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Gómez-Rodríguez, Carola, Timmermans, Martijn J. T. N. ORCID: https://orcid.org/0000-0002-5024-9053, Crampton-Platt, Alex and Vogler, Alfried P. (2017) Intraspeciﬁc genetic variation in complex assemblages from mitochondrial metagenomics: comparison with DNA barcodes. Methods in Ecology and Evolution, 8 (2) . pp. 248-256. ISSN 2041-210X [Article] (doi:10.1111/2041-210X.12667)

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Timmermans ía, UniversidaddeSa ía, , Universidad deSantia , and the Version of Record. Please citethis Please VersionofRecord. and the 1,4

1,5 , Alex Crampton-Platt ntiago deCompostela, ntiago go de Compostela,c/ go 1,3 , and Accepted Article Running title: variabilit Chrysomelidae, intraspecific Keywords: Mitochondrialme complex biodiversity samples tothe estimation of diversity the below species level. thea extend thus The findings approach. nucleotidecomparable variation data well-established tothe specimen-focused Sanger shotg 5. Overall,theassemblage-focused higher. withSanger sequenced correlated site. Thesevalues and tocalculateintraspecific also used reads were 4. Unassembledshotgun correlated were discrepancies greatest the This article isprotected Allrights bycopyright. reserved. synonymous 3 frombothdata barcodes between differences assemblies withlarge fromsamples intra and interspecific variability. Nucleotide in barcode sequenced matches ofaSanger assemblers, the Ignorinvariation. ofintraspecific presence mito 3. Generally, asingle only Illumina sequenced individualsand inthemitochondrial DNA variation (>2600individua 2. Using communities 10natural studies ofpopulationdiversity. mitogenome limitstheutilit assembly, which However, intraspecifi rapididentificati allowing sequences, genome field-collectedsamples mixed assembly of 1. Metagenomicshotgun Abstract Mitometagenomics: Intraspecific genetic variation rd cox1 codon position, although thenumberof codonposition, barcode regions from these as fromthese regions barcode c genetic variability present in present genetic variability c sequencing, using Illumina using sequencing, and technology, nucleotide diversity (pi) fora (pi) nucleotide diversity tagenomics, mitome chondrial contig was was chondrial contig shotgun sequenced specimen pools. specimenpools. sequenced shotgun cox1 y, population genetics, DNA barcodes y, genetics, DNA barcodes population pplication ofmitochondr un sequencing of pooled samples produced pooled samples of un sequencing -5’ ‘barcode’ was compared for Sanger forSanger compared was -5’ ‘barcode’ with poor quality of Sanger sequences. sequences. quality ofSanger withpoor 90.7% of cases, which dropped to 76.0%in which droppedto of cases, 90.7% g ambiguity from the use of two different two g fromtheuseof ambiguity of invertebrates read of invertebrates types were almost exclusively in in exclusively almost were types y ‘mitochondrial of to score single nucleotide polymorphisms polymorphisms nucleotide toscoresingle on and quantification of species diversity. quantificationofspecies on and tagenomics, genome skimming, ls) ofleafbeetle sembliesexact were nucleotide the specimenpoolsislostduring cox1 assembled per species, eveninthe assembled perspecies, affected sites was very low,and affected siteswas variation but were significantly butwere variation ll available populationsateach ll available

ily produce mitochondrial produce ily metagenomics’ for metagenomics’ ial metage s (Chrysomelidae), s (Chrysomelidae), de novo de nomics of genome Accepted that sequence may arecombinant represent ormorecombines closelyfrom two reads relate However, the nucleotidevariation at SNPsmay inconsistently be resolved the assembler if singleat sequence (approximately) th collaps 2013). Assemblerstypically & Pop (Nagarajan rate error sequencing the to beclose may divergence and whosesequence samples, whichcontainclosely posesachallenge This species. different from toseparate isrequired assembly software mitogenomes are reconstructed formultiple species (Crampton-Platt (Gómez-Rodríguez diversity phylogenetic provides informationontherela Article Ji also possiblewithPCR-based (e.g., metabarcoding Tang to amenable mitochondrial genome ispresentinnumerous skimming’ (Straub rapid identification ofthebiological entities ina complex present sample. MMGisa ‘genome Crampton-Platt spee greatly metagenomics, MMG)can reconstruction ofmitochondr of (HTS) sequencing High-throughput shotgun Introduction This article isprotected Allrights bycopyright. reserved. et al. The assembly frommixtures combines reads the shotgun suchseparate that 2014;Crampton-Platt de novo

etal. etal. genomic assembly at fairly 2015;Gómez-Rodríguez 2012) procedure, which takes advantage of the fact thatthe fact ofthe takesadvantage which 2012) procedure, ial genomes(mitochondria related species and haplotypes and species related tive abundance tive abundance of species and a etal. e the genetic variants present in a specimen mixture intoa mixture inaspecimen present genetic variants e the e level of species (Crampton-Platt ofspecies e level 2015).Thecharacterisation d up biodiversity inventories (Tang d upbiodiversity inventories multiple orthologous DNA fragments originatingfragments multipleorthologous DNA etal. does not correspond to a genuine haplotype genuine haplotype correspond toa does not copies per nuclear genome and thusis genome and pernuclear copies for metagenomic assembly etal. mixtures ofspecimens mixtures d haplotypes, and thus the ‘consensus’ contigand thusthe‘consensus’ d haplotypes, 2015) low sequencing depth (Zhou depth sequencing low 2015).Themethodologyallows the etal. l metagenomicsmito- or 2013),butMMG inaddition present in unequal numbers inunequal present llows reliable inference of inference llows reliable of mixed communitiesis of mixed etal. and thebioinformatic etal. of bulkbiodiversity of 2016).Thus,the etal. 2016). etal. 2014; 2013; Acceptedin Schlötterer of poolsofindi sequencing 2013).Thes Ramos-Onsins & Pérez-Enciso nucleotide Hellmann diversity (e.g., & 2010;Zhu Schlötterer Futschik (e.g., frequencies for popula effective technique assembled studies contigs. have Previous illustrated the use as data ofshotgun a cost- affected readsare sample, although theindividual completeboth the interspecific information intraspecific on and genetic variability ofthe base natural communitybe investigated may theseda assembly of potential artefactsinthe population. productscorres it isnotclearhowtheresulting related species. number Asa ofdiscrete limited contigs isassembled primary fromthe reads, Articlecomplicatedapplied when tonatural communities containinggenetic and variants closely (Gómez-Rodríguez the conditions, inparticularwhen under these by the mosaic contigs produced sequencesabsenceof intraspecific inthe variability (Gillett diversity. questions aboutspecies-level and thus barcoding, DNA unlike Sanger-based contig level limitsthe use ofthese sequences quantification forthe ofintraspecific diversity, Arela present inthepopulation. This article isprotected Allrights bycopyright. reserved.

Contigs beenshowntobe fromMMGhave

Comparing the MMGsequences Sang with etal. 2014), suggesting that population ge suggesting thatpopulation 2014), etal. 2015). However, the assembly inevitably becomes more becomes inevitably theassembly 2015).However, viduals of a single species atahi viduals ofasingle species tion genetic analysis, includi tion genetic analysis, ted concern isthatcollapsingconcern ted in silico etal. recombining of genomes of different species are rare rare speciesare genomes of different recombining of 2008; Futschik & Schlötterer 2010; Ferretti, 2008;Futschik& Schlötterer 2010; e studies were based on whole-genome onwhole-genome werebased e studies d on the primary shotgun reads, whichhold the primaryreads, shotgund on ta. In addition,intraspecificvariabilityina ta. assembly uses reads of 250-300 bpinlength 250-300 reads of uses assembly constrains the application of MMGto theapplicationof constrains pond to haplotypes actually present inthe tohaplotypes actually pond by greater noise from read errors than the errorsthanthe noisefromread greater by highly consistent with Sanger-derived consistentwithSanger-derived highly etal. er-derived haplotypes may reveal mayreveal haplotypes er-derived netics parameters can alsobe can parameters netics 2012;Gautier ng the estimationofallele etal. gh depth of coverage (see review review (see ofcoverage gh depth of genetic variation atthe genetic variation of 2014).Equally, chimerical et al. et 2013)and Accepted belowthespecieslevel. MMGstudies scopeof thewider genetics, andfor froma‘bi utility obtained reads ofshotgun estimates fromc comparable to ma read shotgun ofusing and (b)theprospect population withtheassembled addr Herewe genetic variation. st butthe and betadiversity, witheither approach Sequencing (B study from anearlier available were community samples mixed the in each individual for Article PCR-barcodes) (hereafter sequences multiple haplotypes onthe (based mostofwhic perlocality, species few dozen individuals). Thisdataset encompasses large intraspecificvariability, and interspecific witha 10 localcommunitiesofleafbeetles(Chrys specimen lllumina sequencing. Gómez-Rodríguez specimens and species mixed assemblies using copy numbers. estimated foreach species inmixed assemblages iffocused onmitogenomes present inhigh This article isprotected Allrights bycopyright. reserved. This study comparis makesside-by-side udy made no attempt to udy noattempt made consensus haplotypes obtaine consensus haplotypes onventional barcodingmethods.Th ess (a) the sequence similarity of (a) thesequence ess arrived at very similar conc at very similar arrived cox1 -5’ fragment) which were sequenced inbulk.Sanger sequenced whichwere -5’ fragment) aselga, Gómez-Rodríguez & Vogler 2015). & Vogler Gómez-Rodríguez aselga, odiversity soup’inpopulationandcommunityodiversity omelidae) from the Iberian Peninsula (>2600 Iberian the Peninsula from omelidae) h are represented by numerous individualsand numerous by represented are h pping to obtain measures of genetic variation genetic variation of toobtainmeasures pping individual-based Sanger individual-based on of datasets obtained for the same obtainedforthesame on ofdatasets etal. address the question about intraspecific addressthequestionabout (2015) conducted an MMGstudy an on (2015)conducted d from those same specimens, d fromthosesame lusions about species richness lusions aboutspeciesrichness PCR-basedhaplotypes ina e results arecriticalforthe e results sequencing andbulk- sequencing Accepteddatabase.From reconstructed these mitogenomes we hereconsider only the assignment of (v)taxonomic =1%); per read minimumgaps= 99%; Geneious 5.6(minimumoverlap=500bp;minimum overlapidentity of identicalorvery producedby similarcontigs ofnon- removal 1.1.1, followedby Illuminasequencing onan MiSeq sequencer;assemblyIDBA-UD (iii) and withNewbler 2.7 frompool preparation Illumina TruSeqlibraries standard protocol,whic fromshotgun obtained se specimens hadbeen (Gómez-Rodríguez speciesthatex available) in171 barcode is subset oftenlocalassemb Article Mixe Generalized the sequences using Inaddition, species 2015). & Vogler monographs ofWarchalowski sequenced.All specimens had beenLinnaean identifieda to species according totaxonomic specimensfrom20lo Vogler 2015)included4533 (Chrysomelidae)Iberian communities fromthe Peninsula (Baselga, Gómez-Rodríguez & ( PCR-barcodes Sanger sequenced MMG-sequences and library PCR-barcodes ANDMETHODS MATERIALS This article isprotected Allrights bycopyright. reserved. The corresponding Illumina sequences were Illumina were sequences The corresponding etal. h involved these main steps (seeGómez-Rodríguez involved thesemainsteps h 2015). Mitogenome assemblies fromva 2015).Mitogenomeassemblies lages, totalling 2607 (2003) and Petitpierre (2000) (s (2000) (2003) andPetitpierre cox1 mitochondrial andshort(<3000bp) delimitation wasconduc d Yule Coalescent (GMYC) method (Pons d YuleCoalescent(GMYC)method -5’) hibited 1089differentPCR-

from anexisting study leaf of beetle contigsBlast via matche quencing following ofspecimena mixtures specimens (for 88.9% of which aPCR- ofwhich specimens (for88.9% different assemblers through re-assembly in re-assembly assemblersthrough different ed DNA extracts of single individuals; (ii) individuals; ofsingle DNA extracts ed cal assemblages that were successfully successfully thatwere cal assemblages obtained, in a previous study, from a from inapreviousstudy, obtained, ted based on the ted basedonthe ee Baselga, Gómez-Rodríguez rious combinations ofthese rious combinations s to the PCR-barcodes s tothe contigs; (iv)merging contigs; barcodes (haplotypes) (haplotypes) barcodes et al. cox1 cox1 2015):(i) -5’ barcode barcode -5’ etal. -5’ -5’ 2006). Acceptedcomputed with the ofuncorrected and amatrix All PCR-barcodesandMM MMG-barcodes and PCR-barcodes between discrepancies Nucleotide assembly. for available species amongabundance/biomass inthemixture species in anddifference variation nucleotide andinterspecific intraspecific the increased LocL poolstogeneratefrom all combined reads inlength. Third, species of1.5to10.5mm biomassofspecies and variation inabundance and ofintraspecific presence details inGómez-Rodríguez cell(see aMiSeqflow 20%of onapproximately sequenced LocL was haplotypes. Each Articleand 99to156known Linnaean species and 67 27 frombetween specimens drawn localassemblage of10 from each assembly (LocL 1to10) library’ Second, a‘local fortheperforman a benchmark are considered MiSeqflowcell. 117% ofthe sequenced on (includingthe 176focalspecies fivespec aconcentration-adjust of by sequencing shotgun of mitochondrialgenomes.First, will beas referred MMG-barcodes. to consen theassembled are (655bp)which regions This article isprotected Allrights bycopyright. reserved. Using the above methodology, Gómez-Rodríguez above methodology, Using the dist.alignment G-barcodes were aligned with were G-barcodes etal. pairwise distances between between pairwise distances interspecific genetic variation interspecific a ‘mitogenome reference library’ (MitoRL) was generated generated (MitoRL)was library’ a‘mitogenomereference 2015). This set informs us about MMG performance inthe 2015).ThissetinformsusaboutMMGperformance function in seqinr (Charif & Lobry 2007). Thisdistance Lobry& 2007). function inseqinr(Charif was generated and shotgun by sequencing was ies added from another locality), which was fromanotherlocality), ies added an additional assembly was conducted onthe conducted assembly was additional an s, each of which included between 156and336 ofwhichincludedbetween each s, The assemblies ofmitogenomesThe fromthispool ce of MMG under no intraspecific variation. variation. MMGundernointraspecific of ce , which ranged from 1to25individualsper ranged , which a ‘combined library’ (CombL), which further which further (CombL),‘combined library’ a ed mixture of one representative for each of foreach mixtureofonerepresentative ed sus of numerous shotgun reads and hereafter andhereafter reads ofnumerousshotgun sus but also increased the number of reads per ofreads number but alsoincreasedthe etal. both types of sequences was ofsequences both types transAlign (Bininda-Emonds 2005) in a natural community and under communityin anatural (2015) built three different sets different sets (2015) builtthree cox1 de novo de

Accepted the PCR-barcode, closest andthe a MMG-barcode between < 10 BLAST (e-value barcodes using the full for the assessed reads was computed withC were ends. Phredscores with highscore quality(phred inthe >30) sequence consensus after trimming the lowquality either data The quality set. of these changes that may due be toan incomplete fu were MMG-barcodes’) (‘discrepant PCR-barcode intheP thesequences comparison, and99.2%of the correspondingwith length to80%of >524bp, number smallest cases,the Inall CombLall thePCR- and assembly Articlea of MMG-barcode local assemblage;the (c) a LocLgiven assembly and all of construction inMitoRL;(b) theMMG-barcode and thecorresponding barcode mitochondrial contigasn set between twosequences. a di gives matrix distances, whilethediscrepancy calls (s base ambiguous assemblers produced discrepancies between sequences. two This step needed was because ofmultiple the use was a transformed discrepancymatrix ma into cox1 This article isprotected Allrights bycopyright. reserved. MMG-barcodes exhibiting at least one discrepancycompared similar totheir most ma ofthe discrepancy Subsets -5’ reads data set, the shotgun library wa theshotgun library dataset, reads -5’ eeded totestdiscrepancie PCR-barcodes was assessedinGe was PCR-barcodes PCR-barcode of the exact specimen used for library for specimenused exact ofthe PCR-barcode cox1 PCR-barcodes ofthat of discrepancies was selected wasselected of discrepancies barcodes of that morphological barcodes ofthat -5’ dataset as well as for each discrepancy pair. Tobuild pair. discrepancy well asforeach as dataset -5’ -5 trix were extracted for each morphological species and species eachmorphological for extracted were trix ). In the cases where discrepancies were observed observed were discrepancies where In). thecases odonCode Aligner of shotguns v6.Thequality ee below) that complicate the estimateof thatcomplicatethe ee below) given morphological species produced inthe given morphological species trix that summarizes the number of nucleotide thenumberofnucleotide thatsummarizes trix PCR-barcode library or library PCR-barcode CR-barcodes database fu CR-barcodes database s searched against the database ofPCR- database the against searched s rect account of the number ofsitesdiffering ofthenumber account rect a given morphological species produced in produced species morphological a given cox1 rther explored to establish thenatureof toestablish rther explored same species found in thecorresponding foundin samespecies s between: (a) the MMG- MMG- s between:(a)the -5’ fragment, were considered forthis considered were fragment, -5’ cox1 as best match. Only barcodes Onlyas bestmatch. barcodes neious as the number of bases numberofbases neious asthe -5’ reads data set was also data setwas reads -5’ species in thefulldatabase. in species sequencing errors in errors sequencing lfilled thiscriterion. lfilled al. using thevarian fragment) barcode (length of Pi)subsequent (Tajima’s nucleotide diversity areinthepool. species different reads from inreadma value divergence setting amaximum consensus PCR- the from computed hadbeen that species c 3%, minimumoverlapidentity against =97%) 8.0 (customsensitivity minimumoverl settings: shotgun ther reads, from nucleotide diversity in the 1987)fromP (Nei Nucleotide diversity geneticdiv Assessment of need toidentify PCR-polymorphisms. coul PCR-barcode one than more with species invariant inthePCR-barcodesbutshowadi barcodesallowed the identifica custom Rscript.Theassessmentofpolym were fromall identified available PCR-barcodes S3). inAppendix details (see 2012) (Buffalo qrqc Bioconductor package th establishing assessed by those speciesestimate and their quality.The readquality shotgunreads ofthese was then ofthosemorphologi run against thePCR-barcodes

Accepted 2011)forall Article This article isprotected Allrights bycopyright. reserved. pegas Finally, for each morphological species, the polymorphic sitesin the species,thepolymorphic morphological for each Finally, function in the seqinR package (Charif seqinRpackage functioninthe package (Paradis 2010)inR(RDeve (Paradis package cox1 -5’ fragments that had a coverage of >2 reads for >80% (minimum >80% for >2 reads of coverage thathada -5’ fragments e proportion of high quality bases (phred > 30) with the with >30) quality (phred e proportionofhigh bases ersity from shotgunreads tion of ‘MMG-polymorphisms’, i. tion of‘MMG-polymorphisms’, CR-barcodes was computed with the wascomputedwiththe CR-barcodes orphic sites fromboth orphic Mapped reads were expor were reads Mapped fferent nucleotidefferent inthe MMG-contigs.Only ce-sliding perl script perlscript ce-sliding of655bp window forasingle computed ly eads from each LocL were mapped inGeneious LocL mapped were from each eads d be assessed for these sites because of the the because of sites for these d beassessed barcodes ofthat local assemblage using the ap: 100bp, maximum = mismatchesap: 100bp,maximum perread pping, which is a convenient feature when feature convenient whichisa pping, onsensus sequences of each morphological ofeach onsensus sequences

(hereafter ‘PCR-polymorphisms’) using‘PCR-polymorphisms’) a (hereafter lopment Core Team 2015). Tocompute lopment CoreTeam2015). & Lobry 2007). Geneious mappingLobry& Geneious allows 2007). cal species, in order identify the reads for reads the identify cal species,inorder PCR-barcodes andMMG- e. base positions thatare positions e. base of Popoolation(Kofler ted as BAMted as fileand cox1 nuc.div -5’ region region -5’ function function et et Acceptedand 4.1%and8.4%) (between genetic distance In theP thesecases, S1). of (Appendix four outof402)thatr contigs (18 for ninecases the containing species each contig for consensus produced asingle was introducedint IUPAC code ambiguity diffe showedslight IDBAwith the and Newbler dataset, assembled assembled each of10localities, of representatives thesingular derived from:(a) mitogenomehad contigsassembled been that previously fromthree shotgun data sets, the extracting by obtained were MMG-barcodes MMG-barcodes and PCR-barcodes between discrepancies Nucleotide RESULTS Article values ofbothdatasetswas Additionally, aPears factor. between-subjects reads) (PCR-barcodes orshotgun fromthewas value significantly obt different average of value the assesswhether used to mo each for diversity, nucleotide of average mapping (3%). the thresholdsetforread Linnaean withinsome against thePCR-barcodes groups This species. of morphological instead positions.Thesame count=1) oftheirnucleotide This article isprotected Allrights bycopyright. reserved. Repeated Measures ANOVA (RM ANOVA) onna (RMANOVA) ANOVA Repeated Measures altogether (CombL) (see Materials Materials (see (CombL) altogether conducted. Statistical analyses were done inStatistica7.0. done were analyses conducted. Statistical separately (LocL 1 to 10), and (c) all specimens inthe allspecimens 1 to10),and(c) (LocL separately was considered as within-subjects factor and locality as and locality factor as within-subjects was considered ecovered two contigs per morphological species species twocontigs morphological per ecovered hose positions. The re-assembly generally generally There-assembly hose positions. CR-barcodes showed very high within-species very high showed CR-barcodes nucleotide diversity obtained from PCR-barcodes from diversity obtained nucleotide rphological species and local assemblage, was and localassemblage, species rphological ained from shotgun reads. The datasetused fromshotgun The reads. ained on correlation onnatural-logarithm transformed eliminated discrepancies from read mapping readmapping from discrepancies eliminated may correspond to different cryptic species. cryptic species. todifferent correspond may each species (MitoRL), (b) all specimens in in specimens all (b) (MitoRL), species each rences in the re-assembly with Geneious,a inthere-assembly rences analyses were also conducted using GMYC- alsoconducted were analyses cox1 species whose haplotype diversity exceeded exceeded diversity whosehaplotype species -5’ regions fromthe complete orpartial and Methods). Where contigs produced and Methods).Where tural-logarithm tr cox1 -5’ fragment, except except fragment, -5’ ansformed values ansformed = 60). In most cases, the discrepancies inth discrepancies the In= 60). mostcases, but diffe PCR-barcodes required) cox1 discrepancy cases was very (Figure high tothe 1b)and quality similar full observed forthe In S2). 10.5 S.D.,seedetailsinAppendix c proporti (average fulldataset forthe average qua (proportion ofhigh barcodes disc withmore 1b). Thesecases averagedgreatlywereoutliersa numbers inflated differing fewsites by numerous (Figure by These Figure 1b). 655positions(Table1and to 7.8(LocL)from 2.7(CombL) ofthe sites’)w (‘discrepant number ofdiscrepancies Acceptedand 76.0%(CombL) (LocL) 90.7% (MitoRL), 78.8% Article proportionofid sites’,the these ‘ambiguous site (in655positions)wasobservedperMMG-bar MMG-bar all of and 13.5%(CombL) andMe Material (see Geneious re-assembly 1624 sequen by 80 speciesrepresented sequences inthePCR-barcode 1668 by LocL, represented to76morphological corresponding species in MMG-barcodes species;177 inMitoRL, to128morphological corresponding included 128MMG-barcodes the usedfor contigs The finalnumberof This article isprotected Allrights bycopyright. reserved. -5’ reads dataset (proportion ofhigh quality (proportion reads dataset -5’

‘Ambiguous sites’ represented by IUPAC‘Ambiguous by sites’represented We further investigated species exhibiting inPCR-barcodes variation ( database; and database; 82 MMG-barcodes repancies showed notablyrepancies showed lowersequence quality inthe PCR- lity bases [phred >30] ranging from 8.1% to 99%)thanthe from 8.1% ranging>30] bases[phred lity ring from MMG-barcodes for that species (number of cases (numberofcases forthatspecies fromMMG-barcodes ring codes, and in the majority of cases only one ambiguous one cases only themajority of and in codes, ces inthePCR-ba ces analysis of sequence variation inthe variation of sequence analysis ontrast, the quality of MMG reads across all across thequalityontrast, ofMMG reads e MMG-barcodes were observed atbasepositions observed were e MMG-barcodes thods) affected 3.1% (MitoRL), 15.2% (LocL) 15.2%(LocL) (MitoRL), 3.1% affected thods) entical MMG-barcodes and PCR-barcodes was andPCR-barcodes MMG-barcodes entical on of high quality bases [phred >30] =92.2± >30] high bases[phred quality on of ith the closest matching PCR-barcode ranged ranged PCR-barcode closestmatching ith the ambiguity codes and introduced inthe andintroduced ambiguity codes bases = 90%, see details in Appendix S3). inAppendix details bases =90%,see code (Table 1, Figure 1). Allowing for 1). 1,Figure code (Table . Among the divergent pairs, the average average the pairs, divergent Among the rcode database. rcode database. in CombL, corresponding to in CombL, corresponding cox1 ≥ 2 different 2 different -5’ region region -5’ the ten study sites (Figure 3).Nucleotide di the tenstudy sites(Figure datais MMG andSangersequence An exam locality. asampling at species Linnaean ‘popu computed foreach was Nucleotide diversity Assessing nucleotide diversity within populations using shotgun reads 5). including 1 (total numberofMMG-polymorphisms: AcceptedThis type of3 full dataset) and number tothe ofPCR-ba total the num PCR-barcodes dividedby inversely related toboththe relative success ra of codon position=70;Figure 2).Thenumber Article= 1;S.D.4.0) 12 (mean=3.8;median (S The numberofMMG-polymorphisms available fromother localities and thusallo PCR-barcodes since for atotalof66cases, used couldbe MMG-barcodes locality. These they didnothave because MMG-barcodes’ alsowere observed MMG-barcodes notbeen that insix had initially as identified ‘discrepant that for PCR-barcode alignment at nucleotidedifferences exhibited MMG-barcodes MitoRL, inCombL)cases (6in LocL and2 the in15 7in However, morphological species. (i.e.polymor that alsoshowedvariation This article isprotected Allrights bycopyright. reserved. Cryptocephalus rd position SNP appeared to be concentr positionSNPappearedtobe (n=49SNPs), morphological species.Add ber of specimens for a given morphological speciesinthe given morphological fora ofspecimens ber shown for populations offi for populations shown phisms) within the PCR-barcodes for that thePCR-barcodesfor phisms) within and they mostly inthe3 and they occurred Altica NPs uniquetotheMMG-barcodes) st for the same morphological species were were species samemorphological forthe codonposition=7;2 versity from PCR-barcodes was computedfor316 from PCR-barcodeswas versity wed the identification ofPCR-polymorphisms. a corresponding PCR-barcode from thesame from PCR-barcode a corresponding for the assessment of MMG-polymorphisms, the assessmentofMMG-polymorphisms, for SNPs unique to the MMG-barcodes was uniquetotheMMG-barcodes SNPs te of the Sanger-barcoding (the numberof theSanger-barcoding te of (n=6), rcodes available for the species (Figure 2b). (Figure species for the available rcodes ple of measures of nucleotide diversity ofnucleotide in ple ofmeasures lation’, defined as all lation’, definedas sites that were invariable otherwise inthe ated in species from a few genera, genera, a few from ated inspecies Aphthona itionally, unique polymorphismsitionally, unique ve across widespread species (n=6) and (n=6) nd codonposition=2;3 the individualsofa rd Labidostomis codonposition

ranged from 1to ranged (n= rd

Linnaean species (Pearson R = 0.524, p < 0.001, n = 180), due to the presence of some clear ofsome =180),duetothepresence R=0.524,p< 0.001, n (Pearson Linnaean species of intheanalyses only moderate p<0.001,n=178)but groups(Pearson R=0.802, for PCR-barc nucleotide diversity Note that when the natural-logarithm transf methodhadno sequencing between locality and 0.001; GMYCdata:F Acceptedbarcodes (RM ANOVA within-subjects e diversity significantly was whenhigher thenucleotide 4).However, 178)(Figure R =0.776,p<0.001,n GMYC groups(Pearson n =180)or R=0.724,p<0.001, (Pearson morphologicalboth whenassessing species ofthedataset). reads(39.2% populations fromshotgun Article populationsfromPCR- 320 could beobtainedfor popula produced 474local which Linnaean species, groupingon GMYCdelimitation,ratherthan of popula ofavailable thenumber reduced greatly or (n =109) Theabsence = 18.7± 4.3[S.D.]). (40.8%ofthedata computed for186populations populations). Nucl = 22 (n failure sequencing or noP populations) oronly one one from siteswithonly were = 31.6±number ofpopulationsinalocality to95.0%of out of457populations,corresponding This article isprotected Allrights bycopyright. reserved. fromPCR-barcodes values Nucleotide diversity 1, 168 1, =34.01, p < 0.001; Figure 5a, c). =34.01,p<0.001;Figure 5a,c). specimen representing a species at a locality (n=119 at alocality aspecies specimenrepresenting CR-barcode sequence available fo CR-barcode sequence odes and shotgun reads was high in the analyses of GMYC- of analyses high readswas and shotgun inthe odes calculated based on shotgun reads thanPCR- reads basedonshotgun calculated ffect; morphological species: F morphological species: ffect; ormation is not applied, Pearson correlation of isnot applied, Pearsoncorrelation ormation 7.6 (S.D.). Those ‘populations’ notincluded Those‘populations’ 7.6 (S.D.). eotide diversity based on shotgun reads was was reads eotide diversity onshotgun based insufficient number (n=162) of mapped reads of mappedreads (n=162) number insufficient tions. The analysis was also conducted based also conducted based was Theanalysis tions. haplotypes based on assignment toa onassignment based haplotypes significant effect (p>0.05; Figure5b,d). (p>0.05; effect significant set, mean number of populations perlocality populations meannumberof set, barcodes (93.3% of the (93.3% ofthe barcodes PCR-barcodes intheda tions, of which nucleotide diversity data tions, ofwhichnucleotide and shotgun reads were highly correlated correlated highly shotgun were and reads Locality interaction orthe r the species due to Sanger duetoSanger r thespecies 1, 170 dataset) and for 186 and dataset) taset, withamean taset, =12.39,p< probablygreatlyhave benefitted fromincreased sequencing volume ofa (e.g. Miseq 50% (Gómez-Rodríguez community levelanalyses atthepopulati analysis for anexhaustive identified as al. cell corresponding to on 20%ofaflow 10 samp of populationsacrossthe LocLmappingregion inmany tothebarcode However, morphological species. based ontheconsen LocL sample,whichwe referenc required LocL samples.Thisapproach species.In a contrast, mappingcapturingallowed approachgenetic the full variability ofthe 177differ recover LocL that in the analyses same ofthe different haplotypes differentialIntraspecificassembly.was differentiation detectable when tosome extent thedifferent it isnotpossible toreconstruct of forstudies assembled contigs assessment ofspecimen samples. While thischaracteristicMMG approach simplifies therapid biodiversity forthe complex in present variation intraspecific the removing effectively species, per single contig We showthatassembly shotgun from sequen Discussion S4fordetails). Appendix (see defined species by affected outliers inthelatter,whichwere

Accepted (MitoRL between 0.05% 2015),ofwhichonly Article This article isprotected Allrights bycopyright. reserved.

cox1 -5’ with our BLAST procedure. Data ourBLAST procedure. -5’ with ‘soups’ (Crampton-Platt ‘soups’ population genetic variability, as variability, populationgenetic species fromdi were assembled les. The sequencing depth for each LocL library was based wasbased LocL library each depthfor les. Thesequencing the read-based approach was affected by low rates of read low ratesofread by affected was approach read-based the approximately 4,000,000 reads (Gómez-Rodríguez 4,000,000reads approximately on level although it had proven useful for useful on levelalthough ithadproven ent MMG-barcodes assigned to 76morphological to assigned ent MMG-barcodes haplotypes of a single species by means of means by species asingle of haplotypes sus sequence of the PCR-barcodes for each foreach ofthePCR-barcodes sus sequence large genetic distances within morphologically genetic large distances ces of mixed communitiesgenerally mixed producesa ces of etal. populations, limitingpopulations, ouranalyses tojust40.8% e sequences representing the species in each each in species the representing e sequences etal. ) and 0.1% (LocL) of the reads were readswere the of ) and0.1%(LocL) 2015).Asaminimum,this study would 2015),itprecludestheuseof volume used here was notsufficient volume usedhere fferent bulksamples,asseen fferent with the current technology current technology withthe et et Sanger sequences even when closely rela closely even when sequences Sanger pr MMGapproach the showsthat Our analysis Sanger without sequencing benchmar assembly isanatural due also are differences other that conceivable by lo explained were greatest discrepancies differed most ofthediscrepanthaplotypes sp actual PCR-barcode ofthe sites) ambiguous (ignoring consensus sequence in differences assemblers produced types of In S1). (Appendix absence the ofintraspecific variability inthe analysis, MitoRL the two most ofthisvariation bycollapsing all gained assessments canbe S5),r indicating inAppendix that example al. may also for present a challenge pooledsequenceddata from population parameters Enciso 2013;Gautier & Schlö (Futschik each individual of DNA Anderson,Skau &(Futschik Schlötterer2010; th that couldbeincludedin ifthisdoesnotguarantee flow cell),even

Accepted Articlecase, differ 2015).However,inthepresent This article isprotected Allrights bycopyright. reserved. While direct mapping of reads revealed directmappingWhile of etal. 2013) have been recognised as problems for calculating calculating asproblemsfor recognised 2013)havebeen being compromised by intraspecific variability in theassembly. in intraspecific variability being compromisedby depth. at lowsequencing e dataset (i.e. 100% of the popul of e dataset(i.e.100% ecimen used in MMG sequencin ecimen usedinMMG k for assessing differences be differences assessing k for these analyses (see (see Schlötterer analyses these haplotypes whose divergence was below ~4% below was whose divergence haplotypes ted species are present in the sample. sample. the in arepresent species ted a 2.5-fold increase in the number of populations inthenumber increase a 2.5-fold by a single nucleotide only (Figure 1b).The (Figure only a single nucleotide by obust information for population andcommunity obust informationforpopulation w quality in the PCR-barcodes, anditis inthePCR-barcodes, w quality only 4 of the 128 contigs (3.1%). Moreover, the Moreover, (3.1%). 4 ofthe128contigs only tterer 2010; Ferretti, Ra 2010;Ferretti, tterer ent read mappers gave results(see mappers similar ent read to errors in the PCR-barcodes. TheMitoRL inthePCR-barcodes. toerrors g & Barshis 2014) and unequal contributions gBarshisand unequal & 2014) oduces sequences of a quality equivalent to equivalent a quality of sequences oduces SNPs, genomeremoved numerous assembly from both assemblers was identical tothe bothassemblerswas from . The efficacy of current mapping current software of . Theefficacy ations). Low depth ations). sequencing tween Illumina andconventional tween g in 90.7% of the cases and thecases g in90.7%of et al. 2014;Crampton-Platt mos-Onsins &mos-Onsins Pérez- et et Accepted (see Figure SNPs ofMMG-specific presence the with correlates that rate success barcoding bemissing may assembly MMG the by detected many thesinglemay of also explain nucleotid was a tendency towardslower quality inthe PCR-ba (e.g. ofdiscrepancies larger number or asmallnumberof a single mostly affected LocLof contigs in as 21.2%and24% many Ignoring ambiguities the fromdifferentprocedures, assembly we assessed then the PCR-ba conflict between (Crampton-Platt contig obtainlonger to recommended currently from multip of contigssupports there-assembly genetic while inthe variation interspecific mixtures, variation ismaintained. Thisproperty Article& Miller,Koren in approaches of assembly Gr IDBA-UD ortheGreedy in Graph approach assemblers, when even using the ap twoprincipal ambiguous numberofambiguous orthe contigs CombL,more complex LocL assembliestothe betweenassemblers donotincrease the two fromthe intermediate level ofdiversity inthe re twopositionsofthe655-bpbarcode only oneor Gene re-assembly in after library, respectively, 13.5% ambiguitiesways, in15.2%and whichproduces assemblies, the twoassemblers resolved the variability reads inshotgun inslightly different This article isprotected Allrights bycopyright. reserved. variation, as intraspecific facing When etal. 2016). rcodes and MMG-barcodes, whichre rcodes andMMG-barcodes, Clytra quadripunctata Sutton 2010), reproducibly remove intraspecific intraspecific remove Sutton 2010),reproducibly and CombL, but again these discrepancies discrepancies butagain these and CombL, nucleotides. In general, in the cases with a with cases the in Ingeneral, nucleotides. exemplified by the LocL by andCombL the exemplified e differences. In addition, genuine haplotypes haplotypes Inaddition, genuine e differences. neither when considering the proportionof the considering when neither in againdiffer although mostassemblies ious, s and to detect possible assembly errors todetectpossibleassembly errors and s aph-based see assembly inNewbler; details le assemblers in MMG analyses, as itis le assemblersinMMGanalyses, from thePCR-barcode from sites. We thus conclude that thegenome thusconcludethat sites. We proaches for contig building (i.e. de Bruijn (i.e.de building for contig proaches gion (Figure 1a). Notably, the differences thedifferences Notably, 1a). gion (Figure rcodes, and thus errors intheSanger data and thuserrors rcodes, of the consensus sequencesineither oftheconsensus , see details in Appendix S1)there , seedetailsinAppendix vealed discrepancies inas discrepancies vealed dataset duetolow dataset conserved regions that increase these effects of effects these thatincrease regions conserved skimming such of other asgenes, markers nuclear which and include rRNA variable to prone haplotypes highly theyphenomenon, butequally ma Pons (e.g. lineages certain in increased Cryptocephalus wereevident main haplotypes ‘discrepant’ heteroplas to due true variation,e.g. LocL and Atthisstage CombL libraries. we ca only formation isevident whenvery of chimera evident inthe MitoRL assembly composed of heteroplasmy. The possibility of3 result mainly fromlow-qualitycalls base signals, ormixed resulting possibly from MMG assemblies therefore may bethose differentinSanger from barcodes, as the latter intheexistent specimen mixture, evenvariation ifthe itself SNP isgenuine. from The errors related ofclosely the formationofchimeras haplotypes the through forthemethodology, concern greatest perhaps the Accepted Article positions, consistentwith ourfindings of multiple variantsIllumina in sequencing differingprimarily 3 in synonymous wouldleadtobot that heteroplasmy caused by existingbe missing haplotypes ofthe many were 2b), although PCR-barcodes This article isprotected Allrights bycopyright. reserved. An alternative explanation for the large number of ‘discrepant’ 3 ‘discrepant’ of number large forthe explanation An alternative , Altica , in silico Aphthona, in silico recombination amongvariants truein the present mixture, i.e. yalso constitutelocalasse rd available for 88.9% of individuals and thus are unlikely to ofindividualsandthusareunlikely for88.9% available and shuffling. The problem would be aggravated ingenome problem wouldbeaggravated shuffling. The codon shuffling is supported because itismuchless issupportedbecause codonshuffling my. A striking observationwasthat3 Astriking my. etal. Labidostomis low Sanger sequencing success (Figure 2b). 2b). (Figure success sequencing low Sanger ly in a small number of genera, including small numberofgenera, a ly in . Failed barcodes or poor barcodesor . Failed haplotypes to produce sequences thatarenon- sequences toproduce haplotypes 2011), and these genera may be affected by this by affected may be genera andthese 2011), one individual per species, and thusthisform individual perspecies,and one h mixed Sanger signal and the detectionof Sanger andthe signal h mixed nnot resolve these artefactual haplotypes from haplotypes resolvetheseartefactual nnot chimera formation, but if reference sequences sequences formation,butif reference chimera closely related haplotypes are present in the the in arepresent haplotypes related closely is the potential for generating artefactual artefactual is thepotentialforgenerating . It is heteroplasmy . iswellknownthat mblages related closely of quality may also be rd codon positions, and codonpositions, rd codon position rd codon Acceptedgr e.g. GMYC the themselves, sequence data membership in a morphologicallyLinnaean delimited species, onthe ormay be based relies onthematchtohaplotypes whichmay be genetic parametersarecalculated fordifferently defined entities.readmapping The approach cases (see same read Notably, mixtures. inflated ofPCR-barcode values were evident diversity insome 2003;Baselga, Vellend genetic (see diversity nucleotide diversity, oritwillallow the estimation ofcorrelates between species diversity and of structure the into insights additional reveal may analysis A species-per-species S6). nucleotide diversity estimates fromshotgunandreads PCR-barcodes Appendix (see alike ten localitiesisalimiting sa wa with locations iscorrelated nucleotide diversity ineach locality highnucleotide suggests the ten diversity across apreliminary analysis estimation. For example, Article orlocalitie studies among populations different absolute values estimates differ, both are highlycorrelatedand thisenables comparative from PCR-barcodes,an issueIllumina that may related to be Yet, sequencing errors. if even nucleotide diversity estimated was fromshotgunreads significantly than estimated higher fro with thescoresobtained correspondence estima for against thePCR-barcodeswereused diversity possiblewiththeunassemb only are effective. (again approach mapping read the are available, This article isprotected Allrights bycopyright. reserved. Finally, due to the collapse ofvariationin tothecollapse Finally, due Exosoma lusitanicum Exosoma mple sizeforbiological interpre rm climate, cold winters and a marked wet season. Although season.Although andamarkedwet rm climate,coldwinters in Figure 3), which may be due to the fact thatpopulation fact may be due tothe which inFigure 3), m the PCR-barcodes th m the oups from PCR-barcodes. We observedthatthe fromPCR-barcodes. We oups Gómez-Rodríguez & Vogler 2015) fromthe 2015) Vogler & Gómez-Rodríguez led reads. SNPs observed in reads mapped SNPsobservedinreads led reads. ting nucleotide diversity and showed a good showeda and diversity ting nucleotide s using shotgun reads for nucleotide diversity diversity s usingreads fornucleotide shotgun of the climatic correlates of average of the climaticcorrelates of grouped based on external criteria, such as criteria,such external grouped basedon the assembly, estimates of population genetic estimatesofpopulation theassembly, st the mostvariable remains regions) fully tations, this result isfoundusing tations, thisresult emselves. However, emselves. AcceptedRepository: http://datadryad.org/resource/doi:10.5061/dryad.3rh21 KF652242 ( -KF656666 forSanger numbers GenBank accession Data accessibility de Galicia(postdoc (ERDF) CGL2013- Development Fund (grants andCompetitiveness of Economy (to ACP),aNERCPostdoctoral FellowshipNE by Biodive theNHM This workwassupported Acknowledgments community sample. the in species most for diversity ofgenetic measurement accurate the Article diversity, phylogenetic assessment ofspecies toaholisticanalysis final hurdle overcomes the Illuminathe established and data the MMG below potential the species for level now ofintraspe analyses for successful cornerstones de sequencing entities, along withsufficient tothespecies entity corresponds thatroughly related hapl closely only collapsing by effect, from assembly genome The from consideration. mapping inthiscase) (3% that mayeliminate cryptic species exhibiting greater divergence due themorphological species, used, ratherthan groups GMYC are mapping improveswhen geneticfrom read diversity of correspondence This article isprotected Allrights bycopyright. reserved. toral fellowship POS-A/2012/052toral fellowship toC.G.R.). cox1 -5'). MMG-contigs are-5'). MMG-contigs available Digital fromthe Dryad and the European Regional and theEuropeanRegional derived sequences: KF134544 - KF134651 and KF134651 KF134544 - sequences: derived pth and accurate read mapping, willbethe mapping, read accurate pth and otypes and thus producing a consensus foran consensus a andthusproducing otypes placement of taxa, their abundance, and now theirabundance, oftaxa, placement level. Thus,appropriate level. 43350-P and CGL2016-76637-P) andXunta andCGL2016-76637-P) 43350-P rsity Initiative, a NHM/UCL PhDstudentship rsityInitiative,NHM/UCL a cific variationwithMMG.Thehigh of quality cific /I021578/1 (to MJTNT), theSpanishMinistry (toMJTNT), /I021578/1 of community inadditiontothe of diversity, to thethresholdfordi to mixed intraspecificvariantshasasimilar mixed

delimitation ofspecies delimitation vergence used for read for read used vergence

Accepted Gharbi,K.,Céza J., Gautier, M.,Foucaud, & C.(2010)The Futschik, Schlötterer, A. L.,Ferretti, S.E.& Pérez-En Ramos-Onsins, Lobry, (2007)Seqin{R}1.0-2: & Charif, D. J.R. Article Zhou,Crampton-Platt, A.,Yu,D.W., X. Crampton-Platt, G A.,Timmermans, M.J.T.N., A. Vogler, A.,Gómez-Rodríguez, C.&Baselga, & Barshis,D.(2014 H. Anderson, E.,Skaug, References This article isprotected Allrights bycopyright. reserved. genotyping. genotyping. sequenc fromnext-generation frequencies Pudlo, P.,Kerdelhué,C.& Estoup,A. sequenci massively parallel sequencing. M. Porto,H.E.Roman&Vendruscolo),pp.207-232. evolution: Mo approaches tosequence statistical computing tobiolog devoted metagenomics: letting genes the outofthe bottle. Evolution, mitochondrial meta (2015)SouptoTr A.P. Vogler, Khen, C.V.& Ecology andBiogeography, todiscer genetic levels species and pooledsamples. regarding a caveat ecology:

32, Molecular Ecology, Molecular Ecology, Molecular 2302-2316. genomics of a Bornean rainforest sample. rainforest ofaBornean genomics ng of pooled DNA samples. pooled DNAsamples. ng of

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22, 22, & Vogler, A.P. (2016) Mitochondrial & A.P.(2016)Mitochondrial Vogler, n neutral and non-ne n neutral rd, T., Galan, M., Loiseau, M., M., A.,Thomson, rd, T.,Galan, next generation of molecular markers from from markers generation ofmolecular next 3766–3779. 3766–3779. 5561-5576. ciso, M. (2013)Populationgenomics frompool ciso, ) Next-generation sequencing formolecular sequencing ) Next-generation (2013) Estimation of populations networks, lecules, ical sequences retrieval and analysis. analysis. and retrieval sequences ical immel, M.L., S.N.,Cockerill, Kutty, T.D., P. (2015)Multi-hierarchic P. a contributed package to contributedpackage a ing data: pool-versu data: ing Molecular Ecology, Molecular ee: The phylogeny of beetles inferred by inferredby ofbeetles The phylogeny ee: GigaScience, utral processes. utral processes. Genetics,

Springer Verlag, New York. New York. Verlag, Springer population allele Molecular Biologyand Molecular s individual-based

5:15

23,

186, the {R}projectfor the al macroecology at al macroecology . 502–512 502–512 (eds U.Bastolla, 207–218. 207–218. Global Global Structural AcceptedNei, M.(1987) &Nagarajan, Pop,M.(2013) N. Koren,S.& Sutton,G.(2010) Miller, J.R., P.,DeMaio, Kofler, R.,Orozco-terWengel, Y.Q.,Ashton,L.,Ji, Pedley, S.M.,Edwards, ArticleI.,Gu, Z.,Li,Hellmann, P.,Vega, Y., Mang, Gómez-Rodríguez, C.,Crampton-Platt, A.,T Gillett, C.P.D.T.,Crampton-Pla This article isprotected Allrights bycopyright. reserved. 14, data. sequencing e15925. seque generation next analysis of C.(2011)PoP Kosiol, C.& Schlotterer, via metabarcoding. Reliable, verifiable (2013) B.C.D.W. & Yu, Levi,Lott, Bruce, T., M.,Emerson, X.Y., D.S., C.,Wang, Hamer, K.C.,Wilcove, P.,Edwards,F.A., Dolman, P.M.,Woodcock, multiple individuals. ofshotg Population geneticanalysis Evolution, ecology and phylogenetics ofcomplex assemblages. thepowerof A.P. (2015)Validating and Evolution, of weev elucidates thephylogeny Bulk Vogler, A.P.(2014) 157-167. Molecular EvolutionaryGenetics Molecular

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31, Genomics, 2223-2237. Ecology Letters, Ecology Genome Research, tt, A., Timmermans, M.J.T.N., Jordal, B.H., & Emerson,B.C. Jordal, tt, A.,Timmermans,M.J.T.N., Sequence assembly Sequence de novo

95, 315-327. 315-327. ncing ncing data frompooled individuals. ils (Coleoptera: Curculionoidea). ils (Coleoptera: mitogenome assembly from pooledtotalDNA mitogenomeassembly from Assembly algorithms for next-generation Assembly fornext-generation algorithms un assemblies ofgeno un assemblies

mitochondrial metageno mitochondrial 16, N., Pandey, R.V., Nolte, V., Futschik, A., R.V.,Nolte,V., N., Pandey, D.P., Tang, Y., Nakamura, A., Kitching, R., immermans, M.J.T.N., Baselga,immermans,Vogler, A.& M.J.T.N., F.M., Clark, A.G. & Nielsen, R.(2008) Nielsen, Clark,A.G.&F.M., oolation: A toolbox forpopulation oolation: geneticA toolbox 1245-1257. 1245-1257.

18, . Columbia University. Press,New York:. 1020-1029. 1020-1029. and efficient monitoring biodiversityand efficient of demystified. demystified. Larsen, T.H., Hsu, W.W., Benedick,Larsen, S., T.H.,Hsu,W.W., Methods in Ecology and Methods inEcologyand mic sequences from from mic sequences Nature Reviews Genetics, Nature Reviews mics for community mics for Molecular Biology Molecular Plos One,

Accepted S.,Su,X., G.,Yang, Tan, M.,Meng, Tang, M., K.,FiStraub, S.C.K.,Parks,M.,Weitemier, Schlötterer, C.,Raymond Tobler,Kofler, (2015) R DevelopmentCoreTeam Article Fujisawa,Pons, T.,Claridge,E.M.,Savill, J., J Gomez-Zurita, Barraclough,Pons, T.G., J., Petitpierre, E.(2000) for anRpackage Paradis, E.(2010)pegas: This article isprotected Allrights bycopyright. reserved. 42, stepanalysis toward biodiversity using mito-metagenomics. sequencing of Zhou, Multiplex & X.(2014) systematics. Navigatinggenomic tipofthe the iceberg: Genetics, Reviews individuals —mining genome-wide polym Austria. URL https://www.R-project.org/ Evolution, (genus Zealand New from beetles LinneanDeep mtDNAsubdivisionwithin speci insects. ofundescribed DNA taxonomy S., Sumlin, W.D. & A.P.(2006)Se Vogler, Madrid. approach. e166.

Bioinformatics, 59, American Journal of Botany, Journal American 251-262. Coleoptera, ChrysomelidaeI Coleoptera,

15, 749–763. 749–763.

26, R: A language andenvironmentforstatisticalcomputing. R: Alanguage 419-420. . R Foundation for StatisticalComputing,. RFoundation for Vienna, R. & Nolte, V. (2014) Sequencing poolsof V.(2014)Sequencing R. & Nolte, Neocicindela population geneticspopulation withanintegrated-modular shbein, M., Cronn, R.C. & Liston, M.,Cronn,R.C. & A.(2012) shbein, ., Cardoso, A., Duran, D.P., Hazell, S.,Kamoun, ., Cardoso,A.,Duran,D.P.,Hazell, R.A., Barraclough, T.G. & Vogler, A.P. (2011) Vogler, &R.A., Barraclough, T.G. Systematic Biology, Systematic Liu,Li, Q.,Zhang, S.,Song,Wu, A. W., Y.,

Next-generation sequencing for plant plant for sequencing Next-generation 99, orphism datawithoutbigorphism funding. . Museo Nacional de Ciencias Naturales, CienciasNaturales, . MuseoNacionalde quence-based speciesdelimitation forthe quence-based pooled mitochondrial pooled 349-364. ). es in an endemic radiation oftigerin anendemicradiation es Molecular Phylogenetics and Molecular Phylogenetics

55, Nucleic Acids Research, Acids Nucleic 595-609. 595-609. genomes—a crucial crucial genomes—a Nature

Accepted(LocL). libraries:‘mitogenomere shown forthedifferent showingBoxplots base quality(phred score) according toitsposition in the read. Data isalso Appendix S3. score inthe > PCR-barcodes 30) the different of study localities. Boxplots showing the sequence length and pe the Appendix S2. differe twoormore where Description ofcases Appendix S1. Supporting information Article & Petrov,D. J. Zhu,Bergland, A.,González, Y., &Q. Huang,Zhou,L.,J. Liu,Li, M.,Fu, Q.,Su,X., Yang, Zhou, Tang, R., Li, S., Y., X., A.(2003) Warchalowski, biogeo Island Vellend, M.(2003) This article isprotected Allrights bycopyright. reserved. e41901. in populationre-sequencing genome arthropod sampleswithou enables high-fi sequencing (2013) Ultra-deep area. 358-365.

Natura optima dux Naturaoptimadux Problematiccontigs Sequence quality in MMG-barcodes quality inMMG-barcodes Sequence quality in andlength Sequence Chrysomelidae. Thele Foundation, Warszawa. Foundation, Warszawa. graphy graphy of genesand species. t PCRamplification. Drosophila melanogaster PCR-barcodes per locality PCR-barcodesper nt contigs are assembled for the same species. species. same the for assembled contigs are nt af-beetles ofEurope (2012) Empirical validation ofpooledwhole Empiricalvalidation (2012) ference library’ (MitoRL) and ‘local library’ and‘locallibrary’ (MitoRL) library’ ference rcentage of bases with high(phred quality with ofbases rcentage delity recovery of biodiversity for bulk ofbiodiversity recovery delity GigaScience, American Naturalist,

2:4 2:4 and the Mediterranean andthe . Plos One,

7(7),

162,

Accepted climatic factors. Multiple regression oftherelationshipbetween model average nucleotide diversity and ArticleAppendix S6. bbmap) inthe ADSlocality. by ofthenumbermappedreads tw Example Appendix S5 and GMYC-groups. Linnaeanestimatedreads.for fromshotgun Data isshown (morphological species species) Scatterplot ofthe relationship betweendiversity nucleotidePCR-barcodes estimatedfrom and nucleotide diversity estimated fromshotgunreads Appendix S4. This article isprotected Allrights bycopyright. reserved. . Comparison between read mappers . Comparison mappers betweenread Relationship between nucleotidediversityestimated PCR-barcodesand from Climatic drivers ofaverage nucleotide diversity o different mapping so different mapping o

ftware (Geneious and and (Geneious ftware Accepted Article and to re-assembly inGeneious) identicaltoaPCR- Proportion ofMMG-barcodes ambiguous ofmitochondr sitesineachset sitesandaver withambiguous MMG-barcodes withambiguitiesTable 1.Proportionofcontigs This article isprotected Allrights bycopyright. reserved. 1 ±(mean SD) bases #Discrepant contigs Proportion ofidentical ±(mean SD) #Ambiguous sites ambiguous contigs Proportion of MitoRL LocL CombL isn ntersetv aae 1 nMtR,1 nLc n nCmL. inand 3 CombL). LocL in in MitoRL, 12 (10 dataset respective the missing in Comparisons could not beperformed for 25contigs the because correspondingwere PCR-barcodes 1

. . 78±1. 2.7± 5.1 7.8± 14.4 3.8 ± 6.1 1.4± 0.93 76.0% 90.7% 78.8% 2.6± 2.2 4.5 ± 5.2 13.5% 3.1% 15.2% number of discrepant bases. bases. number ofdiscrepant ial genomes (MitoRL, LocL andCombL). age (± standard deviation) number of (± age standarddeviation) introduced by the re-assembly in Geneious. inGeneious. re-assembly the by introduced barcode (excluding the ambiguous sitesdue ambiguous (excluding the barcode Accepted Article than 10 discrepancies, the morphol the than 10discrepancies, This article isprotected Allrights bycopyright. reserved. of contigs number withacertain the re-ass introducedby number ofambiguities datase each showingFigure 1.Histograms for 1 full set. PCR-barcodes quality isthe mean and SD across barcodes, wh discrepancies w discrepancies ogical species isindicated. ogical species t (a) the number of contigs with acertain with the numberofcontigs t (a) embly in Geneious and (b) the numberof and(b)the embly inGeneious ile quality of shotgun reads isa proportion value for the ith aPCR-barcode

1 . In. cases withmore Accepted Article This article isprotected Allrights bycopyright. reserved. species. tothe corresponds ofthecircles The size number ofPCR-barcodes/number fo used sequencing barcoding Sanger-based the numberofMMG the relationshipbetween MMG-specific SNPsfo and thetotalnumberof third codonposition.Note:Thetotalnumberof taking intoaccount SNPs, number ofMMG-specific showing Histogram Figure 2.(a) thenumber of specimens for a givenof specimens a species for dataset). inthe full tal number of sequences available forthe available ofsequences tal number r computing the PCR-barcodes SNPs(i.e. thePCR-barcodes r computing of MMG-barcodes that introduce a certain a thatintroduce of MMG-barcodes -specific SNPs and the success of the ofthe success andthe SNPs -specific MMG-barcodes with specific SNPsis21 withspecific MMG-barcodes r the full dataset is 79. (b) Scatterplot of the fulldatasetis79.(b) r if they occurred in first, secondor infirst, ifthey occurred

Acceptedlusitanicum Cryptocephalus octoguttatus populations ofthespeciespresentinmo Figure 3.Nucleotide diversity estimated fromPCR-barcodes and reads shotgun forthe Article This article isprotected Allrights bycopyright. reserved. ; GonOli: Gonioctena olivacea , CryTib: Cryptocephalus tibialis re than 50% of the localities (CryOct: ofthelocalities(CryOct: re than50% ; SmaCon: Smaragdina concolor ; ExoLus: Exosoma )

Acceptedare transformed. natural-logarithm line(dashedred) Both theregression reads. (b)GMYC-group or morphological species Figure 4.Scatterplot ofthe relationshipnucleotide between diversity estimated foreach (a) Article This article isprotected Allrights bycopyright. reserved. and the 1:1 line (dashed blue) are shown. Values shown. Values are 1:1line (dashedblue) and the from PCR-barcodes and estimated from shotgun estimated fromshotgun

Accepted forthefulldataset (b, d).Resultsarepresented shotgun using reads mo PCR-barcodes andfrom inte 0.95confidence and value Figure 5.Mean Article This article isprotected Allrights bycopyright. reserved. rvals of nucleotide diversity computed from ofnucleotidediversity rvals (a, b) and detailed for b) anddetailed (a, rphological species (a, c) and GMYC-groups c) (a, rphological species each locality (c, d). d). (c, locality each