps, CONICET A. H., G. of Diaz-Soltero, B., member M. a Aguirre, is E.H. CONICET. REFERENCES by awarded M.B.A. and both scholarship PhD Department fellowship Cientifico. 18BR027829/DF). a and Investigador of (41522-16-003), postdoctoral - recipient Service (16BR022349/DF is a Wildlife D.P.M. & IBAMA (OV-1617-15). of Fish the Resources U.S Environmental and the and by Natural (A9273F3) provided of were SisGen Rico the Puerto by from Permits provided were Brazil in , , 16 26 yeotr:eouin idvriyadbooia oto.Fut nentoa Hymenoptera International Fourth control. biological and biodiversity evolution, Hymenoptera: 1,58. (1), eoeResearch Genome 4,1022-1044. (4), , 29 8,1917-1932. (8), 99,1655-1664. 19(9), , eioep armigera Helicoverpa 61,37-48. 16(1), , aainJunlo oetResearch Forest of Journal Canadian Hypogeococcus pungens 12 aueRvesGenetics Reviews Nature . oeua clg,25 Ecology, Molecular p.1119.CIOPublishing. CSIRO 131-139). (pp. snusrco(eitr:Pseudococcidae) stricto)(Hemiptera: (sensu hgltscrs.MlclrEcology Molecular cerasi. Rhagoletis LSCmuainlBiology Computational PLoS 62,337-350. 36(2), , edpinlecontei Neodiprion 2) 5296-5311. (21), , 17 lSOne PloS 2,81. (2), Camponotus ora fInsect of Journal , 3 ). (10). 10(4). , Molecular Molecular (Hy- , 28 Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. uk .J,Nsl . te,W .(06.Eooia iegneehbt ossetypstv associations positive consistently exhibits divergence Ecological taxa. disparate estimation (2006). across J. efficient isolation W. and reproductive Etges, Fast with & P., (2014). Nosil, J., O. studies. D. Francois, Funk, association & ecological G., coefficients. Bouchard, and ancestry T., landscape individual Trouillon, of for F., package Mathieu, R C. E., An Frichot, Wilson, speciation. LEA: & Evolution, A., insect and (2015). Ecology H. initiating O. in Widmayer, in Methods Francois, K., shifts & A. host Ward, E., S., Frichot, of E. role statistics. Tvedte, particular threshold C., the by A. Revisiting limits 1126-1137. Hippee, species’ N., (2017). S. Splits: J. Devine, A., (2009). A. G. Forbes, T. Barraclough, & version T., untangling package Fujisawa, to approach T., integrative Ezard, An (2017). A. Hefetz, the & Evolution in T., Reiner-Brodetzki, delimitation R., species Genome- Seltzer, (2018). A., P. P. Barro, Eyer, De & E., Johnson, K., histories. Gordon, demographic the M., Fumagalli, of T., analyses W. wide Tay, P., Etter, of S., scan Elfekih, genome comparative speciation: ecological a during via differentiation genomic association and host beetles. Selection of (2008). contributions J. the D. R., Funk, isolating & M. P., Nosil, P., Manzano, S. in Egan, C., specialization. speciation host Y. cryptic and Guzman, distance suggests M., geographic Research DNA logical with L. Mitochondrial associated Hernandez, Cecidomyiidae) BEAUti (2018). F., (Diptera: with plant phylogenetics N. M. Host Bayesian Toro-Perea, Castillo-Cardenas, & (2019). (2012). N., R. A. D. Rambaut, J. & Duque-Gamboa, Ott, D., & Xie, 1.7. P., A., BEAST S. the M. Egan, and Suchard, R., J., A. G. Drummond, Hood, herbivore. W., insect specialist Goughnour, R. a 4197-4211. and in & 28(18), Busbee, diversification differentiation , shape C., . to genetic . interact C. . geography plant-associated and H., Nice, associations host T. L., Powell, of P., A. dimensions S. Driscoe, Egan, ecological R., and G. the Geographic Hood, in G., speciation Saint, (2019). Jean B. H., R. Schuler, M., M. Doellman, of analysis global boundaries. Refined genetic (2010). level P. species Barro, De identify & M., Y. Buckley, C., tabaci delimitation. Riginos, L., species Cook, and A., (2011). discordance Dinsdale, G. Mitonuclear McVean, & species? in more . or . Cactaceae . two Ecology A., One, with M. (2019). associated DePristo, L. E., Hemiptera) Despres, Banks, VCFtools. A., and (Insecta: C. format Albers, Coccoidea call G., variant The Abecasis, (2001). A., Auton, E. of P., Danecek, M. Symptomatology Haro, (2015). de A. & Argentina. D. E., Jenkins, L. & Claps, J., Ruiz-Arocho, L., by infestation Aponte-Diaz, R., Carrera-Martinez, Hmpea troryca lyooda lyoia)mtcodilctcrm xds to 1 oxidase cytochrome mitochondrial Aleyrodidae) Aleyrodoidea: Sternorrhyncha: (Hemiptera: , vlto:ItrainlJunlo rai Evolution Organic of Journal International Evolution: 28 , ora ftePoesoa soito o atsDevelopment Cactus for Association Professional the of Journal 115 1) 3845-3847. (17), Hypogeococcus pungen 128-139. , , 0() 739-749. 108(6), , hgltscingulata Rhagoletis 1 1) 2.Aalbefo:ULhttp://R-Forge.R-project.org/projects/splits/ URL from: Available r29. (11), eii tabaci Bemisia oeua ilg n Evolution and Biology Molecular lSOne PloS aalpi bicolor Cataglyphis , () 925-929. 6(8), Genetics 13 pce ope eelcnrsigpten famxueadcomplex and admixture of patterns contrasting reveal complex species :cnrssbtenhs species. host between contrasts s: Dpea ehiia)sbigseisgroup. species sibling Tephritidae) (Diptera: (1). Bioinformatics naso h noooia oit fAeia 103 America, of Society Entomological the of Annals 9() 973-983. 196(4), , eetatcmlxi Israel. in complex ant desert rceig fteNtoa cdm fSine,103 Sciences, of Academy National the of Proceedings 13 , 71) 2156-2158. 27(15), , 29 8,1969-1973. (8), , 62 5,1162-1181. (5), Haseltonia :77–83. 4: , oeua hlgntc and Phylogenetics Molecular eclmssbebbianae Neochlamisus rdpoi longifila Prodiplosis , Insects 2015 ultno Entomo- of Bulletin Evolution oeua Ecology Molecular 2) 14-18. (21), 09,275. 10(9), , 2,196-208. (2), Molecular Bemisia , 71 Gagne (9), (5), leaf R Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. ec´ ,A . uia .K,Mnn .N,&Buket .R 21) pce eiiainuiggenome-wide using delimitation Species (2014). R. R. Bouckaert, data. & N., SNP 7.0 V. K. version Minin, analysis D. K., genetics M. Price, Fujita, evolutionary Leach´e, D., molecular & A. MEGA7: A., P. (2016). datasets. Follett, K. bigger Tamura, M., for & G., S. Stecher, invasive Geib, S., of Kumar, N., diversity Ouedraogo, phenotype K., and M. Jardeleza, genomic Invasions R., Population J. Dupuis, (2020). B., J. Factors. Koch, target Bayes their (1995). and E. agent A. of 795. Raftery, 773– catalogue & world E., A R. weeds. Kass, of Wallingford. control Publishing, Biological edn.CAB 4th 1999. markers. weeds, M.W. genetic Griffiths of new M.H. analysis a multivariate Julien the components: for package principal 1403-1405. R of 24(11), a , analysis adegenet: Discriminant (2008). populations. T. structured (2010). Jombart, genetically F. of analysis Balloux, the & (2017). for filter. I. S., method Birol, Bloom & Devillard, a T., . using . Jombart, . genomes A., S. large Hammond, of S., Yeo, assembly J., 768-777 resource-efficient Chu, H., 2.0: Mohamadi, P., ABySS B. Vandervalk, the D., within S. relationships Jackman, phylogenetic 77 Revised Phylogeny, (2019). and E. Systematics Hasson, Arthropod & S., Revale, buzzatii F., Drosophila agents? Almeida, ineffective P., releasing J. pests: of Hurtado, arthropod risk against agents the control decrease biological to arthropod improved Selecting 255-264. be (2005). science A. the A. butterfly Can Kirk, the (2019). & in A., R. K. discordance Vila, Hoelmer, mitonuclear & striking N., uncover Alvarez, 28 Dinc˘a, Ecology, V., animal genomics C., Molecular new Pitteloud, species: discover sylvestris. A., cryptic to M. of fail Szenteczki, mirage often D., A will Koubinova, barcoding C., one: DNA J. in Hinojosa, (2006). species C. Ten space. Moritz, parameter & (2004). broad P., over W. C. species Hallwachs, Meyer, & J., M. H., Hickerson, Sciences, butterfly D. skipper of Janzen, Academy neotropical M., National the J. in the species of Burns, cryptic H., reveals E. barcoding DNA Penton, D., Agri- P. of Hebert, Department Florida Services Industry, Consumer Plant and of culture Division mealybug, Circular, cactus Entomology Pseudococcidae). A Coccoidea: (1984). B. A. Hamon, de especie Nueva (1981). Pseudococcidae). C. (Homoptera: M. Willink, F-statistics. de hierarchical Granara test and compute to R data. for ecological package a Notes of Hierfstat, analysis dissimilarity-based (2005). for J. package Goudet, ecodist The Software, (2007). Statistical L. of D. Journal Urban, & C., S. Goslee, 3209-3213. () 184-186. 5(1), , 2 1753–1770. 22, , ytmtcBiology Systematic oeua ilg n Evolution and Biology Molecular pce lse Dpea rspiia:Doohl elt ru)uiggnmcdata. genomic using group) repleta Drosophila Drosophilidae: (Diptera: cluster species Neotropica 34,534-542. 63(4), , 27,1-19. 22(7), , 263 1) 3857-3868. (17), ytmtcBiology Systematic . 0(1,14812-14817. 101(41), , (2). yoeccu festerianus Hypogeococcus 27 61-65. , 14 , Hypogeococcus 33 ora fteAeia ttsia Association Statistical American the of Journal , 55 7,1870-1874. (7), 5,729-739. (5), M Genetics BMC rspiasuzukii Drosophila a eTcmn eulc Argentina Republica Tucuman, de Rau LzryTrelles). y (Lizer srpe fulgerator Astraptes 11,94. 11(1), , ilgclCnrl 34 Control, Biological eoeResearch,27 Genome lrd (Homoptera: Florida nHawaii. in oeua Ecology Molecular Bioinformatics . Proceedings Thymelicus Biological 90, , (3), (5), Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. caynR .(2012). E. R. cactus, McFadyen harrisia of Weeds control of Biological Organization.. Control Biological (1981). bug, on J. mealy sium A. the Tomley, Societies. by Science & of Queensland Weed control E., Australian biological of R. Council the McFadyen, 108-112). in success (pp. 1978 of April indications Societies 12-14 Science Preliminary Weed Australian of (1978). Council J. ( A. cactus Tomley, Harrisia & E., R. beetle. critical McFadyen, ladybird the phytophagous as a specialization approach. plant of host populations regression Divergent 24 between Biology, generalized (2011). speciation H. in a Katakura, force & and driving S., clustering Kahono, W., Seal, disease K. Matsubayashi, & of detection . The . . Research H., (1967). Delatte, N. R., Mantel, in analyses. Krause-Sakate, associations RNA-Seq host-plant by E., revealed and Sharon, perspective diversification empirical detoxification E., Species-complex the Feldmesser, in (2018). D., term S. the Santos-Garcia, misinterpreting O., ‘generalism’debate: The Malka, of The insects. potential in (2009). (2016). breadth evolutionary dietary A. R. The on J. focusing Durbin, Harvey, (2020). literature & S. & Clusella-Trullas, D., & H. . Loxdale, M., . change. . K. climate Keegan, N., under invader A., Homer, insect I. J., an Minnaar, L., Ruan, M. T., Logan, Fennell, SAMtools. A., and Wysoker, format alignment/map B., sequence Handsaker, H., construction. network Li, haplotype for software Evolution full-feature and Popart: Ecology (2015). D. in Bryant, & W., J. Leigh, detecting for approaches alternative data. and genetic test of Mantel analysis 831–844 the spatial of the in Comparison relationships (2010). multivariate J. complex M. Fortin, & P., Legendre, oer,N . esh . utd,J,Amia . ard,C,&Hso,E 21) htdoes What (2019). E. Hasson, & C., Laprida, F., the Almeida, of history J., evolutionary of Hurtado, the inference about for J., us Gateway tell Mensch, Science mitogenomics N., CIPRES the N. Creating Moreyra, (2010). T. In Schwartz, trees. & phylogenetic W., large Pfeiffer, A., M. Miller, Australia in weeds of control tetragonu oi,P,Gmet . aks .E,Cmal,A . ee,J . url,C . acmn .L. T. Parchman, & A., C. insect. Buerkle, stick L., a J. in Feder, processes A., effective speciation Sciences A. Biological and multiple Comeault, B: fast of Society E., a consequences T. IQ-TREE: Genomic Farkas, (2012). Z., (2015). Gompert, Q. P., B. Global Nosil, Minh, (2015). & J. phylogenies. Day, A., maximum-likelihood & Haeseler, 268-274. estimating Von 32(1), . . for A., . A., algorithm H. R. Schmidt, stochastic Senior, T., I., biodiversity. L. Lysenko, terrestrial S., Nguyen, local Contu, on L., use S. land Hill, of N., effects L. Hudson, T., Newbold, One 14(11). , , L)Hmeiksodpa,p.2421 nuinMMFde ulnJ(eds), J Cullen R McFadyen M InJulien 274–281. pp. pear, Hummelink—sword (L.) s 27 7,1421-1432. (7), 2Pr ) 209-220. 1), Part (2 roeesmartinii Eriocereus arsa(roees martinii (Eriocereus) Harrisia () 1110-1116. 6(9), , 00gtwycmuigevrnet okhp(GCE) workshop environments computing gateway 2010 SR ulsig olnwo,Australia. Collingwood, Publishing, CSIRO . , yoeccu festerianus. Hypogeococcus 279 14) 5058-5065. (1749), a. nQenln.In Queensland. in Lab.) p.5954.Cmowat cetfi n nutilResearch Industrial and Scientific Commonwealth 589-594). (pp. . Evolution eda ainlSineCnr,Prvle itra Australia, Victoria, Parkville, Centre, Science National at held ilgclJunlo h ina Society Linnean the of Journal Biological iifrais 25 Bioinformatics, Nature 41,132-144. 74(1), , 15 Lbu. rto—arsacactus Britton—Harrisia (Labour.) oeua Ecology Molecular 2(55,45-50. 520(7545), , rspiabuzzatii Drosophila In rceig fte5hItrainlSympo- International 5th the of Proceedings rceig fteFrtCneec fthe of Conference First the of Proceedings 1) 2078-2079. (16), oeua clg Resources Ecology Molecular oeua ilg n Evolution and Biology Molecular eii tabaci: Bemisia , 27 lse rpeagroup)?. (repleta cluster 2) 4241-4256. (21), rceig fteRoyal the of Proceedings ora fEvolutionary of Journal roeesmartinii, Eriocereus p.1-8). (pp. , 119 plant-defence, A Acanthocereus 2,265-282. (2), Biological Methods Cancer 10(5), , PloS in , Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. mue .E,Ln,J . oa,R .(96.Mlil ersinadcreainetnin fthe of extensions correlation and regression Multiple (1986). R. R. Sokal, correspondence. & matrix C., of J. test reaction Mantel Long, chain E., polymerase phy- P. conserved and of weighting, Smouse, Evolution, compilation a (1994). P. and (2005). Flook, sequences & M. gene H., primers. Dicke, mitochondrial Liu, of B., & Crespi, utility J., A., Global logenetic Beckenbach, J. F., Frati, (2019). C., Loon, Y. Simon, van Li, B., & Demand. Loon, on Press Van . University . . M., Dinc˘a, V., practice: L. 116 G., to Schoonhoven, Sciences, Talavera, promise of Academy R., From butterfly National Vila, pest the (2015). A., agricultural of Proceedings A. the Espeset, of E. E., history Johnson, Lombaert, invasion & F., D., S. P. conservation. Ryan, in Etter, genomics D., with sampling M. non-invasive & Waterhouse, pairing S., A., datasets. large Ramos-Onsins, M. a P., Russello, of Librado, analysis polymorphism S., sequence Huelsenbeck, Evolution, Guirao-Rico, DNA & and C., v6: Biology model DnaSP J. large . (2017). . Sanchez-DelBarrio, a . A. A., across Sanchez-Gracia, S., choice Ferrer-Mata, Hohna, model J., A., and Rozas, Darling, inference Available: L., phylogenetic Bayesian D. [Software]. efficient Ayres, P., 3.2: v1.6. space. MrBayes Mark, Tracer Der (2012). P. Van (2014). J. M., A.J. Teslenko, F., Drummond, Ronquist, & D., Xie, designed BEAST M.A., pipeline http://beast.bio.ed.ac.uk/Tracer variant-calling Suchard, RADseq, a A., dDocent: Rambaut, organisms. (2014). multilocus non-model R. of using J. Gold, genomics structure & population population M., for C. of Hollenbeck, Inference reproduc- B., J. and (2000). Puritz, P. adaptation Donnelly, Ecological & of M., data. (2014). races genotype Stephens, L. host K., J. native J. Feder, for Pritchard, & evidence phenotypic R., and G. 23 genetic Ecology, Hood, Molecular sympatry: A., in evidence. A. isolation genetic Forbes, tive and E. Hasson, associations H., & plant T. host . Powell, . on . based S., A. Hight, Rico Vogler, A., Puerto Colina, & and la Australia, de L., . the . Calderon, . Untangling G., insects Logarzo, S., (2019). B., undescribed Hazell, M. of P., Aguirre, taxonomy D., (2011). D. Poveda-Martinez, DNA G. Duran, the H. A., for Zimmermann, Cardoso, delimitation 55 & J., species Biology, S., Gomez-Zurita, Sequence-based Neser, G., (2006). W., T. P. C. Barraclough, Mathenge, J., Africa. Pons, H., South Tran- Klein, BMC in (2018). cactaceae H., species. R. of J. related control Streiff, closely Hoffmann, Biological & two D., in plants I. . . non-host . Paterson, and M., host Vabre, 145. S. to A., 18(1), A. moths Pommier, biology, M. adult F., evolutionary Suggests, female Dorkeld, & of S., J., response Nidelet, M. scriptomic P., Oksanen, Audiot, H., M., H. Orsucci, M. package. Stevens, vegan B., The O’Hara, P., (2007). Legendre, S. R., Kindt, Press. University J., Oxford Oksanen, speciation. Ecological (2012). P. Nosil, ytmtcBooy 61 Biology, Systematic naso h noooia oit fAmerica of Society Entomological the of Annals 4,595-609. (4), Genetics 3,688-704. (3), Hypogeococcus pungens 4 3299-3302 34, 5() 945-959. 155(2), , omnt clg Package Ecology Community 3,539-542. (3), ytmtcZoology Systematic pce ope Hmpea suoocde o Argentina, for Pseudococcidae) (Hemiptera: complex species fia Entomology African PeerJ, irsrapae Pieris 16 4) 20015-20024. (40), ,e431. 2, , , 87 35 , 10 6,651-701. (6), PeerJ 4,627-632. (4), eeldwt eoisadctznscience. citizen and genomics with revealed 631-637. , 92,230-246. 19(2), , , 3 e1106 , netpatbiology Insect-plant lSOne PloS hgltspomonella. Rhagoletis 14(7). , Systematic . Molecular Oxford . Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. aa ...... md edcleto.GL,SDH,H.D.S., S.D.H., G.L., collection. field made H.D.S. S.V.T., analysed M.V.D., and research S.D.H., performed G.L., M.B.A., D.P.M., E.H., project. M.B.A., the designed data, E.H. S.D.H., G.L., M.B.A., D.P.M., under GenBank in deposited CONTRIBUTION were AUTHOR the haplotypes MT138931. - mtDNA under MT138921 (NCBI), New Information numbers: JAAOIU000000000. access Biotechnology FuEDEI as for number: available Centre is accession genome National under Reference the PRJNA593002. at number accession available BioProject are data sequence American Raw South The (2010). J. STATEMENT Golubov, ACCESSIBILITY DATA & cacti. C., American isolation: M. North Mandujano, temporal threatens M., or that Cuen, Habitat mealybug Sandi Perez (2018). G., J. H. Zimmermann, B. Sharanowski, & RADseq. C., digest with 9803-9816. double D. method (19), using Fernandez, delimitation patterns I., species speciation A. herbivore–parasitoid general Unraveling Bass, A M., (2013). Y. A. Zhang, Stamatakis, & placements. P., plant phylogenetic Pavlidis, to host P., applications Kapli, and J., Timing plants. J. flowering (2017). Zhang, and H. beetles Letsch, of & pattern B., Evolution co-diversification Gottsberger, ancient tribe J., an weevil the J. indicate of Astrin, evolution the L., in of associations A. origin Friedman, Pseudococ- the S., Coccoidea, Deciphering (Homoptera, Winter, Argentina (2017). from F. mealybugs Leese, cactus-feeding & Two cidae). C., (1973). Zhang, J. herbivo- L., D. species. Yu, insect Williams, caddisfly Geography Y., generalist sibling (2019). Li, a two M. in H., of S. discordance Cai, structure Murphy, mito-nuclear M., genetic & Weiss, population M., H., the R. Weigand, for Tinghitella, use O., plant J. host III, than re. Stireman important W., success the more T. and is Quinn, variation genetic C., Reduced M. (2000). J. Vidal, T. Case, & species. A., invasive D. Holway, an V., of and A. Suarez, primary D., N. A., on Tsutsui, V. notes Trjapitzin, taxonomic C., and J. other identification and Rodrigues, 1-23. mealybug Rico: Verle cactus B., Puerto Harrisia parasitoids. the M. in of secondary Aguirre, enemies Pseudococcidae) Natural A., (Hemiptera: (2020). G. Y. Rodr´ıguezspecies Logarzo, Reyes, & D., . S. . . Hight, V., S. & Triapitsyn, F., P. Rugman-Jones, and A., Encyrtidae M. (Hymenoptera: Ciomperlik, parasitoids Entomologist, secondary D., and S. primary Hight, of of Complex A., Signiphoridae) G. (2018). C. Logarzo, J. B., Rodrigues, Vancouver M. Verle cactus, Canada, Aguirre, Harrisia Agriculture V., Weeds. of S. of Triapitsyn, control Control Biological Biological of Symposium (1984). International mealybug, E. VI (2017). the R. C. by McFadyen, Queensland Lin, & central & J., . . A. . Tomley, J., Hulcr, complex. the T., of species phylogeography Thibault, Scolytinae) invader: A., cryptic idae: Eskalen, a Q., of P. origin the Thu, Tracing P., Rugman-Jones, R., Stouthamer, oeua Ecology Molecular ultno noooia Research Entomological of Bulletin , 107 101 179-190. , Hypogeococcus 3,411-434. (3), , rceig fteNtoa cdm fSciences of Academy National the of Proceedings etoia Entomology, Neotropical 28 1) 4317-4334. (18), p.mayus(eitr:Pedccia)i h e ol.Florida World. New the in Pseudococcidae) (Hemiptera: mealybugs spp. giutrladFrs Entomology Forest and Agricultural yoeccu festerianus Hypogeococcus Bioinformatics, , 62 Apionini nieFrt https://doi.org/10.1007/s13744-019-00754-w, First: Online 4,565-570. (4), atsadScuetJournal Succulent and Cactus 17 Ainne rnia,Cruinie,Coleoptera) Curculionoidea, Brentidae, (Apioninae, 9 2869–2876. 29, oeua Ecology Molecular ualcafornicatus Euwallacea ieyasatrrlae In release. after years nine , , , 97 19 1) 5948-5953. (11), 4,366-375. (4), 62) 5705-5715. 26(20), , oeua hlgntc and Phylogenetics Molecular , 82 clg n Evolution and Ecology Clotr:Curculion- (Coleoptera: roeesmartinii, Eriocereus 3,105-107. (3), HPun ..ai NCBI in 1.1.fa p.843-847). (pp. rceig of Proceedings Hypogeococcus , in 8 Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. acl ii Vitorino Diniz Marcelo Triapitsyn Serguei Hight D. Stephen Aguirre Belen Maria Poveda-Martinez Daniel interest. of conflict no ORCID have they that declare authors The INTEREST OF CONFLICT S1 Figure S5. Table S4. Table areas. invaded and S3. Table S2. Table S1. Table INFORMATION Brazil; SUPPORTING southeastern from States. United mealybugs of the Br-A, Mealy- and dataset BrPR-C). plant: Rico (clade the host Argentina, Rico Portulacaceae from on Puerto Ar-A, and mealybugs and based clade ArPaAu-C, Amaranthaceae Brazil clade SNAPP on southeastern from feeding plant: in mealybugs host bugs method and Cactaceae Australia, on coalescent and feeding for using Paraguay Mealybugs mtDNA reconstructed (C). and SNPs tree 1,679 SNPs Species on (B). Cactaceae based haplotypes on hypotheses feeding complex. Phylogenetic Amaranthaceae. Rico species on Puerto feeding and 3. Brazil Brazil southern Figure southern from feeding from specimens Australia specimens Br-A: and BrPR-C: Paraguay blue: the Amaran- Argentina, on and in blue: from feeding light and States specimens Amaranthaceae; United Cactaceae; analyses ArPaAu-C: the on on clustering and feeding red: Rico both Argentina Portulacaceae; Puerto from in and/or Brazil, specimens northern thaceae codes Ar-A: from Color yellow: specimens BrPRUS-AP: same: cluster. green: the corresponding are the network SNPs joining to 1,707 neighbor individual on each F based (C). of methods and ability data (B) E mtDNA DAPC D, on and C, based (A) while sNMF 141 distribution, using range analyses complex. from native Clustering species the the 2. of with Figure distribution correspond range represent B non-native circles the and yellow with while A plants, correspond host plants. Amaranthaceae host represent circles Portulacaceae purple collected; were mealybugs iue1 oain fmayu olcinstsi ohtentv n o-aierange non-native and native the both in sites collection mealybug of Locations of 1. the to Figure equally contributed co-authors all CAPTIONS and FIGURE draft first the wrote E.H. revisions. M.B.A., D.P.M., funding. obtained yoeccu pungens Hypogeococcus arieFtetmtsfor estimates Fst Pairwise arieFtetmtsfrtegop endb lse analysis. for cluster scenarios by delimitation defined Species groups the for estimates Fst Pairwise step. filtering quality each after identified loci of of Number origin geographic and use plant Host ucmso igelcsseisdlmtto analyses. delimitation species locus single of Outcomes . yoeccu pungens Hypogeococcus .pnessnustricto sensu pungens H. https://orcid.org/0000-0003-0832-394X http://orcid.org/0000-0002-5086-7847 hlgntcteswr nerdb LadB ae n177SP A n mtDNA and (A) SNPs 1,707 on based BI and ML by inferred were trees Phylogenetic https://orcid.org/0000-0002-3773-8805 https://orcid.org/0000-0002-9537-6275 https://orcid.org/0000-0003-3657-8421 pce complex. species iulrpeetto ffiecutr K )aogwt ebrhpprob- membership with along 5) (K= clusters five of representation Visual ld ld rRSA,mayusfo otesenBai,Puerto Brazil, northeastern from mealybugs BrPRUS-AP, clade clade ; Hypogeococcus pungens pce ope pcmn n egbrjiignetwork joining neighbor and specimens complex species Hypogeococcus pungens Hypogeococcus pungens 18 re ice ersn atca otpat where plants host Cactaceae represent circles Green pce ope ouain ape nnative in sampled populations complex species pce complex. species pce ope samples. complex species yoeccu pungens Hypogeococcus Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. cactus-mealybug-pest-and-implications-for-biological-control diversification-by-host-plant-use-in-an-herbivorous-insect-the-source-of-puerto-rican- Table_2.docx file Hosted cactus-mealybug-pest-and-implications-for-biological-control diversification-by-host-plant-use-in-an-herbivorous-insect-the-source-of-puerto-rican- Table_1.docx file Hosted Hasson Esteban vial at available at available https://orcid.org/0000-0003-3326-9877 C E A https://authorea.com/users/318612/articles/448543-species-complex- https://authorea.com/users/318612/articles/448543-species-complex- F 19 F D B C D A E B Posted on Authorea 6 May 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158879241.12522183 — This a preprint and has not been peer reviewed. Data may be preliminary. B A A Ancestry proportions 1.0 0.8 0.6 0.0 0.4 0.2 3 3 100/1 Amaranthaceae ArPaAu*-C Br-A 0.08 1 1

Native range host BrPR-C Ar-A 100/1 Amaranthaceae/Portulacaceae 100/1 BrPRUS-AP host 4 4 Br-A Non-native rangeNon-native BRAba055 BRAes565 BRAes564 BRAes563 BRAes562 BRAes1561 ARAlr642 ARAlr641 ARAlr675 ARAlr673 ARAlr674 ARAlr672 ARAlr671 ARAse522 USA17511 USA17510 USA1759 USA1758 USA1757 USA1766 USA1765 USA1764 USA1763 USA1762 USA1761 BRAbap103 BRAbap102 BRAbap101 BRAba136 BRAba114 BRAbap712 BRAba073 BRAba072 BRAba071 PRAvi39 PRApm362 PRApm361 PRAhp6252 PRAhp6251 PRAh623 PRAvi622r PRAvi622 PRAvip618 PRAvip6172 PRAvip6171 PRAvip612 PRAvip611 PRAc6026 PRAc6023 PRAc6025 PRAc6022 PRAc6024 PRAc6021 PRAh053 PRAh052 PRAh051 PRAc044 PRAc043 PRAc042 PRAvi2b6 PRAvi2b4 PRAvi2b3 PRAvi2b2 PRAvi2a1 BRCes702 BRCrj515 BRCrj514 BRCrj517 BRCrj516 BRCrj512 BRCrj511 BRCbz607 BRCbz606 BRCbz605 BRCbz604 BRCbz693 BRCbz692 BRCes704 BRCes502 BRCes501 BRCes708 BRCes708b BRCes714 BRCes706 PRCg35 PRCcr37 PRCcr38 PRCcr302 PRCg33 PRCg32 PRCg31 PRCpp275 PRCpp274 PRCpp262 PRCPC176 PRCpp185 PRCpp184 PRCpp173 PRCpp172 PRCpp261 PRCpp171 PRCg16a6 PRCg16a5 PRCcr29 PRCg16a4 PRCg16a3 PRCg16a2 PRCg16a1 PRCcr126 PRCcr125 PRCcr124 PRCcr123 PRCcr121 PRCcm1b5 PRCcm1b4 PRCcm1b2 PRCcm1b3 PRCcm1b1 AUC1774 AUC1773 AUC1772 AUC1771 PAC1331 PAC641 ARCsl682 ARCsl1784 ARCsl681 ARCsl1783 ARCsl1782 ARCsl1781 ARCco1695 ARCco1694 ARCca1795 ARCca1794 ARCca1793 ARCca1792 ARCca1791 ARCsa566 ARCsa565 ARCsa563 ARCsa562 ARCsa561 BrPRUS-AP 2 2 Ar-A Br-A BrPR-C BrPRUS-AP ArPaAu-C Ar-A 5 5 20 BrPR-C Cactaceae 0.004 host B 99/1 C 81/0.7 Native range H4 ArPaAu*-C 156_3_Br_EsS_Am 156_1_Br_EsS_Am 705_Br_Bh_Am 701_Br_EsS_Am 700_Br_EsS-V_Am 7OV.4_AR_Cat_Ca 56.1_AR_Sal_Ca 17.10_AU_Que_Ca 40.12_AR_Crr_Ca 40.11_AR_Crr_Ca 133_2_PY_Ca 68.2_AR_SaLap_Ca 711_Br_Bh_Am Haplo_105_6_PR_Huc_Po_USA_A 43.6_AR_SJu_Am 30A.1_AR_Cba_Am 30A.10_AR_Cba_Am 27A.3_AR_SEs_Am 66.1_AR_LRi_Am Haplo707-2_Br_Bh-B_Am_PR_A_ARA 24A.5_AR_Cat_Am 22A.1_AR_SEs_Am 43.10_AR_SJu_Am 22A.5_AR_SEs_Am 71.2_AR_SaLap_Am Haplo_151_1_Br_rj_Ca_PR_C 150_2_Br_EsS_Ca C H2 ArBrPRUS-AP BrPR-C Br-A ArPaAu-C Br-A BrPr-C ArPaAu-C Ar-A BrPRUS-AP H3 H1