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Kajtoch, L.; Kubisz, D.; Heise, W.; Mazur, M.A.; Babik, W. 2015. - herbivorous networks: molecular characterization of trophic ecology within a threatened steppic environment. Molecular Ecology, 24 (15). 4023-4038. 10.1111/mec.13278

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type Article Article :Original Accepted Date : 15-Jun-2015 Revised Date :12-Jun-2015 Received Date :10-Jan-2015 This article This protectedis by Allcopyright. rights reserved. 10.1111/mec.13278doi: lead to differences betweenversionthis and the Versionof Record. Please cite this article as through thebeen typesetting,copyediting, pagination andprocess,which proofreading may This article hasbeen accepted for publicationand undergone fullpeer review but has not

Chrysomelidae,DNACurculionidae, barcoding, words: Key Sciences, Sławkowska 17, 31-016, Krakow, , [email protected], +48fax. 12 3 1 Center for Biodiversity DepartmentStudies, of Biosystematics, Opole University, Opole, Instituteof Systematicsof Evolution and An Plant herbivorous – beetlenetworks: Molecularcharacterization trophicof Łukasz Kajtoch, Institute ofSystematics andEvolution ofAnimals Polish Academy of 2 Centre Ecologyfor and Hydrology, ECW, Deiniol Road, Bangor LL57 UK 2UW, 4 Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland Kajtoch Ł. Kajtoch ecology within a threatened steppic environment 1* , KubiszD., *corresponding author:*corresponding 1 , Heise W. , Heise 422-42-94 422-42-94 Poland Poland

imalsPolish Academyof Sciences, Krakow, host plant,host cpDNA, xerothermicgrasslands 2 , Mazur M.A. 3 , Babik W. , Babik 4 Accepted Article . belonging to seven genera feed on the same or related plants. Their withplants.Webetween host and interactions families) foundlinks 224 the beetles theand in differ food selection as a resultoftheir phylogeneticrelationsgenera (within betweenand steppic beetles plants, theirhost and we tested Illumina polyphagousfor taxa). To better understand theecological associationsbetween rbcL) twoand (trnL and sequencing strategies(Sangerfor mono/oligophagous and beetlesleaf (55species) andweevils (59)knownCentralusing from Europebarcodes two steppicEuropean grasslands. We determinedho ecological associations between herbivorous beetles and host plants from seriously threatened of examination dietary the of herbivor diversity ABSTRACT beetlePlant of networks environment steppic Running head: This article This protectedis by Allcopyright. rights reserved. immediately useful forpurposes. conservation basicexpand knowledge aboutthetrophic relations ofsteppic beetles andplants andare observations are generallyconcordant with host beetles. Finally, we found thatthe previous identifications ofhost plants based ondirect specieson particularfeeding plantsfor polyphagous taxa, butnot non-polyphagous for defenses. We foundsignificant correlations betweennumberstheof leafbeetle andweevil intrageneric competition duepossibly andto adaptation an different toplant chemical Beetles chemistry. genera four from plant’s preferences were probably inherited from comm DNA barcoding facilitates many evolutionary and ecological studies, including the feedondifferentplants, possiblyreducing the hypothesis that leaf beetles and es. Inthis paper, present we survey a of st the plants of steppic (65%)for majority onresulted and/or ancestors thehostfrom plant barcoding from gut. Our results Accepted Article DNA of otherDNA organisms present inside the bodi diversity (Farrell 1998),(Farrell diversity 135,000as of 360,000 beetle speciesphytophagous are 2005; (Gillot herbivorous beetles and flowering plants have (Ibanez Orthoptera2013) and et al. 2013; studiespioneering in this field were performed on Coleoptera (Jurado-Rivera associations between host plants andinsects (Matheson theseaddition, are methods time-consuming. power, as theyrarely allow forthe identification of host plants atthe species level.In 1998). Allof thesehave methodsserious limitations with regard totheir discriminatory morphological or chemicalapproaches (Dove& Mayes 1996; Dahle Johnson1982; & 2001) Nicolson or gu herbivores (Symondson Harper 2002; studies, suchas the examination ofthe dietar al. &CiceroMoritz 2004; Pons be assigned to the appropriate species relatively simply and quickly (Hebert INTRODUCTION This article This protectedis by Allcopyright. rights reserved. Pinzón-Navarro 1998; Novotny the directobservationof feeding al. 2009a,b; Taberlet 2009a,b; Taberlet In theIn lastyears, few significantbeen has progress inmade barcoding the of The development recent oftheconcept of et al. et al. 2002, 2006; Dyer et al. et al. 2010; Kitson 2012). This could evolutionary many facilitate and ecological 2012). Previousused methods the forstudy of herbivores included et al. 2006).Italso providesfor identifying opportunity an the et al. et al. t content(Otte &Joern 1976;Fry et al. 2013;Garcia-Robledo (Sandholm &Price 1962;Dieckmann 1980;Barone Avanesyan 2014). Interactions between 2005; Sheppard &Hardwood 2005;Sheppard Valentini 2005; 2007) and the analysis offeces(Holechek y diversity of predators, fungi-eaters, and and fungi-eaters, predators, of y diversity been postulated driversmajor asof beetle esthe of examined specimens(Valentini barcoding enables examinedspecimensto etal. 2008).The ofthemajority et al. et al. 2013;Kishimoto et al. 1998; Foley et al. et al. 1978) using 2003; 2009; et al. et al. et al. et et

Accepted Article forests (Jurado-Rivera (Jurado-Rivera forests 2002). Itis notsurprisingthat ofall these studi Warchałowski 1991; Mazur 2001). The primary aim of this study was to test hypotheses that basedon direct observations describe as usingbarcoding data, the accuracy ofinferen closelyto relatedinitialessential AnEurasianof the steppes. step ourstudy to was evaluate, grasslandsxerothermic fromw CentralEurope inpolyphagousproblematic taxa (seeKajtoch 2014). these studies alsoused single individuals thefor identificationof hostplants, which could be databasebarcode localfor which flora, often limitedidentification to the genusfamily or highlysuccess reducingthe barcode of amplification, or(ii) withoutthe developmentof a often hadlimitations as they wereperformed verybevaluable for purposes conservation in threatened environments. Previousstudies have ecological associations betweenherbivores and plants and herbivores,to expandour knowledge ofthe evolutionary interactions and inperformed areas other habitats, and particular tropical plants and insectsNovotny and (e.g., plants tropical haveplants beena target many other studieof Robledo et al. the twomost speciosegroups: weevils (Curculionoidea; >62,000 known species, Oberprieler Zhi-Quiang2013). Sofar, all hostplant barcoding onstudies beetles have been performed on This article This protectedis by Allcopyright. rights reserved. level (Jurado-Rivera 2007)and leafbeetles (Chrysomelidae; In this paper, we focused on plant and beetle assemblages of steppic habitats— steppic of assemblages beetle and plant on focused we paper, this In et al. 2013; Kishimoto et al. et al. 2009; Pinzón-Navarro 2009;Pinzón-Navarro et al. 2013), as interactions between tropical and d in (Szymczakowski the literature 1960; et al. ces about the feeding preferences of beetles (i) on beetle samples collected from traps, collected on traps, samples beetle from (i) around 35,000 knownspecies; Jolivet& Verma s, due tothe extremelyhigh diversity ofboth 2002; 2006;2007). Similar shouldstudies be es focused on species associated with tropical ith anextrazonal threatened plant community ly those that sustain diverse assemblages of of assemblages diverse sustain that ly those plants. The results of such studies could also et al. et et al. 2010; Kishimoto 2010;Kitson et al. et al. 2013; Garcia- 2013; 2013). Most of Accepted Article of relatively well-preserved steppichabitats with communitiesrich inplant and insect Festuco-Brometea database plant a barcode of and development the sites Sampling METHODS andthe managementof rare and threatenedsteppic habitats. results arediscussed in lightof theirreleva Schultz 1988; Becerra 1997; Hartley & Jone improve insects’ ability to deal with host plant chemical defenses (Ehrlich & Raven 1964; 2006; Agrawal 2007): co-evolution, competition for scenarios of(Jermyinsect-plant 1976; interactions 1984;Futuyma1996; &Mitter Janz relatedsame or host plants. We discuss these hypotheses in the context of macroevolutionary Intrageneric comparisons test the hypothesis that phylogenetically related species feed on the polyphagous beetles tend to favor similar plants (Bernays & Chapman 1994; Jolivet 1998). weevils andleaf beetles differentutilize host plants theyas consume plants selectively,but on feedingspecialization. Inwords, other ourhypothesis was that mono/oligophagous taxanon-polyphagous to thetest hypothesis that Farrell 1991; 1998).Farrell We addressed this these twoexophagous imago) (as of beetles groups differedin selection& (Mitterfood and intrageneric. The purpose of a comparison on the family-level was to examine whether compared the diet of beetles on two taxonomic levels: (weevils inter-family vs. leaf beetles) explain theecologicalcould associations be This article This protectedis by Allcopyright. rights reserved. The sampling was performedin thesteppic (xerothermic, calcareous) grasslandsof phytocoenoses locatedin CentralregionThis . sustains a network nce for theconservationof decliningpopulations tween herbivores and their host plants. Wehost plants. their tween herbivores and s 1997; Becerra & Venable 1999). our Finally, considering separately polyphagous taxa and differencesin foodselection are dependent food resources,food natural and selection to et al . Accepted Article 2006; Mazur & Kubisz 2013). species,including very diverse assemblages ofbeetles (e.g.,Mazur2001; 2002; W This article This protectedis by Allcopyright. rights reserved. Ceynowa1968; Warchałowski 1976;Mazur 2001; 2006; W in studied CentralEurope (e.g., Preuss 1912;Kuntze 1931; Szymczakowski1960; 1965; andsteppic beetles plants communitie their and even endangered (Binot endangered even highlevelof threat and conservationneeds—many steppicspecies are rare,threatened, or Zaj andassociated species-rich communities of plants andbeetles in Central Europe (Zaj Nazarenko 2009;Mazur Kubisz& 2013);ii) Itcont most species-rich in steppic habitats and were ob Apionidae,Anthribidae, )and and leaf beetles(Chrysomelidae).These are Beetle sampling species, constituting85% approximately steppic ofthe plantspecies Europe.Centralfrom includes trnLandsequences rbcL 128plant for speciesandsequences matK 115plant for basis ofsteppicsampling (xerothermic)plantPoland in (Heise fordirect, the accurate, and efficient identificationof hostplants. steppic plants Central from Europe has recently been developed (Heise 1960; Warchałowski 1991; Mazur 2001); and v) A multilocus database of barcodes for been havethem studied, butonly onbasis the ofdirectobservations (e.g., Szymczakowski et al ą . 2005);iv)is There an availability datathe of diet about ofsteppic beetles—some of c 2001; Mazur 2001;2002;c 2001;Mazur2005; Matuszkiewicz2013); Mazur&Kubisz iii)Ithas a This typeThis ofhabitat was chosen for severa The target selectedstudy this for was two groups beetles:of weevils (Curculionoidea: A databaseplant of barcodes rbcL, (trnL, andmatK)was developed in2014 onthe et al . 1998; Pawłowski s, as theys, have already been intensively et al jects of many previous studies, both classical l reasons:l have i)We good a knowledge of ains all major types of steppic grasslands . 2002;Holecová &Franc 2001;Farka ą sowska 2006; Chytrý 2007; et al. 2015). The database et al. 2015), allowing ą sowska ą c & c & č

Accepted Article coordinates of the center of southern Poland (in the uplands localized between the cities of Kraków and Kielce; development). Therefore, the majority (> 90% southernPoland(whereof plantmajority the species were collected for the databasebarcode species ondiversefeed plants differentfrom taxonomicgroups. plant species or closely related members of th are knowneither orassumed to feed on fewaspecies related a from single family,on a single 17% of all leaf beetlesincountry; the Borowiec & Mokrzycki 2005) and 85 species inhabiting this environment (approximately grasslands in (approximately11%of Poland 2012; Mazur well 2013) as phylogeographicas(e.g., Kajtoch zoogeographical and ecological (e.g., Mazur 2001; 2002; W This article This protectedis by Allcopyright. rights reserved. species,examined only 1–2 specimens couldbecollected and usedfor barcoding. Forseveral degradation. Samples were kept frozen until DNA isolation. Due to the rarity of most of the specimens were then immediately preserved inthe field in ethanol (96%)tominimize DNA and amplification is decreased in starving individuals (Kajtoch & Mazur 2015). The toconditions collecting avoid starvingspecimens, since the efficiency plant of isolation DNA several fieldinMay trips Juneand 2011–2014. Beetles wereonly collectedgood in weather in western ; see Data Accessibility). Beetles were collected in sweep-nets during (inMoravia Europe in the CzechRepublic, southernSlovakia,northern , andPodolia extinct inthisregion were collectedin neighboring theCentral of regions easternand Central could notbefound due totheir ins rarity We aimedWe to onlysamplespecies beetle known to the inhabitsteppic grasslandsof et al. 2014). There are around 114 known species associated with steppic this area are 50.374°N andbeetleSome 20.407°E). species which outhern Poland orbecause theirpopulations are all weevilsin thecountry; Mazur 2001;Wanat e same , whereas lessthan quartera of ) of thebeetle species were collected in et al. et al. 2009; 2012; 2013; 2014a; Kubisz 2011). The majority ofsteppic beetles ą sowska2006; Mazur &Kubisz et al.

Accepted Article following following primers: rbcL-F1 and rbcL-724R (Fay amplification of two chloroplast barcodes, thei.e., rbcLgene and the trnL intron, using the alsoused in further phylogenetic analyses (seebelow). Next, DNA isolates were used for the (Taberlet (Taberlet onstudies beetles (C1-J- TL2-N-3014;2183 andSimon amplifying the COI gene mitochondrial usingprimer the InNanodrop. of quality addition, DNA the isolates the from beetles was checked by samples. TheDNAquality concentration and AXkit(A&ASherlock Biotechnology)dedicated tothe isolationof DNAtraces low- from Laboratory procedures were analyzed(10-16) (details in Table1). least 10specimens, preferablyeach from a differentlocality, larger a number ofspecimens species, especially those known tobepolyphagous and which for we were tocollectable at This article This protectedis by Allcopyright. rights reserved. collected andimmediately specimens preserved byand usingtwo barcodes. samplessome (Kajtoch &Mazur 2015), butwe reduced theof risk byusingthis freshly 650–680 bpofrbcL the gene. This potentiallycould lead toan absencePCR of products for amplifying of longerselected parts barcodes, possible taxonomic level (preferably to the species level), we decided to use standard primers (see alsoLittle 2014). As theof purpose research this wastoidentify hostplants to thelowest do nothavepower sufficientallow rarely discriminatory identification and for species-level Hofreiter 2015). We didnotuse developed primers to am amplification and sequencingwere problematic for somesteppic plants(see Heise Whole beetles Whole were digested withproteinase K andDNA was isolated usinga

et al. et al. 1991, c primers andd). We didnot analyzethe matK barcodebecause its 2000 for rbcL 2000 Taberlet and

et al or approximately350–640 bpintronand oftrnL purity ofallisolates was assessedusing . 1991, 2007as for trnL), these shortmarkers plify short barcodes (minibarcodes; e.g., et al et s thathave frequentlybeen used in other . 1997), and A49325and B49863 et al. 1994). These sequences were et al.

Accepted Article equimolarly (separately for each species; all rbcL PCRs were pooled separately from trnL of both rbcL andwere trnL) checked onfirst agarosegelandthen pooled approximately specimens of each species were used (see Table 1 for details). All amplicons (small volumes unequal concentration of plant DNA inisolates from weevilbodies. Between 10 and16 were amplified separately for each individual to avoid problems and errors caused by an 1), Table see wasrepeatedprocedure under modified PCR cond CapillaryAutomated DNA Sequencer. In ofcasessequences, unreadable the sequencing KitSequencing (Applied Biosystems,Carlsbad, CA, USA)and run onan ABI 3100 Cycle v.3.1 Terminator BigDye a and primers forward using sequenced Sanger then were products were purified using the ExoProStar observed, bandswere extracted from the gel This article This protectedis by Allcopyright. rights reserved. oftrnLandmostlength of length the ofrbcLbarcodes. MiSeqa 2x paired-end 300bp run, which allowed sequencing for of theor full, almost full, step, i.e., adaptors were ligated to the amplicon ends. Thelibraries were sequencedas part of werelibraries prepared using NEBNext DNA li contamination.of None thesecontrols negativeresulted in PCRa product. barcoded The includedblank samples (with all reagents butwithoutDNA totest templates) for possible PCRs) and purified using the Nucleospin DNA Extraction Kit. Each batch of PCR reactions For seven speciesFor weevils of and eight speciesof leafbeetles polyphagous, (mostly PCRAll products werevisualized onan gel,agarose and if morethan one band was

another another method of host plant identification was used. Barcodes of rbcL and trnL usingNucleoSpin Gel andPCR Clean-up.PCR kit(GE Chemicals). DNA Purified products braryprep without theDNA fragmentation itions withitions theof use reverse primers.

Accepted Article 10 2015) usingMegablast (Altschul gutswere compared withthe available databasesof xerothermicplant barcodes (Heise model. Next,model. used we PhyML3.0 (Guindon 2002)PAUP* wereused towith (Swofford determine thesubstitution best-fitting nucleotide The Akaike (AIC)inMrModeltest Information 2.3Criterion (Nylanderin 2004) conjunction databasebarcode theand dataset was using aligned (KatohMAFFT & v.7 Standley 2013). intron. All rbcL sequences generated from the beetles were added to rbcL sequences from the gene as thisidentification could be andtrnLaligned,easily reliably contrary indel-rich to the the beetles andfrom the database. We selectedtherbcL barcode phylogeneticfor hostplant databaseof steppicplants, we constructedphylogenetic ausing tree sequences obtained from beetlethe twothe in context families ofpresentin thepreviously the species compiled 1983; Mitter identification Sanger from sequences wasbased onphylogenetic analysis & (Mitter Brooks vari genetic intraspecific and errors sequencing for allow to chosen was 99% least at of identity An PCR. during generated been have coverageof atleast 95% required was to exclude, for example,chimeric sequences thatmay to theofofarbitrarily maximize stringency the identification plant Query host species. in Sanger sequenced samples. First, Sanger sequences of both barcodes obtained from (rbcL),were used for further analysis. Twoapproaches were usedfor hostplant identification qualitygood sequences 350than longer(trnL, bp thanlonger mostly bp) 500 or600bp Sanger sequences plant Host identification Data analysis This article This protectedis by Allcopyright. rights reserved. -200 and>95% query coverageretainedwere Sanger sequences were checked visually using v.7.0.5.2BioEdit (HallOnly 1999). etal . 1991; Miller &Wenzel 1995). To visualize plants featured in the diets of etal. 1990). Onlyhits with atleast 99%identity, E-value< et al . These thresholds were set somewhat ation. An alternative approach for host plant .reconstruct 2010)toa Maximum

beetle et al et .

Accepted Article et al technologies (e.g., Soininen host plantperformed identification using plantbarcodes next-generationand sequencing alignment approach. Identification ofalignment approach.Identification plant the was performedby a comparison ofthe the middle ofreads theshould notsignificantly affect blast sensitivityit as uses a local amplicons shorter than 600 bp which occur in some plant species). Duplicated fragments in in duplications the middle ofthe sequence joined ifthe paired-endreads overlappedtrnL (for readsboth from amplicon ends did notoverlap.Thisprocedure have may resulted in rbcL andinmany plant species also trnLamplicons longer are 600bp,than so the 300 bp than300larger were bp used in analyses. wasnecessary further joining End-to-end because paired readswere Both species. beetle join sequencesIllumina colors.distinct withwerebeetles marked leaf and weevils, plants, from directly wastree visualized andedited with FigTre supportnode was assessedwith thebootstraptechnique using 1,000 pseudoreplicates. The phylogeneticLikelihood PhyML tree. waswith run model substitution appropriate an and This article This protectedis by Allcopyright. rights reserved. previous study (Heise was estimatedon the basisof divergence analyses made forall available steppicplants in a to 98% least identity at leastoneplant species performed with the maximum of 10 hits retained For each byperformed comparing sequencing reads withsequences in the database ofplant barcodes. readswith sequencesin thesequencing databasePlant ofplantwas identification barcodes. .read 2015). A was consideredto have a uniquematch ifonly singlea washit reported We usedWe the following approachto analyzethe Illuminasequences obtained 15from ≥ 300 bp read, an ungapped Megablast search with the cutoff E value of 10 et al . 2015). Moreover, 98% identity was used in other studies that et al. 2009;Valentini e v1.3.1e (Rambaut 2009).Sequences generated ed end-to-end andonly joined readsof length in the database were retained. This threshold thein retained. were database . . Only readswith the best hits showing at et al . 2009b; Hajibabaei et al . 2011; Heise -150 was Accepted Article interactions, we decidedway, ininteractions, tovisualize networks simplified these connecting a the information across barcodes and sequencing technologies). Due to a large number of such interactionsvisualizing all identifiedbetween the theirhost plantsand beetles (combining showingtrees relationships ofbeetle species were used then for the of preparation networks representatives of distant (in respecttoweevils andleaf beetles) beetle families.The COI Platycerus virescens conjunction with PAUP*. Phylogeneticconjunction withPAUP*. trees usingMaximum Likelihood the were approach nucleotidefitting substitution modelwas2.3 using determined in AIC MrModeltest in Beetle-host plantanalysis sequenced, and analyzed describedas above. pratensis, Rosa canina, Arenaria serpyllifolia, each family: sample. Amplicons ofboth barcodesobtained for the eightselected plant species(one from successfully testedon a polyphagousbeetle (seeHeise wereonlybeen identifiedtheofThis procedure species has basis on these reads. recently pairs [hsp]occurred high-scoring multiple for a given query, these were plantHost combined. or ifthe bitscore ofsecond-bestthe hit was nobe This article This protectedis by Allcopyright. rights reserved. communis EU037102), (Tenebrionidae; used out-groups as downloaded(sequences from GenBank): separatelyconstructed in 3.0 PhyML weevilsfor leaf and beetles. Five beetle specieswere COI sequences generated for beetles were aligned using MAFFT v.7, and the best- the and v.7, MAFFT using aligned were beetles for generated sequences COI alsoWe validatedmethodthisthe identification for ofknown plant species ina mixed (Elateridae;EF424474), Eryngium planum, Inula ensifolia, Onobrychis viciifolia, Adonis vernalis, Salvia (Lucanidae; AB609585). These species were randomly selected among elatus Arachnodes emmae (;JQ303296), and tter than0.95× the bitscore ofthebesthit; if Elymus repens et al (Scarabaeidae;and GQ342139), . 2015). Nyctoporis carinata ) werepooled,Illumina Melanotus Melanotus

Accepted Article one identifiedone host plant. Tocheck the correlation between thenumberof beetle species particular plantspecies. This analysiswasperformed onall beetle speciesthat had at least [monophagous and oligophagous], and simultaneously for leaf beetles) feeding on a number of beetle species (separately for weevils polypagous and mono/oligophagous the onbasis of barcodespecies identification. used matrix The wasthen to calculate the preferencesof beetles.Steppic plant specieswere assignedas host plants for certain beetle barcodes andsequencing technologies, were used for generala analysis feedingof the between current andolder knowledge about beetle feeding preferences. 1992, 1995, 1997, andother workscited above)was analyzed to evaluate thecongruence from the (basedliterature on information collected from Burakowski based on plant DNA barcoding using a comparison of data from the barcodes to information presented in tables).additional Each species beetle for which hosta plantwas identified beetle species hostto plants their at the family level (details about the host plant species are This article This protectedis by Allcopyright. rights reserved. weevils,and (iv) polyphagous mono/oligophagous and leaf beetles. weevilsmono/oligophagous andleaf beetles,(iii) polyphagous andmono/oligophagous & (BC) (Bray 1957)Curtis polyphagous betweenweevils (i) leafand beetles, (ii) Finally, using EstimateS (Colwell2013), we calc variance (ANOVA).All statistical analyses were byconsumed the aboveof beetles fourgroups the areaof feedingon particularplant species.Differences in the of plantscomposition Analysis was implementedtovisualize relative clustering offour definedgroups of beetles in feeding on a particular plant species, we used Next, dataNext, about the host oftheplants steppic beetles, which were combined from Pearson’scorrelation (R).Additionally, Cluster were also tested with use of analysis of usingperformed Statistica 10.0 (Statsoft). ulatedthe Bray-Curtis dissimilarityindex et al . 1990a,b; 1991,. 1990a,b; Accepted Article Supplementary TableSupplementary 22details). andSupplementary File for Illumina generatedin sequences blastedagainst thereferencebarcode database (see Figure1). Alleight speciesplants of preselected for the validationprocedure were identified 62%identification for beetle of species leaf weevil65%and of species (see Supplementary details).the phylogenetic Similarly, approach based onsequences rbcL allowed hostfor plant identification of the host(seeSupplementary plants1 Table Supplementaryand 1 File for of thebeetles of (66% leaf beetlesand67% ofweevils), Sanger sequencingallowed thefor General diet characterizations of steppic beetle species local populations (Kajtoch conservation status, as the collection of even si of oftheir some speciesregardless omitted fromthese ourstudy, We intentionally localities). another in western Ukraine; weevil the (e.g., that couldnot be collected included extremelyrare beetlesoften restricted single to localities species Europe; Centralfrom Mazur 2001; MokrzyckiWanat2005; & see 1). Table Species Borowiec Europe; leaf beetles (i.e., 65%of species associated with steppic grasslandsinPoland and Central Sampling efficiency RESULTS This article This protectedis by Allcopyright. rights reserved. PCR failure rates were 14.9% forleaf beetles,and 15.7% for weevils.For the majority Despiterarity the of manysteppic beetle species, we managed to collect55 species of nidensis, et al . Schmitt2011; & Rönn 2011)and 59species ofweevils (52% of et al Timarcha rugulosa . 2014b). known only in one steppic patch in southern Poland and ngle individuals becould detrimental for their , a very speciesknown rare onlyfrom a few Accepted Article genera (see Supplementary Table 2). exception of polyphagousby observations direct were confir the particular beetle (Supplementary Table 1). the same plantgenus ortoone of the species belongingplantto the hostsfamily known as for speciesbeetles (91%of leaf andof 90% weevils) were foundon tofeed plants belonging to observations (Supplementary Table 1). Similarly, approachidentified the same hostplant thatwas previously on thebasis reported of direct ofmajority monophagous beetles (94%of leaf beetlesand 100% ofweevils), the barcoding Barcodingvs. direct the obse (15). bameuli Polydrusus inustus were: weevils: 6.7 ±0.64(5–10); leafbeetles: 7. weevils: 7.4 ± (SD) 1.02 (range 3–12); leaf of hostnumbersplants species per identified were byIllumina sequencing: basedon trnL, generated for the beetle species) are presented in Supplementary File 3. The following beetlesexamined andrelative frequencies ofidentified hostplants ingroups of sequences results of host plant identification (number of This article This protectedis by Allcopyright. rights reserved. Centricnemus leucogrammus For species whose host plants were identified unambiguously, including the vast The efficiency of Illuminasequencing onplant DNAisolated from beetles and the Cryptocephalus violaceus, (11), and (27), Eusomus ovulum Cryptocephalus pygmaeus rvation ofrvation feeding beetles (16 hostplants), which iswhich apparently associatedwithonly two plant 8 ± 1.97 The most(3–18). polyphagous weevils beetles: 6.1 ± 1.83 (2–18);based onrbcL, hits to particular host plants host plants for particularhits identified to Moreover, almost allspecies classified as (10). The most polyphagous beetlesleaf were med towith feedonsingle the multiple hosts, anoverwhelming majorityof oligophagous Argoptochus quadrisignatus (18), and fornicata (12), Accepted Article (31 for leaf(31 for beetles 34 and weevils; for see Supplementary1,TableFigures 1 and 2). we exclude polyphagous taxa, the number ofinte interactions (117 for leaf beetles and 107 weevils;for see Figures 1 and 2). However, when were plants to host their befound complex.In total, weidentifiedbeetle-host plant 224 Differences diet in composition betweenbeetle families(weevils vs.leaf beetles) This article This protectedis by Allcopyright. rights reserved. viciifolia plant of6% plant beetles), of weevils and 3.4% of weevils and 3.4% of leaf beetles) scabiosa, Campanulaglomerata, Salvia pratensis, Plantago lanceolata, Asparagus officinalis cyparissias mostoften consumed by monophagous groupsthese two was 0.39. feeding onparticular plant species (R=0.704, P<0.001). TheBCdissimilarity index between between correlationsignificant the numberof (5.1% and8.5%), and Lamiaceae(both6.0%; see Figures 1 and2).There was a (14.5% of weevils and 23.9% of leaf beetles), (20.5% and 17.1%, respectively), 2details). Figure Atthefor level,family the most commonlyhost eaten were plants The polyphagous species most commonlythe ate following hostplants: Networks ofinteractions weevilsbetween andtheir hostplants and leaf beetles and A different pattern was observed whenpolyphagous specieswere omitted. The plants Salvia pratensis (the hostplant 6–7%oftheof studied beetles), (16.1%ofleaf beetles), leaf beetles), of leaf beetles), (6.5% of leaf beetles each), (2.6% of both weevils and leaf beetles), Lotus corniculatus , Cirsiumpannonicum, Linumflavum, Genistatinctoria, monogyna and and oligophagous and beetles were: Sarothamnus scoparius polyphagous beetleleaf and speciesweevil (4.3% of leaf beetles), ractions decreases substantially to only65 Medicago variaMedicago (3.4% of both weevils and leaf leaf and weevils both of (3.4% (3.4% of weevils; see vulgaris vulgaris Filipendula (8.9% of weevils), Prunus spinosa Euphorbia Onobrychis (the host (4.3% (4.3% (2.6% Accepted Article host plant), and food selection(ANOVA=24.78, P<0001; see Supplementaryalso 2). Figure (R=0.152, P=0.201; BC index=0.74). All defined groups of beetles differed significantly in (BC index=0.79). The samewasobserved for polyphagousmono/oligophagous and weevils correlation was found ( R=0.204, P=0.092), as both of these groups were highly dissimilar groupsthese two was 0.75. feeding onparticular plant species (r=0.107, P=0.380). TheBCdissimilarity index between significant correlationbetween numbers ofmono/oligophagousbeetle leaf andweevil species no1was There 2). of weevils;details inFigures and (8.8% Lamiaceae and beetles), and14.7%of weevils), Hypericaceae(16.1% of leafbeetles), Rosaceae (9.7% of leaf weevils were alsoobserved at thehostplant family Asteraceaelevel: (16.1% of leaf beetles Trifolium arvense lychnitis, Verbascum CoronillaLathyrus varia,tuberosus, Onobrychis viciifolia, This article This protectedis by Allcopyright. rights reserved. Asteraceae), Dianthus, e.g., related, beetle generathat feed ontwo ormore generaof thatplants often are phylogenetically plants: could be identified. The firstcontainsof members genera the that feedon thehost same Differences diet in composition amongcongeneric species (feeding mostly on mostly(feeding Fabaceae).Genera belonging thirdto thegroup include beetles on feeding Crioceris When analyzingWhen the feeding of preferences congenericbeetle species,three groups Moreover, when comparing polyphagous and mono/oligophagous leaf beetles, no both fromCaryophyllaceae), Tychius Apthona Pseudoprotapion Pseudoprotapion (feeding exclusively on (5.9% of weevils seeeach; Figure2). Differences leaf between beetles and (feeding on (feeding mostly on Trifolium (monophages of Asparagus Euphorbia or Melilotus Melilotus ( ,Centaurea, and Onobrychis ), , butsome on Cleopomiarus – Fabaceae), ). The second groupincludes (with the the (with Sibinia ), and ( Silene Campanula and —all from from —all and Accepted Article also found some discrepancies in hostpl 1991, 1992, 1995, 1997), correctly identifiedhost toplants thesespecies. beetle However, we CentralEurope. Comprehensive analysesusing two DNA barcodes and twosequencing Herepresent we the first analysis of ecological associationsbetween herbivorous beetles andtheir hostplants steppic from grasslands, a highly threatenedenvironmentin DISCUSSION considering genera represented by at least two species in our study). observedpoliphagywas in 30%of genera(4/12of leafbeetles and 9/12 of weevils, only , Larinus, Miarus, Pseudoprotapion, ( monophagous/oligophagous Sitona, monophagous species (e.g., (generally polyphagous), different, unrelatedplants: This article This protectedis by Allcopyright. rights reserved. beetlesthe in field (Freude This finding conf preferences. barcoding generallyDNA withpreviously agreed publishedinformation about their feeding Accuracy and reliability of directobservations of feeding ecologicalguild of beetles. technologies have significantlyexpanded knowledge about feeding preferences thisfor Some generaof steppicSome weevilsandleaf beetles containbothpolyphagous and For monophagousFor oligophagous most and species,host plant identification onbased and Trichosirocalus and Polydrusus and Cryptocephalus et al ). In other). In genera, allof the examinedsteppic species are either Cryptocephalus, Cassida,, Pachybrachis, Sibinia, Apthona, Dibolia, Gonioctena, Longitarsus, Hemitrichapion, irms that traditional studies, mostly observations direct of Tychiu . 1966;. 1981;Dieckmann 1980; Burakowski , and ). Overall, a transition between mono/oligophagy and s) or polyphagous ( Trichosirocalus. ant identificationbased onobservations and (with some polyphagous species), , ,

et al Polydrusus . 1990a,b, Accepted Article striatellus presumablysome polyphagous species arerather oligophagous (e.g.,weevil the sequencing ofbarcodes generated severalfrom randomly pickedindividuals showed that ecologically and showsimilar feeding habits (mainly leaf-eatersas imago). Data collected for Mazur&2011; Kubisz 2013). They are phylogeneticallydistant but closely linked steppic grasslands (Mazur2001;Wanat &Mokrzycki2005; W Does diet compositiondifferbetween weevils and leaf beetles? Supplementary 2). Table Fabaceae, leaf beetles weevils: (like families or only plants.a few species Other polyphagous, are butwith a dietrestricted tosomeplant beetle leaf unknown diets.Examplesthe are weevil Novel findings on diet preferences of steppic beetles include some Rosaceae (e.g., et al oligophagous andfeed exclusively onFabaceae ( Warchałowski1991). Previous on studies limited samples indicatethat this species is Anthyllis,Onobrychis, the leafbeetle isofOneinterestinghost ofthe multiple plantsfor the findings most barcodes. identification This article This protectedis by Allcopyright. rights reserved. . 2013; Heise Weevils halfthanand leafWeevils more beetles of allbeetleconstitute species with associated One interesting result was the identification of host plants resultplants species waswith theforOne interesting ofhost identification previously and leafbeetles Cryptocephalus violaceus, Cheilotoma musciformis, musciformis, Cheilotoma et al Gonioctena fornicata . 2015),.whereas current the study extendsthe listofhost its plants to Polydrus inustus and Rumex Prunus (Szymczakowski 1960;Gruev & Tomov 1984; and which feeds on – Rosaceae, which, based onobservations, should onlyon feed Omias globulus, Crataegus – Rosaceae and Fabaceae;see also Onobrychis, and ) and ) and Eusomus ovulum ovulum Eusomus Onobrychis Smaragdinaaffinis Hypericum. Hypericum. which feeds on

Lotus, ą sowska 2006; Borowiec and and Hypericum – Rosaceae and and – Rosaceae Oxytropis Elymus repens, ) and two onfeed Sitona . Illumina ; Kajtoch et al and . Accepted Article of either weevilsor leaf beetles.This suggests somedietary niche displacementbetween both of whereas groupsplants for other beetles, and leaf beetles. Only members of Asteraceae analyzed, significant differences in host plant composition were detected between weevils betweenthe links hostplants beetles.and When only mono/oligophagous beetles were specifically adapted. Polyphagouswerespecies responsible for of two-thirds approximately who than are adapted probably toare specialists, that less generalists repellents implies which beetles is thatall of these species feed onplants whichhave less effective chemicaldefenses, Another explanationthisdifference for lack of species could simply follow theabundance and constancyof plants in the environment. steppic beetles feeding(Bernaysare generalists &1997). MinkenbergThe polyphagous beetles feed on nearly the same plantspecies. polyphagousand mono/oligophagous species. Inpolyphagous species, weevils leaf and comprehensively compare these groups. two Two patternsopposite are observed for dozens of steppic species from both families gave us a unique opportunity to This article This protectedis by Allcopyright. rights reserved. &SegarraPetitpierre beetles (e.g., in particularly observed This has been plants. host their Bothprocesses not are exclusive andhave oftento led betweenco-evolution herbivores and species,leaf beetles and weevils could be adaptedto feedon plants with different repellents. Jones& 1997;Aniszewski 2007). However, due tothe different physiologyof particular avoidancesome of host plants possessing efficient chemical defenses1988; (Schultz Hartley et al ofonebe theof major forces beetle speciationand insectspeciation ingeneral (Thorsteinson accelerated when seedplant radiation began,host as plantselection is currently consideredto resulted from availablefor competition foodresourcestheduringevolutionof both groups.It groupsthese two of beetles caused byhost plantspecificity. Such specificitycould have . 1960; Ward et al . 2003;Grimaldi & Engel 2005). Anotherprobable explanationis the and and similarlyLamiaceae are important host as This simplyresult confirms that polyphagous in thediets ofpolyphagous and weevils leaf plant families were more frequent in the diet the in were morefrequent plant families Accepted Article these genera apparently maintainedgeneral feeding preferences fromcommon ancestors, as the same hostplants (e.g., assemblages. On the otherhand,only some beetle genera were tofound feed exclusivelyon these However, 1996). transitional eventscould pre-date the offormation steppic happened; similar shifts were reported for some other beetles (e.g., ofthird thestudied beetle genera, someshiftbetween monophagy, oligophagy, andpoliphagy present inplants.Moreover, wefound inthat plants),nutritious or again, couldberesult a of todifferent adaptation insectrepellents which also re could plants, host multiple genera,preferences the feeding of particularspecies probably evolved as a way to feed on all orsomeof these beetles are polyphagous (e.g., different chemical defenses. In some genera, species feed on different, unrelated plants and This can also be explained as either food competition avoidance or adaptation to hosts with should be observed mainly between closely related species (Petitpierre &Segarra1985; Intrageneric competitionfor food-plant resources 2011). Trichosirocalus, Metcalf 1986; Anderson 1993; Farrell 1998; Oberprieler genera feedgenera on different usually from otherplants, genera or (e.g., families thatfind theof diets related speciesare substa onlyHowever, insome genera thatwere represented bymultiple species in the studydidwe 1985; Metcalf 1986; Anderson 1993; Farrell 1998; Oberprieler This article This protectedis by Allcopyright. rights reserved. This dietaryThis displacement couldbeassociated withbeetle phylogeny, so,and, if it andto a lesser extent also Crioceris , Cleopomiarus, duce congeneric more competition(e.g., for Apthona the the evolutionary history of approximately one- ntially different. Species belonging to the sameto the belonging ntially Species different. Cryptocephalus Cryptocephalus , and Larinus, Sibinia Pseudoprotapion et al . 2007;Lawrence et al and Oreina . 2007; Lawrence , Sitona, Sitona, Polydrusus ). Species from from Species ). , Dobler Cassida and et al Tychius ). In these these In ). et al and . 2011). et al et

. ,). . Accepted Article the trophic relationsthe trophic between steppicbeetles theirand host plants. caused bythese technical constraints.In ouropinion, thepresented results adequately reflect 2009; Pinzón-Navarro were also database re barcode the reference polyphagousspecies. PCR sequencing failure, problems errors, or with species assignmentto groupinglevels such as the analysesof weevils vs. leaf beetles and mono/oligophagous vs. oligophagous species,butthis shouldnot affectmo sampling couldhaveresulted in the underestimation ofhost plantdiversity inthe dietof species and some technical constrains (see Supplementary File 4 for details). Limited of hostplantbarcoding Limitations Ophraella, was shown for some othergenera beetle (e.g., This article This protectedis by Allcopyright. rights reserved. semiquantitative atbest. amplification, most likely considerableintroduce species in the rates ofdigestion, the efficiency of extraction,DNA and the process ofPCR to species the dietofbeetle particular species considered asi.e., quantitative, to actualcorresponding thecontributionof plantvarious It is alsotoIt important thatemphasize da We are aware of the limitations of this study, are for sampling includingof somelimited aware ofthis We the limitations Futuyma Futuyma et al. et al. 1995; 2010;Kishimoto Anthonomus grandis portedsimilar studies in (see Jurado-Rivera . Multiple factors, such asvariation plantamong . Multiple factors, et al. Phyllobrotica, ta in Supplementary Tableinta Supplementary 2should notbe bias, andthus the data can be regardedas 2013), andwe tried to minimize biases st ofthe results, especiallyon those higher speciesgroup, Jones 2001). Farrell & Mitter 1990; & Farrell et al.

Accepted Article sites). However, effectivethe protection ofsteppic patchesshould notonlyinclude mainly protected as localities that are important under localor international (e.g.,European Union) laws. Consequently, steppic grasslands are of steppicecology invertebrates.Despite their knowledge about local flora and needed; isfauna little however, relativelyknown is about the conservationof populations steppic and management of steppic habitats, an extensive populationsboth steppic of plants and animals are highlythe threatened.effective For condition (Eriksson the gene betweenflow populations is(seeKajtoch limited presently highlyfragmented. Patches ofthisha the Apart from ecological implicationsof th beetlesfor couldbe crucial from a point ofconservation view. grasslands Steppic are implications Conservation This article This protectedis by Allcopyright. rights reserved. grasslands mostly asor herbs fruits). Knowle food domesticfor preferred mammals andalso are utilizedby humans (collected in Lamiaceae, andHypericaceae, which are most frequently eatenby beetles, arethe most which depend on single or several host plants. Some plants from Fabaceae, Rosaceae, their tosustain populations certainplants. This concerns mainly mono/oligophagous species, the inhabiting area, including beetles, herbivorous it ascould be crucial forsurvival the of actions in steppicgrasslands needs torooted be in basic knowledge about thespecies issue other for habitatsspecies; and seeplanning CardosoThe 2012). of anyconservation the beetles examined in thisstudy), but also for allother steppic organisms (alsoa common prioritiesconservation for these “flagship” plants Only some steppic patches in protected areas (usually very small) remain in good et al. 2002; Janišová et al. dge host theabout plants ofbeetles and other 2011; Wesche rarity, steppicbeetlerarity, species are notprotected bitat are usually isolated from oneanother and for other taxa, such as orchids (Natura 2000 (which are notfound tobe hosts for anyof is study, the identification of host plants theis study, of host identification et al. et al. 2014a). 2012). Consequently, Accepted Article performed). performed). study is under protection in Poland and other central European countries where sampling was andNaturalfrom Sampling PopulationsExperimentation beetle (none of species usedin the commentson the previous version ofthis article. Thisstudycomplieswith Laws onAnimal sequencing. We thank Brent Emerson and four anonymousreviewerstheir for helpful We would like to thank K. Dudek for perf -2011/01/B/NZ8/01491, Investigator Principal Ł.). Kajtoch study was This by funded the grant ofNational Science (UMO- Centre, Poland Acknowledgements translocatedare plants alongthe beetles. with host the preferred plants confirmedare in the patchesused beetlefor settlement or these orpreserve restore or at-risk locallyextinct steppic species couldbe evenmore important This article This protectedis by Allcopyright. rights reserved. SocietyEntomological of Canada of HostEvolution Plant Associations Anderson RS (1993)Weevils andPhylogenetic Plants: versus Ecological Mediationof tool. Altschul SF, Gish W, MillerMyers W, Li EW, Evolution Agrawal AA (2007) Macroevolution of plant defense strategies. References Authors declare no conflictsof interests. Journal ofBiology, Molecular , 22 , 103–9 , 165

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47

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Accepted Article 10.1093/ee/nvv019 Conservation endangered leaf-beetle Kajtoch Ł,Korotyaev B, Lachowska-Cierlik(2012)D Genetic distinctness of ofbarcodesutility andsequencing techniques. Kajtoch Ł(2014)A DNA metabarcoding studyof apolyphagous beetledietary diversity: the plant DNAbarcoding for polyphagousbeetles. MazurMA Ł,Kajtoch (2015) Theimpact ofenvironmentalconditions on host efficiency of Europe. Polydrusus inustus Lachowska-CierlikKajtoch Ł, D, MazurM (2009) Kajtoch Ł, KubiszD, Lac Science ofEuropean parthenogenetic forms endangered habitat. and limited connectivity in xerothermic beetles: Implications for the conservation of an MazurM,Kajtoch Ł, KubiszD, Mazur MA,Babik effective(2014b) Low W population sizes This article This protectedis by Allcopyright. rights reserved. :(Curculionidae: Cratopus). Molecular characterization oftrophic ecologywithin JJN,Kitson Warren BH, VincentFlorens FB,Baider C, D, Strasberg Emerson BC (2013) ofLibrary Science ONE (2013) M Kishimoto YK, Kamiya MelengP,K, Diway B, H,Kaliang ChongL, ItiokaT, Sakai S, Ito inperformanceimprovements andusability. K, StandleyDMKatoh(2013) sequencemultiple MAFFT alignment software version 7: , EuropeanJournal ofEntomology, 19

Wide Host RangesHerbivorous of Bee , 183–194. , 17 , 67–77. and Animal Conservation

Cheilotoma musciformis Centricnemus leucogrammus , 8(9) howska-Cierlik D,Mazur MA ( , e74426. e74426. ,

Polydrusus Molecular Ecology , 5 106 , 454–466. Molecular Biology and Evolution Folia Biologica (Krakow) , 325–334.

Environmental Entomology. populations in Poland. weevils ofthesubgenus tles? tles? Insights from DNA Bar Coding. Genetic diversity of xerothermic weevils an island radiation of insect herbivores (Coleoptera: Curculionidae) in central 2013) Conservation2013) genetics of , 22 ,

5441–5455. Journal ofInsect Journal

Scythodrusus. Insect , 62 DOI: , 223–234. , 30 , 772–780. Public Accepted Article und Ökologieder Tiere, Berlin Fauna in Podolien, Brandenburg, Österreich und der Schweiz. Kuntze R (1931) Vergleichende Beobachtungen und Betrachtungen über die xerotherme Chrysomelidae). Little DPLittle (2014) Zoologici Phylogenyof thebased Coleoptera on morphological charactersof adults and larvae. JF, Lawrence isolated populations of xerothermic Kubisz KajtochD, Ł, MazurMA,A, LisHolecová M (2012)Conservation ofgenetics highly This article This protectedis by Allcopyright. rights reserved. populations inpopulations central-east Europe. distinctiveness ofsteppic MA,Mazur KubiszD, Kajtoch Ł(2014) Restricted geographic distribution andlow genetic of Animals PolishEvolution Academy ofSciences, Krakow,Poland (in polish). (Coleoptera) inthe Vistula Rivervalley].Faunistic Monographs (Coleoptera) w dolinie Wisły M,Mazur Kubisz(2013) D polish). Publisher, plant Poland]. in (in communities Scientific of Poland Warsaw, identification Matuszkiewicz W(2005) Organisms& Diversity Evolution (2008) A YSchlein PCR method detectionfor of plantmealsfromguts of the insects. GC,K, JunnilaGreenblatt A,CD, Vernon A,C, MA, Muller Miller Matheson Hausmann 437–446. ,

61 , 1–217. Ś lipi

A DNA mini-barcode for land plants. Molecular Ecology Resources, Resources, Ecology Molecular plants. land for mini-barcode A DNA Invertebrate Biology ń ski A,SeagoThayerAE, MK, AF, Newton AE Marvaldi (2011) Crioceris quinquepunctata Przewodnik do oznaczania zbiorowisk ro Rozmieszczenie imigracje kserotermicznychchrz [Distribution and beetles migration ofthexerothermic[Distribution , 21 , , 629–690. 7 Entomologica Crioceris , 294–303. , 131 , 333–344.

quatuordecimpunctata Fennica, (Coleoptera: Chrysomelidae) 25 , 103–111. Zeitschrift für Morphologie Zeitschrift für , Institute of Systematics Institute ofSystematics and ś linnych Polski (Coleoptera: ą szczy szczy [Manual to [Manual Annales 14 , Accepted Article Entomology Miller JS, Wenzel JW (1995) Ecol Chrysomelidae) to cucurbitacins. Metcalf RL (1986) Coevolutionary adaptati G ( Curculionidae, Rhynchitidae) ofsel MAMazur (2006) 213–244. , Apionidae, Curculionidae) in western Ukraine. M (2002)Thedistribution Mazur and ecology PolishAcademyAnimals ofSciences, Krakow, (in Poland polish). Zoogeographic studies]. Faunistic Monographs Poland (Curculionoidea: , Attelabidae, Apionidae, Curculionidae). Apionidae,Attelabidae, Curculionidae). Studium zoogeograficzne M Mazur (2001) This article This protectedis by Allcopyright. rights reserved. Biology DNA C (2004) Cicero C, Moritz diversity. into the genesis of insights Mitter C, FarrellB, Futuyma DJ (1991)Phylogen Bemays E ( Mitter C, FarrellB (1991)Macroevolutionaryas S. ( S. Mitter C,DR Brooks(1983 eds eds )Biodiversity of quarries 145–163. pits: and Opole Scientific Society, Opole,Poland. .), , 2(10) Coevolution ed , 40 ), ), , e354. , e354. , 389–415. Insect-Plant Interactions Insect-Plant Ryjkowce kserotermiczne Polski (Curculionoidea:Nemonychidae, Weevils (Coleoptera:Curculionoidea: Anthribidae,Apionidae, . Sinaner Associates,. Sunderland, UK. ) Phylogenetic aspects of coevolution Barcoding: Promise and Pitfalls. Pitfalls. Barcoding: Promise and Journal of Chemical Ecology ogical characters andphylogeny. ected ected excavations ofOpole Silesia Trends in Ecology andEvolution , v. 3. CRC Press, Boca Raton, USA. ons ofrootwormbeetles(Coleoptera: of weevils (Coleoptera: Nemonychidae, , Institute of Systematics and Evolution of pects of insect-plant relationships. [In:] etic studiesetic ofinsect-plant interactions: Acta Zoologica Cracoviensia , 12 . [In:] Futuyma. [In:] DJ,Slatkin [Xerothermic weevils of Public Library of Science , 1109–1124. Annual Review of . [In:] Nowak A, Hebda A, Hebda Nowak . [In:] , 6 , 290–293. , 45 , Accepted Article Novotny V, Drozd P, Miller SE,Kulfan M, Janda BassetM, Y specificity ofherbivorousinsects intropical a forest. LLBremer GD, B, Miller Cizek Y, SE, V,Weiblen Basset Novotny ukrainian). Pawłowski J,KubiszPawłowski D, M (2002) Mazur 126. many species of herbivorous insects in tropical rainforests? specialized diets. Otte D,Joern(1976) On A patternsfeeding in desert grasshoppersand the evolutionof Zootaxa RG,Oberprieler Marvaldi AE, Anderson RS Biology Centre, Uppsala University,Sweden. JAANylander (2004) forests. tropical Novotny V, Miller SE, Basset Y „Mykhajlivska tsilyna” and adjacent territories. Nazarenko VYu(2009)of Weevils theof branchthe Ukrainian Steppe Natural Reserve This article This protectedis by Allcopyright. rights reserved. ofhostprofiling herbivore interactions in tropical forests. JA, SP,Jurado-Rivera Pinzón-Navarro Gomez-Zu 403-426. (Coleoptera), particularly that of and Palearctic Alticinae. Segarra E, C (1985)Chromosomal variability Petitpierre and Chrysomelidaeinevolution Kraków, Poland. threatenedanimals in Poland , 1668 , 491–520. Nature Proceedings of theAcademyof SciencesNatural ofPhiladelphia MrModeltestv2. Programdistributed bythe author , 448 , 692–696. . Polish Academyof. ofNature Institute Conservation, Sciences, et al. (2007)diversityLow beta herbivorous of insectsin Coleoptera (2007) Weevils, weevils, weevils everywhere. Vestnik Zoologii rita J, rita Lyal CHC,Vogler APDNA (2010) . [In:] . [In:] Głowaci Nature Ecological Entomol Science , 416 et al. et , , 841–844. 22(supl.) , ń (2006)Whyare there so 313 et al. ski Z ( , 1115–1118. (2002)Low host . Evolutionary Entomography , 36–50. (in ed o .) gy Red list of of list Red ,

, 35 128 , 18–32. , 89– , 3 , Accepted Article Systematic Biology Sequence-based species delimitation for the DNA of undescribed insects. through predator-prey food-webs. SK, HarwoodAdvancesSheppard JD (2005) in molecular ecology:trackingtrophic links inCentral(Chrysomelidae) Europe. M,SchmittTypes(2011): RönnTh distribution ofgeographical ofleafbeetles Minnesota mosquitoes. HA,Sandholm RD Price observations (1962)Field http://treebioedacuk/software/figtree/. Accessed 2012 April 20. Rambaut A (2009) Naturdenkmalpflege HPreuss (1912)Die pontischenPfla J,Pons TG, Barraclough J,Gomez-Zurita CardosoA,Duran DP, Hazell S This article This protectedis by Allcopyright. rights reserved. Ecology Symondson(2002) WOC Molecular identification ofpreyinpredatordiets. Sinauer Associates, Sunderland, Massachusetts, USA. DLSwofford (2002) E Valentini EM, Coissac A, Soininen polymerase chain reaction primers. andphylogenetic utility ofmitochondrialgene sequence and compilationconserved of Ch,FratiSimon F, Bechenbach A, Crespi B, LiuH, FlockP(1994)weighting,Evolution, the composition ofthe composition complex plantmixtures. efficiency ofDNA barcoding coupledwith central. is 1988Schultz J.C. Manyinfluence factors the evol , 11 Ecology , 627–641 , , 69 FigTree v1.3.1:Tree Figure Drawing Tool 55 , PAUP*. Phylogenetic Analysis UsingParsimonyPAUP*.Analysis (*andOther Phylogenetic Methods) , 896–897. 2 , 595–609. , 350–517. Mosquito News, Mosquito Functional Ecology Annals Entomological Society America ZooKeys nzenbestandeWeichselgebiet. im et al.

22 (2009) Analysing (2009) diet ofsmall herbivores: The , 846–849. high-throughput pyrosequencing deciphering for Frontiers in Zoology , 157 ution ofherbivore diets,plantution but chemistry , 131–158. on the nectar feedinghabitsof some , 19 , 751–762. . Available from , 6 , 16. Beitrage zu

et al. et , Molecular 87 , 651–701. (2006) . Accepted Article Ecology &Ecology Evolution A,Valentini Pompanon Taberlet F, P (2009b)DNA Barcoding for ecologists. Resources, barcoding DNA andparallel pyrosequencing: the trnLapproach. 5 Thorsteinson AJ (1960) noncoding regionsof chloroplast DNA. L, P,GiellyTaberlet Pautou G, BouvetJ(1991) primers for Universal of amplification three 2045–2050. plantDNAintron for (UAA) barcoding. Upland]. [MaterialeMałopolskiej toknowledge ofxerothermophilous faunabeatles of on Małopolska Szymczakowski W (1960) This article This protectedis by Allcopyright. rights reserved. Provinz (Coleoptera, Chrysomelidae). WarchałowskiA (1976) BiogeographischeStudi Curculionoidea). M,WanatT Mokrzycki (2005)Anewchecklistof theweevilsof Poland(Coleoptera: A,Valentini Miquel C, Nawaz MA generation biodiversity generation assessmentusing DNA metabarcoding. Brochmann P, E E,(2012)Coissac next-Taberlet F, C, Pompanon Towards Willerslev F E, Pompanon P, Coissac Taberlet SzymczakowskiMateriały do W (1965) poznania chrz communities in Poland]. in Poland]. communities kserotermicznych Polski [Materiale to knowledg , 193–218. Polish Journal Entomologyof

9 , 51–60. Genus , 24 Host selection in phytophagous insects. , , 110–117. 16 Polish JournalPolish of Entomology

Materiały do poznania kserotermofilnej fauny chrz : 69–117. et al. et al. Polish ofEntomology Journal , Plant Molecular Biology Nucleic Acids Research 30 (2009a) New perspectives indiet basedanalysis on (2007) Powerand limitations ofthechloroplast trnL , 173–242(in polish). en über die Blattkäfer der Pontischen e ofbeatles (Coleoptera)plant xerothermic ą , szczy (Coleoptera) siedlisk 35 , 225–257 (in polish). Molecular Ecology , Annual Review Entomology

, Molecular Ecology

35 17 , e14. , , 1105–1109 46 , 29–94. ą Trends in szczy Wy , 21 , ż yny yny , Accepted Article Linnean Society phytophagousinsects andmites reflect past evolution with plants. LK,Ward HackshawA, Clarke RT(2003) Do fo polish). ( Data Accessibility Zootaxa Outline ofHigher-level Classification and Zaj Chorology, Institute ofBotany, Institute Jagiellonian University,Chorology, Poland(in Krakow, polish). Plants AtlasofVascular Poland].in [Distribution plants European grassland Large vegetation: losses inspecies richness and animal-pollinated WescheK, Krause B, Culmsee H,Leuschner (2012) C years Fifty ofchange inCentral Bratislava,Biologia, grasslands ( W Warchałowski A (1991) This article This protectedis by Allcopyright. rights reserved. COI: KP306793-KP306891 trnL: KJ746323–KJ746436 KJ746209–KJ746322matK: rbcL: KJ746116–KJ746208 - raw DNAsequences: Genbank accessions: ZZhi-Quiang (2013) subfamilies: Clythrinae and ą ą sowska,(Chr M(2006)Chrysomelid communities c A,Zaj . Biological Conservation , 3703 Inuletum ensifoliae ą c M ( , 17–26. , 78 eds , 51–83.

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Kraków: Laboratory ofComputer ( ysomelidae, Coleoptera)ofxerothermic Insecta: Coleoptera ś lin naczyniowych w Polsce. naczyniowych lin Ed .) Animal An Biodiversity: Biological Journal of the ) . Part II Part .

Accepted Article Figure captions of themanuscript. analysis, Ł. Kajtoch and W. Babik wrote the paper, all authors contributed to the final version andŁ. Kajtoch collectedsamples, beetle Ł.Kajtoch and W. Babik performed molecular All authorsdesigned the research. W. Heise collected plantsamples, D. Kubisz, M.A. Mazur Author Contributions Dryad: http://dx.doi.org/10.5061/dryad.26h4v trnL files rbcLsequencessamp andIllumina and ofrbcLalignments sequences for phylogenetics and Likelihood Maximum rbcLfile; trees of- alignments COIsequences phylogenetics for COI andMaximum Likelihood trees files; ) http://onlinelibrary.wiley.com/doi/10.1111/boj.12261/suppinfo et al. 2015, Botanical Journal of Linnaean Society; -plant Final barcodeassemblies available as supporting information in separate article(Heise This article This protectedis by Allcopyright. rights reserved. by eaten of any marked beetlesarefamilies in available (Heise bracketsalongnamesin presented of all with IOxidase mitochondrial (onlygene bootstraps wi phylogenetic LikelihoodMaximum trees reconstructed from sequencesof the Cytochrome grasslands in Central Europe and theirhost plants. Weevils and leaf beetles are presentedin 1.Networks ofFigure interactions identified amongherbivorousbeetles steppic inhabiting for beetlesfor with Illumina datasequencing (mostly polyphagous taxa)in aremarked blue. sequencing data (mostly monophagous oroligophag et al., 2015) express the numbers of plan

steppic plant forfamilies which barcodes were frames. Interactions frames. Interactions fo ling(table locations and maps googlefile): thvalue a of >50% arepresented). Numbers ous taxa) are marked in red, interactions t species in the Plant families. r beetles Sanger r with Accepted Article beetles: Images ofweevils: generate a homologous sequence species.thisfor Labidostomis longimana This article This protectedis by Allcopyright. rights reserved. Genistatinctoria, 49- Lathyrus tuberosus, 50 -Lotus corniculatus, 51- Medicagofalcata,52 Astragalus45 arenarius,46 - vulneraria, danicus,47 - Astragalus - Coronilla - varia, 48 spinosa, 41 - Rosa canina, 42 - minor, 43 - Cuscuta epithymum, 44 - Anthyllis vulgaris, 37- viridis, 38-Potentilla39 alba, - argentea, 40- Prunus 33 - Linum hirsutum, 34 - eupatoria, 35 - Crataegus monogyna, 36 - Filipendula 29 -Euphorbiacyparissias, 30 -Berberis vulgar Silene nutans,26 - Rumex 27acetosella, - Si lychnitis, 22 - Plantago lanceolata, 23 - Linaria vulgaris, 24 - Dianthus carthusianorum, 25 - - Thymus19 pannonicus, -Thymus 20 pulegioides, -Clinopodium vulgare,21 -Verbascum 14 -Galium15 mollugo, Salvia pratensis,- - 16 Chrysanthemum corymbosum,12 -Campanula glomerata, 13- Vincetoxicum hirundinaria, pannonicum,- Hieracium 8 pilosella, 9- Inula 10ensifolia, - Picrishieracioides,- 11 Carlina onopordifolia, 5 - Centaurea stoebe, 6 - Centaurea scabiosa, 7 - Cirsium speciesfollows: as drawings). Colors(bottom particularto plantcorrespond numbersplant indicate families; examined beetles (top drawings) and for only species with single host plants identified Figure2.Host plant compositionsteppic of beetles (leafbeetles and weevils) showed for all University of Wroclaw, Poland)). Borowiec, Wrocław 2007-2014, Department of from ICONOGRAPHIA COLEOPTERORUM POLONIAE (© Copyright by Prof. Lech asclepiadeus 1- , 2 - Artemisi millefolium,21- Achillea Eusomus ovulum leaf beetles are presented outside the COIas it was notpossible to (top) and (top) and Gonioctena fornicate symbrium loeselii, 28 - Hypericum perforatum, 28-Hypericum perforatum, loeselii, symbrium Trichosirocalus troglodytes Biodiversity and Evolutionary Taxonomy, Stachys recta, 17- Teucriumchamaedrys, 18 is, 31- acris, Ranunculus - 32 Linumflavum, a campestris, 3 - Carlina acaulis, 4 - acaulis, - 3aCarlina campestris, (bottom) (photographs are (bottom) (photographs (bottom) (bottom) leafand This article This protectedis byAllcopyright. rights reserved. Accepted Articleavellana 67 -Koeleria macrantha, 68- Stipa Joannis, 69 - officinalis,Asparagus70 -Corylus cervaria,Peucedanum 64- Pimpinella saxifraga,65repens, -Elymus 66 -Festucarupicola, arvense, Trifolium 60- Viciatenuifolia, 61-Eryngiumplanum,62 -Seseli libanotis, 63- viciifolia, 56 - Ononis spinosa, 57 - Oxytropis pilosa, 58 - Sarothamnus scoparius, 59 - 53 lupulina,- Medicago -Medicago Melilotus5455 -officinalis, varia, - Onobrychis

Table 1. Efficiency of host plants amplification and sequencing for examined steppic beetle species (ordered according to systematics). Abbreviations: M – monophagous, O – oligophagous, P – poliphagous, U – unknown diet, number of specimens (no. sp.), PCR and sequencing success (√ - PCR & sequencing successful, X – unsuccessful, U - sequences unreadable, P - sequences of poor quality, L - lack of BLAST hits) and sequencing technique used for amplification of host plants DNA from the beetles (S - Sanger and I – Illumina)

no PCR & sequencing seq. no PCR & sequencing seq. species phagism species phagism sp. COI rbcL trnL tech. COI rbcL trnL tech. sp. CHRYSOMELOIDEA CURCULIONOIDEA CHRYSOMELIDAE ANTHRIBIDAE

Article Criocerinae Urodontinae Crioceris quatuordecimpunctata (Scopoli, 1763) M 2 √ √ √ S Bruchela rufipes (Olivier, 1790) M 1 √ L L S Crioceris quinquepunctata (Scopoli, 1763) M 2 √ √ √ S APIONIDAE Apioninae Cassida lineola Creutzer, 1799 M 1 √ √ √ S Hemitrichapion pavidum (Germar, 1817) M 2 √ √ √ S Cassida margaritacea Schaller, 1783 O 1 X X X Cyanapion platalea (Germar, 1817) M 1 √ √ √ S Cassida panzeri Weise, 1907 M 1 √ L L S Mesotrichapion punctirostre (Gyllenhal, 1839) M 2 √ √ √ S Cassida pannonica Suffrian, 1844 M 1 √ √ √ S Pseudoperapion brevirostre (Herbst, 1797) M 2 √ √ √ S Hypocassida subferruginea (Schrank, 1776) M 1 √ L L S Pseudoprotapion elegantulum (Germar, 1818) M 2 √ √ √ S Chrysomelinae Pseudoprotapion ergenense (Becker, 1864) M 2 √ √ √ S Chrysolina cerealis (Linnaeus, 1767) O 1 √ √ √ S Squamapion elongatum (Germar, 1817) M 2 √ P √ S Chrysolina sanguinolenta (Linnaeus, 1758) M 1 √ √ √ S Exapion elongatulum (Desbrochers, 1891) M 1 X X X Entomoscelis adonidis (Pallas, 1771) O 1 X X X CURCULIONIDAE Gonioctena fornicata (Brüggemann, 1783) P 10 √ √ √ I Entiminae Gonioctena olivacea (Forster, 1771) O 1 √ √ √ S Argoptochus quadrisignatus (Bach, 1856) P 12 √ √ √ I Phyllobius brevis Gyllenhal, 1834 P 12 √ √ √ I Galeruca pomonae (Scopoli, 1763) U 2 √ L L S Centricnemus leucogrammus (Germar, 1824) P 16 √ √ √ I Galeruca tanaceti (Linnaeus, 1758) U 1 √ U U S Cycloderes pilosulus (Herbst, 1795) P 1 X X X Calomicrus circumfusus (Marsham, 1802) O 2 √ √ √ S Eusomus ovulum Germar, 1824 P 16 √ √ √ I Luperus xanthopoda (Schrank, 1781) O 2 √ √ √ S Omias globulus (Boheman, 1843) U 1 √ √ √ S Alticinae Omias puberulus Boheman, 1834 O 1 √ √ √ S

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Accepted Podagrica fuscicornis (Linnaeus, 1767) M 1 X X X Otiorhynchus fullo (Schrank, 1781) P 2 √ U U S beckeri Jakobson, 1896 M 1 √ √ √ S Paophilus afflatus (Boheman, 1833) P 1 √ U U S Aphthona cyparissiae (Koch, 1803) M 2 X X X Parafourcartia squamulata (Herbst, 1795) P 16 √ √ √ I Aphthona czwalinai Weise, 1888 M 1 √ √ √ S Philopedon plagiatum (Schaller, 1783) O 1 √ √ √ S Aphthona euphorbiae (Schrank, 1781) M 1 √ √ √ S Polydrusus confluens Stephens, 1831 P 1 √ √ √ S Aphthona lacertosa Rosenhauer, 1847 M 2 √ √ √ S Polydrusus inustus Germar, 1824 P 16 √ √ √ I Aphthona ovata Foudras, 1861 M 1 √ √ √ S Sitona humeralis Stephens, 1831 M 1 √ √ √ S Aphthona pygmaea (Kutschera, 1861) M 2 √ √ √ S Sitona inops Schoenherr, 1832 M 2 √ P √ S

Article Aphthona venustula (Kutschera, 1861) M 2 √ √ √ S Sitona languidus Gyllenhal, 1834 M 2 √ √ √ S Dibolia cryptocephala (Koch, 1803) M 1 √ √ √ S Sitona lateralis Gyllenhal, 1834 O 2 √ √ √ S Dibolia schillingii (Letzner, 1847) M 2 √ √ √ S Sitona longulus Gyllenhal, 1834 M 1 √ √ √ S Longitarsus exsoletus (Linnaeus, 1758) O 1 √ P P S Sitona striatellus Gyllenhal, 1834 P 12 √ √ √ I Longitarsus quadriguttatus (Pontoppidan, 1763) O 1 X X X Sitona waterhousei Walton, 1846 O 2 √ √ √ S Longitarsus tabidus (Fabricius, 1775) M 1 √ √ √ S Strophosoma faber (Herbst, 1784) P 1 √ U U S Neocrepidodera ferruginea (Scopoli, 1763) O 1 √ √ √ S Lixinae Phyllotreta nodicornis (Marsham, 1802) M 1 √ L L S Larinus obtusus Gyllenhal, 1836 M 3 √ √ √ S Podagrica fuscicornis (Linnaeus, 1767) O 1 √ √ √ S (Fabricius, 1792) O 1 √ U U S Psylliodes cucullata (Illiger, 1807) M 1 √ P √ S Larinus sturnus (Schaller, 1783) O 2 √ √ √ S testaceum (Fabricius, 1775) O 1 √ √ √ Larinus turbinatus Gyllenhal, 1836 O 2 √ √ √ S Cryptocephalinae Hyperinae Cheilotoma musciformis (Goeze, 1777) O 12 √ √ √ I fuscocinerea (Marsham, 1802) O 2 √ √ √ S Coptocephala unifasciata (Scopoli, 1763) P 1 √ √ √ S Curculioninae Labidostomis humeralis (Schneider, 1792) M 1 X X X Cionus clairvillei Boheman, 1838 M 2 √ √ √ S Labidostomis longimana (Linnaeus, 1760) P 12 X √ √ I Mecinus pascuorum (Gyllenhal, 1813) M 1 √ √ √ S Lachnaia sexpunctata (Scopoli, 1763) O 1 X X X Rhinusa tetra (Fabricius, 1792) M 3 √ P √ S Smaragdina affinis (Illiger, 1794) P 10 √ √ √ I Cleopomiarus distinctus (Boheman, 1845) M 1 √ √ √ S Smaragdina aurita (Linnaeus, 1767) O 1 √ U √ S Cleopomiarus graminis (Gyllenhal, 1813) M 2 √ P √ S Cryptocephalus bameuli Duhaldeborde, 1999 P 16 √ U √ I Sibinia subelliptica (Desbrochers, 1873) M 2 √ √ √ S Cryptocephalus bilineatus (Linnaeus, 1767) P 2 √ √ √ S Sibinia tibialis (Gyllenhal, 1836) O 1 √ √ √ S

Cryptocephalus chrysopus Gmelin, 1790 O 2 √ √ √ S Sibinia vittata Germar, 1824 M 1

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Accepted Cryptocephalus flavipes Fabricius, 1781 P 2 √ √ √ S Tychius aureolus Kiesenwetter, 1851 O 2 √ X X Cryptocephalus fulvus (Goeze, 1777) U 2 √ L √ S Tychius crassirostris Kirsch, 1871 O 1 √ √ √ S Cryptocephalus pygmaeus Fabricius, 1792 P 10 √ √ √ I Tychius schneideri (Herbst, 1795) M 2 √ √ √ S Cryptocephalus quadriguttatus Richter, 1820 U 1 √ U U S Tychius sharpi Tournier, 1873 M 2 √ √ √ S Cryptocephalus violaceus Laicharting, 1781 U 14 √ √ √ I Tychius medicaginis Brisout, 1862 M 1 √ X X Cryptocephalus virens Suffrian, 1847 O 2 √ √ √ S Smicronyx jungermanniae (Reich, 1797) M 1 √ L L S Cryptocephalus vittatus Fabricius, 1775 O 2 √ √ √ S Pseudorchestes ermischi (Dieckmann, 1958) M 1 √ √ √ S Pachybrachis fimbriolatus (Suffrian, 1848) P 12 √ √ √ I

Article Pachybrachis hippophaes (Suffrian, 1848) O 1 √ L L S Mogulones geographicus (Goeze, 1777) M 1 X X X Pachybrachis tesselatus (Olivier, 1791) O 1 √ L L S Mogulones javetii (Gerhardt, 1867) M 1 X X X Phrydiuchus tau Warner, 1969 M 1 √ √ √ S Chrysochus asclepiadeus (Pallas, 1773) M 1 √ P √ S Stenocarus ruficornis (Stephens, 1831) M 1 √ L L S Thamiocolus signatus (Gyllenhal, 1837) M 1 √ √ √ S Trichosirocalus barnevillei (Grenier, 1866) O 1 √ √ √ S Trichosirocalus troglodytes (Fabricius, 1787) M 2 √ √ √ S Zacladus geranii (Paykull, 1800) M 1 √ L L S

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