Bulk De Novo Mitogenome Assembly from Pooled Total DNA ... · quencing (NGS) technologies a nd...

Article

Bulk De Novo Mitogenome Assembly from Pooled Total DNAElucidates the Phylogeny of Weevils (ColeopteraCurculionoidea)Conrad PDT Gillett12 Alex Crampton-Platt13 Martijn JTN Timmermans14 Bjarte H Jordal5

Brent C Emerson26 and Alfried P Vogler14

1Department of Life Sciences Natural History Museum London United Kingdom2School of Biological Sciences Centre for Ecology Evolution and Conservation University of East Anglia Norwich United Kingdom3Department of Genetics Evolution and Environment University College London London United Kingdom4Department of Life Sciences Silwood Park Campus Imperial College London Ascot Berkshire United Kingdom5The Natural History Museum University Museum of Bergen Bergen Norway6Island Ecology and Evolution Research Group Instituto de Productos Naturales y Agrobiologıa La Laguna Tenerife Canary IslandsSpain

Corresponding author E-mail cgillettueaacuk avoglernhmacuk

Associate editor Stephen Wright

Abstract

Complete mitochondrial genomes have been shown to be reliable markers for phylogeny reconstruction among diverseanimal groups However the relative difficulty and high cost associated with obtaining de novo full mitogenomes havefrequently led to conspicuously low taxon sampling in ensuing studies Here we report the successful use of an eco-nomical and accessible method for assembling complete or near-complete mitogenomes through shot-gun next-gener-ation sequencing of a single library made from pooled total DNA extracts of numerous target species To avoid the use ofseparate indexed libraries for each specimen and an associated increase in cost we incorporate standard polymerasechain reaction-based ldquobaitrdquo sequences to identify the assembled mitogenomes The method was applied to study thehigher level phylogenetic relationships in the weevils (Coleoptera Curculionoidea) producing 92 newly assembledmitogenomes obtained in a single Illumina MiSeq run The analysis supported a separate origin of wood-boring behaviorby the subfamilies Scolytinae Platypodinae and Cossoninae This finding contradicts morphological hypotheses propos-ing a close relationship between the first two of these but is congruent with previous molecular studies reinforcing theutility of mitogenomes in phylogeny reconstruction Our methodology provides a technically simple procedure forgenerating densely sampled trees from whole mitogenomes and is widely applicable to groups of animals for whichbait sequences are the only required prior genome knowledge

Key words next-generation sequencing genomics MiSeq mitochondria phylogenetics wood-boring

IntroductionWith the advent of high-throughput next-generation se-quencing (NGS) technologies and their ability to generatelarge amounts of data suitable for genomic assembly system-atists are increasingly adopting such methods to reconstructcomplete mitochondrial genomes (mitogenomes) to inferphylogenies across a diverse range of taxa Such researchhas provided compelling insights in studies ranging fromthe investigation of deep-level metazoan relationships(Osigus et al 2013) to those within single phyla (egCnidaria Kayal et al 2013) orders (eg PrimatesFinstermeier et al 2013) families (eg Braconidae waspsWei et al 2010) and genera (eg Architeuthis giant squidWinkelmann et al 2013) Mitogenomes have an intrinsic suit-ability for phylogenetic analysis due to their unambiguousorthology (Botero-Castro et al 2013) phylogenetic signal atdiverse taxonomic ranks (Bernt et al 2013) broadly uniform

rate of molecular evolution (Papadopoulou et al 2010) anduniparental inheritance consistent with bifurcating phyloge-netic trees (Curole and Kocher 1999) even if phylogeneticanalyses may be confounded by inconsistencies of the coa-lescent history near the species level (Funk and Omland 2003)and by lineage-specific compositional and rate heterogeneityat higher hierarchical levels (Sheffield et al 2009 Bernt et al2013 Cameron 2014) In addition the fact that mitochondrialDNA (mtDNA) is present in multiple copies per cell facilitat-ing its amplification and sequencing has undoubtedly con-tributed to the wide use of mitochondrial markers inphylogeny reconstruction However in spite of these advan-tages complete mitogenome sequencing has been compara-tively labor intensive and costly resulting in oftenconspicuously few newly generated mitogenomes per study(eg 17 bird mitogenomes in Pacheco et al [2011] four com-plete Cnidarian mitogenomes in Kayal et al [2013] and

The Author 2014 Published by Oxford University Press on behalf of the Society for Molecular Biology and EvolutionThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) which permits unrestricted reuse distribution and reproduction in any medium provided theoriginal work is properly cited Open AccessMol Biol Evol 31(8)2223ndash2237 doi101093molbevmsu154 Advance Access publication May 6 2014 2223

at University of E

ast Anglia on July 24 2014

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1 cockroach and 13 termite mitogenomes in Cameron et al[2012]) Techniques have almost always included either shot-gun sequencing of expensive multiple-indexed libraries(Botero-Castro et al 2013) or a target-enrichment stepsuch as primer walking using standard polymerase chain re-action (PCR) amplification of overlapping fragments (Botero-Castro et al 2013) long-range PCR followed by eithersequencing-primer walking (Roos et al 2007) or shot-gunsequencing (Timmermans et al 2010) and hybrid-captureusing sheared long-range PCR products as ldquobaitsrdquo immobi-lized on magnetic beads (Winkelmann et al 2013) Althoughthese techniques can generate full mitochondrial genomeseach of them has limitations that generally restrain thenumber of taxa or samples that can be incorporated econom-ically within a study

This study aims to address this sampling bottleneck bytesting the possibility of parallel de novo mitogenome assem-bly from a single library of pooled genomic DNA from a bulksample consisting of many species This method has recentlybeen applied to sequencing of environmental samples of ar-thropods from a rainforest canopy (Crampton-Platt ALTimmermans MJTN Gimmel ML Kutty SN Cockerill TDKhen CV Vogler AP unpublished data) Here we apply thistechnique to investigate the higher level phylogeny of an ex-tremely diverse superfamily of insects the weevils(Coleoptera Curculionoidea) Mitogenome sequences inthe Coleoptera have to date been accumulated graduallyfor major lineages including the four suborders mostlyusing Sanger sequencing (Sheffield et al 2008 2009 Ponset al 2010 Song et al 2010 Timmermans et al 2010)These studies consistently encountered difficulties in resolv-ing basal relationships in Coleoptera due to apparent com-positional heterogeneity (Sheffield et al 2009 Song et al 2010)and markedly different rates of molecular evolution (Ponset al 2010) However it is not known whether heterogeneitythat confounds deep-level divergences also affects subcladesfor example at the level of superfamilies and families(Cameron 2014) In addition the effect of different data par-titioning schemes remains to be investigated across taxo-nomic levels (Cameron 2014)

The Curculionoidea are composed of no fewer than 62000described species distributed wherever terrestrial plants grow(Oberprieler et al 2007) The current higher level classificationproposed by Bouchard et al (2011) recognizes nine extantfamilies among which the Curculionidae s str is by far thelargest containing at least 51000 species in 17 subfamilies and292 tribes and subtribes The phylogenetic classification of theweevils was recognized by the eminent beetle taxonomistCrowson (1955) as ldquo probably the largest and most impor-tant problem in the higher classification of Coleoptera rdquoSince that time there have been considerable advances in ourunderstanding of the phylogeny of this group with significantmorphological analyses by Kuschel (1995) and Marvaldi(1997) More recently molecular data have contributed to-ward reconstructing weevil higher level relationships includ-ing studies by McKenna et al (2009) Hundsdoerfer et al(2009) and Jordal et al (2011) which each incorporated be-tween two and six gene markers A recent analysis of 27 weevil

mitogenomes using 12 protein-coding genes (Haran et al2013) supported the paraphyly of Curculionoidea s str ascurrently defined because the subfamily Platypodinae wasrecovered in a distant position in a clade with the familiesDryophthoridae and Brachyceridae that together were sisterto all other Curculionoidea Although undertaken with lim-ited taxon sampling within the Curculionoidea s str (18tribes) this last study also supported the division of thefamily into two large clades One comprising the ldquobroad-nosedrdquo weevils (subfamilies Entiminae Cyclominae andHyperinae) and another containing the remaining subfamilies(except for Platypodinae) In the same study a tRNAAla totRNAArg gene order rearrangement was identified in a clusterof six tRNA genes located between nad3 and nad5 whichappears to be a synapomorphy for the ldquobroad-nosedrdquo weevilsubfamilies further supporting their monophyly This topol-ogy was consistent with that proposed by McKenna et al(2009) who concluded that the initial diversification of wee-vils occurred on gymnosperm plants during the Early to earlyMiddle Jurassic

The Platypodinae is one of several weevil subfamilies thatare specialist wood-borers together with the bark-beetles(Scolytinae) and the subfamily Cossoninae although othersubfamilies also contain xylophagous members (egMolytinae Cryptorhynchinae and Conoderinae) The evolu-tion of wood-boring behavior was investigated in detail byJordal et al (2011) whose analyses incorporated morpholog-ical characters together with molecular data concluding thatboth Scolytinae and Platypodinae are derived lineages withinthe Curculionoidea sensu Oberprieler et al (2007) Howeverseveral important head characters that underpin this relation-ship are likely to be homoplasious and associated with tun-neling habit (Jordal et al 2011) Thompson (1992) identifieddistinct characters of the platypodine eighth abdominal ster-nite and male genitalia which indicated a distant relationshipto Scolytinae and a possible justification for their inclusion ina separate curculionoid family Therefore the question aboutthe polyphyly of wood-boring lineages remains open and thefailure of previous mitogenome studies to recover the platy-podine and scolytine lineages as monophyletic (Haran et al2013) may be due to limited taxon sampling The issue there-fore may only be resolved if Jordal et alrsquos (2011) comprehen-sive taxon sampling of wood-boring lineages could bematched using mitochondrial genomes

Results

Mitogenomic Assembly

Specimens were selected to represent a wide taxonomic cov-erage and included 173 species from six different families ofCurculionoidea and 16 subfamilies and 104 tribes ofCurculionidae They were acquired from various sourcesand in different stages of preservation leading to variableDNA quality as is common in phylogenetic studies that in-volve lineages for which DNA-ready material is difficult toobtain Individual DNA extracts were not characterized ingreat detail but based on bait PCR success they are likelyto differ in the degree of degradation and purity All DNA

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ownloaded from

extracts were included in a single sequencing pool at equi-molar concentrations although for several including aliquotsfrom 31 specimens already extracted for a previous study(Jordal et al 2011) the available amount of DNA fell shortFollowing sequencing with an Illumina MiSeq approximately5 of the reads resembled mitochondrial sequences afterBLAST filtering (from a total of 18341901 paired-end readsobtained in a single MiSeq run) Assemblies constructed withthe Celera and IDBA-UD assemblers resulted in 338 and 336assemblies of more than 1000 bp respectively rising to 361assemblies when combined using Minimus2 Of these 105were more than 10 kb in length and potentially represented(largely) complete mitogenomes The cumulative distributionof the assemblies by sequence length is shown in figure 1whereas figure 2 represents the frequency distribution of as-sembly lengths for each of the Celera IDBA-UD andMinimus2 assemblies The latter produced a shift towardlonger contigs especially for the critical contig length ofmore than 15 kb that corresponds to the full length ofinsect mitogenomes All subsequent analyses were conductedon the Minimus2 assemblies We were able to newly assembleand identify a total of 92 complete or near-complete mito-genomes comprising at least eight genes including 75 (43 ofall pooled samples) containing the full complement of 15genes a further 15 (87 of pooled samples) containingmore than or equal to 12 genes (supplementary table S1Supplementary Material online) and two assemblies contain-ing eight and nine genes respectively Those falling short of afull-gene complement were mainly lacking the ribosomalRNA (rRNA) genes in particular rrnS which was the leastcommon gene present in only 56 of the assemblies whereasnad6 and cytB were present in all 92 assemblies A majority of86 assemblies contained a portion of the noncoding controlregion whose exact length is difficult to ascertain because ofreduced sequence complexity due to the presence of re-peated regions The mean estimated length of the controlregion was 1190 bp whereas in those 33 mitogenomes thatcould be circularized the length varied between

approximately 200ndash2780 bp (supplementary table S1Supplementary Material online)

Identification of Mitogenomic Assemblies Using ldquoBaitrdquoSequences

From the set of 361 partial and complete contigs obtainedwith Minimus2 a total of 163 cox1 (529ndash1560 bp) 154 cytB(218ndash1147 bp) and 162 rrnL (211ndash1340 bp) gene sequenceswere extracted Sequences from each gene were grouped intolibraries and used as queries in a BLAST search against eachcorresponding bait sequence reference library The latter wascomposed of all successful PCR-based sequences from the 173original DNA extractions and included 84 cox1-50 115 cox1-30132 cytB and 107 rrnL sequences (fig 3) All samples used inthe bulk sequencing were represented by at least one bait (36samples) whereas 42 57 and 36 samples were represented bytwo three and four bait sequences respectively Matchingthese bait sequences to the 92 long mitogenomic assemblies16 assemblies showed a match to one bait 30 assembliesmatched two baits 32 assemblies matched three baits and14 assemblies matched all four baits Four of the completeand near-complete mitogenomes contained sequences fromtwo nonoverlapping assemblies that each matched at leastone bait from the same specimen Out of the remaining 81weevil samples there were 37 instances where baits hit a shortcontig that was not included in the collection of near-com-plete or complete mitogenome assemblies but in 44 in-stances the baits did not hit any of the assembled contigsAdditionally one divergent assembly was rejected because itwas found to match Coleoptera other than weevils in thereference database possibly present in the sample due to acontamination Supplementary table S2 SupplementaryMaterial online summarizes the bait-matching identificationresults by bait for each pooled sample with matching contigsgiven by their unique number and with reasons for identifi-cation failures listed Overall the different baits contributedfairly equally to the final identifications with 56 of all cox1-30

baits leading to a successful identification 53 of cytB 50 ofrrnL and 45 of cox1-50 Proportions of total number of baitsbait hits and hits leading to assembly identifications by geneare illustrated in figure 3 A further 50 short contigs (1025ndash6437 bp mean 2472 bp) matched single baits but were notincorporated in the analyses because they contained only amaximum of four complete protein-coding or rRNA geneseach Their inclusion would have considerably increased theamount of missing data in the matrix

The total number of reads making up each of the 92mitogenomes (which were made up of 96 separate contigs)was used to calculate the sequencing depth (fig 4) The ma-jority of sequences showed a 10ndash50 coverage that generallyresulted in contigs of 15ndash20 kb Coverage reached over 200in a few cases but this did not appear to closely correlate withcontig length For example two contigs of high coverage wereless than 5 kb in length and corresponded to two noncontig-uous fragments from the same species (Dryocoetes autogra-phus) linked by multiple baits obtained from a singlespecimen In addition read coverage was not closely

0

5000

10000

15000

20000

25000

Asse

mbl

y le

ngth

(bp)

Cumulave number of assemblies

IDBA-UD

Celera

Minimus2

FIG 1 Cumulative distribution of assembly lengths from the CeleraIDBA-UD and the combined Minimus2-generated assemblies

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correlated with the initial DNA concentration in the sequenc-ing pool Most samples were present at 10 ng yet their cov-erage varied by more than an order of magnitude whereascoverage for samples present at a concentration up to 4lower varied over the same range (fig 4) Twenty-one of the31 nonassayed genomic samples resulted in assemblies ofmore than or equal to eight genes (of which 17 assembliescontained all 15 genes) We found no taxonomic correlate

IDBAminusUD

IDBAminusUD assembly length

Freq

uenc

y

1000 5000 9000 13000 17000

020

4060

8011

0

Celera

Celera assembly length

Freq

uenc

y

0 3000 7000 12000 17000

020

4060

8011

0

Minimus2

Minimus2 assembly length

Freq

uenc

y

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

020

4060

8011

0

FIG 2 Frequency distribution of assembly lengths from the Celera IDBA-UD and the combined Minimus2-generated assemblies

0

5000

10000

15000

20000

25000

0 50 100 150 200 250 300

Con

g le

ngth

Coverage

0

25

5

75

10

1 10 100 1000

ng g

DNA

Coverage

A

B

FIG 4 Mean sequencing coverage versus (A) assembly (contig) length(bp) and (B) approximate mass of genomic DNA in the sample pool foridentified mitogenomic assemblies Thirty-one samples that were notassayed for DNA concentration are shown at bottom of graph B

0

20

40

60

80

100

120

140

cox1 5 cox1 3 cytB rrnL

Total baits

Total bait hits

Bait hits leading to idenficaon

FIG 3 Relative proportions by gene of total ldquobaitrdquo sequences availableldquobaitrdquo sequences with matching ldquohitsrdquo to the assembled genes andmatching hits that contributed to a successful mitogenome identifica-tion following a BLAST search

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with sequencing or assembly failure because representativesof all six pooled families and 13 of the 16 included subfamiliesof Curculionidae resulted in long assemblies (the three miss-ing subfamilies were represented only by a total of five speci-mens) Specimen size is also unlikely to be the dominantlimiting factor in determining sequencing success becausemany of the small-sized (~2ndash5 mm) Scolytinae producedfull assemblies

Phylogenetic Analyses

The 92 new assemblies were combined with existing data foran aligned data matrix of 122 samples and 13792 positionsOf the final set of mitogenomes 2 belonged to the familyAnthribidae 5 to Attelabidae 3 to Brachyceridae 4 toBrentidae 4 to Dryophthoridae 1 to Nemonychidae and101 belonged to 67 identified tribes within theCurculionidae including 19 tribes of the wood-boringScolytinae The optimal partitioning scheme was establishedusing PartitionFinder starting with a total of 39 partitions (41partitions with the two rRNA genes included) that split all 13genes (15 in data sets A C and E) and three codon positionsin each protein-coding gene PartitionFinder selected five par-titions for the ldquoonly protein-coding genesrdquo data set and sixpartitions for the ldquoall genesrdquo data set whereby the two rRNAgenes were grouped with the first codon positions of nad2nad3 and nad6 and the second codon position of atp8(table 1) For both data sets the first and third codon posi-tions on forward and reverse strands were split into separatepartitions whereas all second positions were collapsed into asingle partition Forward and reverse genes mainly differed inbase frequencies with a shift from A to T and G to C in thereverse strand partitions and rates shifted accordingly (nor-malized to the time-reversible G-T changes supplementaryfig S3 Supplementary Material online) The data set contain-ing ldquoonly protein-coding genes R-Y codedrdquo resulted in onlytwo partitions separating first and second codon position for

both strands combined (third positions are removed fromthis data set) The findings are in accordance with previousobservations on Curculionoidea that also showed a great im-provement in likelihood values when partitioning by bothcodon position and strand (Haran et al 2013) reflectingthe great differences in codon usage in genes coded oneither strand (also see Pons et al 2010) However this doesnot extend to produce differences in variation in amino acidchanges as forward and reverse strands were consistentlygrouped into a single partition for the data set using secondposition only and for the R-Y-coded matrix (eliminating firstcodon synonymous changes)

The maximum-likelihood (ML) trees were greatly im-proved using six partitions over an unpartitioned analysisbut the benefit of using a model with 41 or 39 separatepartitions was low as seen from the small additional improve-ment in the Akaike information criterion (AIC) values(table 2) Interestingly the improvement in ML from usingthe partitioned models was very similar whether the treeswere obtained directly under the partitioned model or ob-tained under the unpartitioned model but with the likelihoodcalculated under partitioning (table 2) Hence despite thegreatly improved likelihood scores after partitioning the re-sulting trees differ only slightly in parameters of greatestimpact on the likelihood Indeed the topologies are littlechanged between searches using the unpartitioned model6-partition model (5-partition model without rRNA genes)and the 41 (39) partition model and hence there was only asmall increase in likelihood if the simpler model is imposed onthe tree obtained with the more complex model

ML trees obtained with the various coding schemes(including or excluding rRNA genes R-Y coding presenceof third codon position supplementary table S4Supplementary Material online) also resulted in highly con-gruent topologies based on strongly supported (gt80 boot-strap analysis [BS]) nodes Figure 5 depicts the best RAxMLtree obtained with the ldquoall genesrdquo data set under six partitions

Table 1 Partitioning Schemes and Nucleotide Substitution Models Selected by PartitionFinder for Two Data Sets According to Gene and toCodon Position (Numbered 1ndash3) in Protein-Coding Genes

Partition Nad2 cox1 cox2 atp8 atp6 cox3 nad3 nad5 nad4 nad4L nad6 cytB nad1 rrnL rrnS

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

All genes

P1 X X X X X X

P2 X X X X X X X X X X X X

P3 X X X X X X X X X

P4 X X X X X

P5 X X X X

P6 X X X X

Only protein-coding genes

P1 X X X X X X X X X X



P4 X X X X

P5 X X X X

NOTEmdashReverse strand transcribed genes are indicated in light gray and the rRNA genes in dark gray Separate partitions are numbered P1ndashP6 and allocated positions to eachpartition labeled X

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Indicated on this tree are nodes that are retained in the strictconsensus of trees obtained from all different treatments ofthe data and those nodes unresolved in the strict consensusthat is the nodes whose resolution is consistent with thestrict consensus Nodes with high nodal support (80ndash100BS) occurred throughout the entire span of nodal ages andthis pattern is found across all analyses (supplementary fig S5Supplementary Material online) Results obtained from thethree additional smaller subsets of data indicate that the treesobtained using the plus- and minus-strand-encoded subsetsof genes (supplementary figs S8 and S9 SupplementaryMaterial online) agree well with the full matrix-derivedtrees but importantly those constructed using only the ldquobaitrdquosequences (supplementary fig S6 Supplementary Materialonline) contain much lower nodal support than any of themitogenomic trees This is expected from a data matrix thathas much missing data which consequently does not allowfor robust inference of relationships

The data set also allowed us to address the question aboutthe hierarchical level at which the confounding effects ofcompositional heterogeneity may be encountered (Sheffieldet al 2009 Song et al 2010) The w2 test of base heterogeneity(Swofford 2002) revealed that with only one exception (atp8)the data are heterogeneous by this test (supplementary tableS7 Supplementary Material online) In contrast the R-Yrecoded data stripped for third positions indicated thatmost genes are homogeneous by this test although not forthe concatenated complete matrix However the more de-fensible test of Foster (2004) showed that only cox3 cytb andnad1 are homogenous in composition Hence the issues ofheterogeneity persist at a much lower hierarchical level thanthe subordinal and superfamily-level relationships investi-gated previously (Sheffield et al 2009 Song et al 2010)

Family-Level Relationships

All 15 analyses recovered the monophyletic ldquoambrosia bee-tlesrdquo Platypodinae (100 BS) outside the other ldquotrue weevilsrdquo

(=Curculionidae sensu Bouchard et al 2011) which wouldotherwise be monophyletic In most analyses except thoseincluding R-Y-coded protein-coding genes Platypodinae wasplaced in the sister clade to the rest of Curculionidae togetherwith the Dryophthoridae (palm weevils) and the brachyceridgenus Ocladius with moderate to strong support forthis adelphic relationship (62ndash95 BS) In all analyses themonophyletic Brentidae (100 BS) were recovered as thesister taxon to a Curculionidae + Dryophthoridae +Brachyceridae clade with very strong nodal support (100BS) The sister relationship between the monophyletic(100 BS) Attelabidae (leaf-rolling weevils) and this latterclade plus Brentidae was similarly very strongly supported(100 BS) across all analyses The Nemonychidae was consis-tently recovered as sister to the clade containing Attelabidaeand all other weevil families mentioned so far Support for thisrelationship was very high ranging from 98 to 100BS across analyses The two taxa belonging to theAnthribidae were always recovered as monophyletic (100BS) Within the Attelabidae the subfamilies Apoderinae andRhynchitinae were recovered as monophyletic with BS sup-port of 100 and 83ndash97 respectively across analyses

Relationships within Curculionidae s str

In most analyses the subfamily Bagoinae represented only bya single Bagous was recovered as the sister to all otherCurculionidae (excepting Platypodinae as noted above)with BS support between 66 and 91 Similarly most anal-yses resulted in the recovery of both a monophyleticEntiminae + Cyclominae + Hyperinae clade (marked A infig 5 100 BS) and a strongly supported sister relationshipbetween this clade and a second clade (marked B in fig 5)containing all other Curculionidae subfamilies (100 BS)Within the entimine clade the Entiminae itself is not recov-ered as monophyletic because the tribe Sitonini is consistentlyrecovered (100 BS) either as sister to the clade containingHyperinae + Cyclominae + the rest of Entiminae or in a

Table 2 ML of Trees under Different Partitioning Schemes

Data Set Partitioning Scheme TopologicalConstraint

Number ofPartitions

SubstitutionModel

Number ofParameters

Ln L AIC AIC

All genes Unpartitioned (one partition) None 1 GTR 8 787773 1575562 62885PartitionFinder (six partitions) On one partition tree 6 GTR 48 758061 1516219 3349Genecodon-position (41 partitions) On one partition tree 41 GTR 328 756379 1513414 737Genecodon-position (41 partitions) On six partition tree 41 GTR 328 756272 1513199 522PartitionFinder (six partitions) On 41 partition tree 6 GTR 48 758010 1516116 3439Genecodon-position (41 partitions) None 41 GTR 328 756010 1512677 naPartitionFinder (six partitions) On one partition tree 6 GTR 48 758061 1516219 3542

Protein-codinggenes

Unpartitioned (one partition) None 1 GTR 8 684161 1368339 34473Genecodon-position (39 partitions) On 1 partition tree 39 GTR 312 666834 1334219 425PartitionFinder (5 partitions) None 5 GTR 40 668480 1337039 3173Genecodon-position (39 partitions) On five partition tree 39 GTR 312 666678 1333981 115PartitionFinder (five partitions) On 39 partition tree 5 GTR 40 668523 1337127 3261Genecodon-position (39 partitions) None 39 GTR 312 666621 1333866 naPartitionFinder (five partitions) On one partition tree 5 GTR 40 668567 1337213 3347

NOTEmdashTrees were obtained under no partitioning under the six- or five-partition schemes selected by PartitionFinder and by the maximum number of partitions tested(partitioning by gene and codon position) Each of the resulting trees was then assessed for their likelihood under the alternative models Note the comparatively small differencein likelihood (AIC) under each partitioning scheme regardless of the model used in the tree search

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Phy

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Mon

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Mel

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Mag

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Mag

dalis

sp

(Ital

y)M

ES

O L

aem

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Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

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ni C

ampt

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nus

sp (A

ustra

lia)

BA

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Mel

anob

aris

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(Fra

nce)

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Cop

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hina

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sp

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sp (I

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Lep

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pyru

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na)

MO

LY H

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turb

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CO

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mat

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porc

atus

(Fra

nce)

CU

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min

i A

ntho

nom

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omor

um (F

ranc

e)C

UR

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erat

opin

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erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

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2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

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ther

curc

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veth

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bels

colo

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Num

bers

adja

cen

tto

nod

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eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

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hi

ghlig

hted

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dT

heth

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pri

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pal

woo

d-bo

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bfam

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are

rep

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nte

dby

dash

edbr

anch

esan

dth

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odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

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nid

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indi

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dto

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ith

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rear

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gem

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are

indi

cate

dSc

ale

bar

rep

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subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

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triac

us (F

ranc

e)AT

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ae B

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li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

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AC B

rach

ycer

inae

Bra

chyc

erus

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us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

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inae

Cos

mop

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s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

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hync

hoph

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ae S

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gran

ariu

s (F

ranc

e)D

RYO

Orth

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thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

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i D

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s un

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neus

(PN

G)

PLA

T P

laty

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ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

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Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

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s sp

(Aus

tralia

)E

NTI

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piph

orin

i C

atas

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s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

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xan

thog

raph

us (R

SA

)E

NTI

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tini

Oph

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sp (U

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)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

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min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from

sister clade also containing the Hyperinae (with generallyweak nodal support for this relationship) Three entiminetribes are consistently recovered as monophyletic withstrong nodal support the Otiorhynchini (100 BS)Brachyderini (100 BS) and the Naupactini (100 BS) Thetribe Tropiphorini is apparently paraphyletic because a well-supported clade (95 BS) containing two monophyleticAustralian members (Catasarcus and Leptopius) is itselfsister to the Naupactini with strong support (96 BS) andis only distantly related to the other Tropiphorini species inthe data set (Tropiphorus) which is sister to theOtiorhynchini with strong nodal support (100 BS) AllEntiminae (except Sitona) are marked by an ARNSEF toRANSEF rearrangement in the tRNA cluster discovered inearlier studies (Song et al 2010 Haran et al 2013) and cor-roborated here (fig 5) One taxon Dichotrachelus manueliclassified in Cyclominae by Alonso-Zarazaga and Lyal (1999)also possesses this same rearrangement whereas the remain-ing Cyclominae taxa possess the common gene orderARNSEF Sitona and Hypera were characterized by uniqueRNSAEF and REANSF gene orders respectively first observedby Haran et al (2013) and hypothesized to constitute aninitial step in the evolution of the derived gene order of theEntiminae Here Hypera + Sitona form a clade that is sisterto all others in clade A whereas the Cyclominae (minusDichotrachelus) not represented in Haran et al (2013) andexhibiting the ancestral gene order occupy the next node assister to the remaining Entiminae characterized by the derivedgene order This demonstrates that the gene order changes inHypera and Sitona are independent of those in Entiminae

Within the second main curculionid clade the scolytinetaxon Coptonotus (Coptonotini) is never recovered togetherwith the bulk of the scolytines which except for Scolytini(monophyletic with 100 BS) are consistently recovered ina clade with moderate to high support values of 66ndash100The scolytine tribes Corthylini and Ipini are always recoveredas monophyletic (100 BS support) within this The followinghigher level taxa from the second main Curculionidae cladeare recovered as monophyletic across all analyses (BS sup-ports follow taxon name) Ceutorhynchinae (100) Lixinae(100) Conoderinae Lobotrachelini (100) andCurculioninae Cionini (100) The Cryptorhynchini appearsto be paraphyletic owing to the presence of a sample(Cryptorhynchini sp from Cameroon) falling outside thewell-supported clade (98 BS) comprising all four othergenera analyzed

Discussion

Contig Formation from Pooled Total DNASequencing

Our results provide a clear demonstration of economic effi-cient and reliable sequencing assembly and identification oflarge numbers of mitogenomes from a pool of total DNA ofnumerous samples without any enrichment or PCR amplifi-cation We obtained a complete or near-complete set of pro-tein-coding genes for well over 50 of all samples attemptedOther recent papers attempting to generate full

mitochondrial genomes from total DNA either generated aseparate library for each taxon (Williams et al 2014) or pooledonly a small number of distantly related taxa (Rubinstein et al2013) We have been able to employ the resulting sequencedata to reconstruct a higher level phylogeny of the superfam-ily Curculionoidea that is highly congruent with recent mo-lecular phylogenies and provides additional evidence for theconvergent evolution of specialized wood-boring behaviorand morphology in weevils The method has been exploredpreviously for the analysis of bulk insect samples from a forestcanopy (Crampton-Platt AL Timmermans MJTN GimmelML Kutty SN Cockerill TD Khen CV Vogler AP unpublisheddata) applied to nearly 500 individuals from more than 200species They found that the assembly of mitogenomes frombulk samples is hampered by substantial differences in DNAconcentration for species in the pool due to variation in bothbody size and number of specimens representing a species Inaddition intraspecific variation was found to cause difficultieswith assembly due to polymorphisms mirroring the well-known problem with genome assembly from heterozygotes(eg Langley et al 2011) The design of this study was ex-pected to avoid these problems by normalizing the DNAconcentration in the pool and by selecting a single individualper species However we find that there is no close correlationof sequencing depth and assembly success (fig 4) in accor-dance with Crampton-Platt AL Timmermans MJTN GimmelML Kutty SN Cockerill TD Khen CV and Vogler AP (unpub-lished data) Our study excludes the presence of intraspecificvariation but indicates that there is a sequencing depth atwhich assemblers no longer operate optimally possibly due tothe larger numbers of individual sequencing errors contrib-uted by overlapping reads

A concern of pooled assemblies is the formation of chi-meras by the miss-assembly of different mitogenomes Thepotential for this is expected to increase if closely relatedsamples that may not differ in conserved regions of the mito-genomes are included in the pool The prevalence of chimeraswas tested using 77 taxa for which multiple baits were avail-able In many cases these tests involved both the cytb or rrnLand the two fragments of the cox1 gene that map to distantpositions in the mitogenome We did not observe a singlecase of chimera formation In addition the tree topology gaveno reason to suggest chimeras because of the monophyly ofthe smaller families of Curculionoidea whereas chimera for-mation would also have produced great differences in thelength of terminal branches which were not observed

Phylogenetic Analysis from Densely SampledMitogenomes

Together with existing mitogenome sequences a total of 120terminals were included in the phylogenetic analysis As mito-genome data sets increase with the numbers of taxa neededfor dense sampling this may produce problems with treesearches and model choice Specifically the most complexmodels such as the amino acid-based CAT model used byTimmermans et al (2010) that was required for resolving thedeep-level relationships within the Coleoptera are not

2231


niversity of East A



ownloaded from

practical when the number of taxa becomes larger This raisesthe question of what is the value of using complex modelsHaran et al (2013) have shown that likelihood trees of weevilscan be substantially improved under model partitioning ac-cording to 1) codon position and 2) forward versus reversestrand the latter presumably due to the well-established dif-ferences in codon usage on either strand We conducted aformal analysis to test whether this partitioning scheme bystrand and codon captures the most important aspects of thenucleotide variation using the PartitionFinder software start-ing from 41 potential partitions of each codon position withineach gene This could be reduced to the codon positions forall genes on either strands similar to Haran et al (2013) butmaintaining a single partition for the second codon positionon either strand while adding a separate partition for therRNA genes not included in that study The use of these sixpartitions over the full set of 41 partitions led only to a smallreduction in likelihood whereas the unpartitioned modelswere substantially worse (table 2)

A general difficulty for comparing models is that compar-isons are only possible for a single topology but searchesunder different partitions favor different topologies Wetherefore used the optimal trees obtained under no partition-ing and the 6- and 41-partition schemes to assess likelihoodsof the alternative partitioning schemes on those three topol-ogies The likelihoods on all trees for the three models werealmost identical (table 2) indicating that tree topology is nota major deciding factor for the best model Taken at facevalue the 41 partition wins out over the 6 partition schemein all three analyses but the likelihood gain is minor As like-lihood values become very large with the use of numerouswhole mitogenomes AIC values may not be an appropriateapproach to avoid overparameterization unless they are nor-malized for the total likelihood values (Castoe et al 2005) Wetherefore believe the 6-partition scheme is fully adequate Inaddition the practicalities of tree searches on increasinglylarge data sets from full mitogenomes as generated withthe proposed methodology also strongly argue for parameterreduction

Trees obtained from analysis of full mitogenomes were themost robust but those obtained using the subsets of protein-coding genes resulted in good topological approximations tothem (supplementary figs S8 and S9 Supplementary Materialonline) suggesting that phylogenetic signal is largely uniformacross genes and is strengthened with additional data Thiscan be seen by the recovery of certain monophyletic groupssuch as the Cyclominae only possible with the full matrixHowever trees constructed from the ldquobaitrdquo sequences alonewere the least robust due to both the reduced informationcontent (comparable to the reverse strand genes) and toconsiderable missing data

Implications for the Systematics of Weevils

The close relationship linking Platypodinae withDryophthoridae as sister to the Curculionidae s str hasbeen demonstrated multiple times (Marvaldi 1997McKenna et al 2009 Haran et al 2013) and indicates that

the family Curculionidae as presently classified is paraphy-letic The simplified classification system proposed byOberprieler et al (2007) recognizing a broaderCurculionidae also containing the presently definedBrachyceridae and Dryophthoridae as respective subfamilies(sensu Alonso-Zarazaga and Lyal 1999) would be consistentwith our family-level results Our results strongly support therelationships among the curculionoid families at the base ofthe tree which are consistent with most previous molecularanalyses with the exception of the placement ofNemonychidae This family has previously been suggestedto be split off at the most basal node (eg McKenna et al2009) as opposed to Anthribidae in our results but our sam-pling lacks two of the ldquoprimitiverdquo weevil families (Belidae andCaridae) prohibiting a definitive conclusion Our resultsare also consistent with the previously suggested hypothe-sis that the Brentidae are the sister family to all the ldquotrueweevilsrdquo Curculionidae if we include Brachyceridae andDryophthoridae in the latter

A previously described deep split within the true weevilswas confirmed by our substantially increased samplingOne strongly supported clade contains theEntiminae + Cyclominae + Hyperinae and represents themonophyletic and diverse ldquobroad-nosedrdquo weevils so namedbecause of their relatively short and blunt rostrumsRearrangements within the cluster of six tRNA genes are re-stricted to this clade even with our increased taxon coveragefurther supporting its distinctiveness The cyclomine genusDichotrachelus containing the same RANSEF rearrangementas all other Entiminae (except Sitona) in our analysis has beentreated as belonging to the Entiminae by some authors(Meregalli and Osella 2007) on morphological groundsCombined with the low nodal support for its inclusion in amonophyletic Cyclominae (lt50 BS) our tRNA rearrange-ment data are consistent with this opinion The second cladecontaining all other curculionoid subfamilies with the excep-tion of Bagoinae which is placed outside of the two mainclades is much less satisfactorily resolved with only two of itsconstituent subfamilies (Lixinae and Ceutorhynchinae) beingmonophyletic It contains a number of very large subfamiliesincluding the Curculioninae Molytinae BaridinaeCryptorhynchinae and Conoderinae whose relationshipsremain obscure due to a lack of strong nodal supportAlthough the recovery of two tribes within this group beingmonophyletic (Lobotrachelini and Cionini) is encouraging tofurther investigate the confusing topology of this clade sig-nificantly more representative taxon sampling will be re-quired Indeed limitations in taxon sampling are often citedas potentially limiting factors in higher level phylogenetics(Franz and Engel 2010) and this is certainly an importantconsideration in such a large group as the Curculionoidea

An interesting finding is that strong nodal support spansthe full depth of the tree and differing taxonomic ranks (fam-ilies subfamilies and tribes supplementary fig S5Supplementary Material online) This pattern was seen inanalyses of all data sets and under all partitioning modelsA potential criticism of mitochondrial sequence data is thatdue to accelerated evolutionary rates saturation of sites may

2232


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obscure or distort phylogenetic signal at deeper nodes(Talavera and Vila 2011) It is clear from our data that atleast at the intrasuperfamily level in weevils this is not nec-essarily the case with phylogenetic signal being evenly distrib-uted across the estimated 170 My diversification history ofthe weevils (McKenna et al 2009)

Evolution of Wood-Boring Behavior

The wood-boring weevil subfamilies are highly adapted toexcavate galleries either subcortically or in woody tissueand feed on ligneous matter directly or cultivate symbioticfungi in the tunnels as a food source and for this reasonmany are widespread pests of forestry (Oberprieler et al2007) The taxon density of the current analysis nearlymatched the extensive sampling of the wood-boring groupsby Jordal et al (2011) a study that is the basis for suggestingtheir close affinity However in contrast to Jordal et al (2011)our results support the conclusions of Haran et al (2013) andMcKenna et al (2009) indicating that wood-boring lineagesare clearly not monophyletic with Platypodinae consistentlyretrieved as closely related to the Dryophthoridae (andBrachyceridae) in a clade sister to all other Curculionidaesensu Bouchard et al (2011) Although our analyses recoveredneither the Scolytinae nor the Cossoninae as monophyleticand they were never recovered as sister taxa or nested withinthe same clade we cannot confidently conclude as to therelationship between them because only a series of weaklysupported nodes separate the cossonine taxa and Coptonotusfrom the rest of the Scolytinae The latter genus is interestingfor consistently not being recovered in our analyses within thegenerally well-supported Scolytinae clade (exceptingScolytini) Based on morphological characters Coptonotushas been considered to be a transitional taxon betweenPlatypodinae and other Curculionidae (Jordal et al 2011) oralternatively as an intermediate form between Cossoninaeand Scolytinae (Thompson 1992) while also containing mor-phological characters linking it with Cossoninae Thompson(1992) has suggested a close relationship betweenCoptonotini and the scolytine tribe Hylastini based on struc-tures of the aedeagus However our results argue against thisbecause the Hylastini sample (Hylastes opacus) was retrievedwith strong support as the sister of Tomicini and this cladeitself was strongly supported as sister to the Hylesini withinthe main Scolytinae clade

ConclusionsWe have demonstrated the relative ease of efficiently andeconomically obtaining a large number of mitogenomeDNA sequences from a pooled mixture of DNA extractswithout the need for enrichment or species-specific taggingprior to genome pooling Mitogenome sequences are confi-dently identified to specimen with a limited amount of priormtDNA sequence data for each sample and exhibit no errorwith regard to these bait sequences Our mtDNA genomedata yield phylogenetic relationships that are highly congru-ent with prior expectations and provide phylogenetic signalwith robustly supported nodes across a broad range of lineage

divergence times and taxon diversity from family level togeneric level which are consistent across different data par-titioning schemes

It is evident that the efficiency of our approach will be afunction of the relative concentration of mitochondrial tonuclear DNA within a focal group The average coleopterangenome size is estimated to be approximately 065 Gb plusmn 005(httpwwwgenomesizecom last accessed May 10 2014)Under the assumption that the copy number of mtDNAgenomes does not differ substantially across organisms ourapproach should be of broad utility within insect phyloge-netics where mean nuclear genome size is estimated to be122 Gb plusmn 005 However it may be less efficient for taxa withlarger average nuclear genome sizes (eg crustaceans meannuclear genome size = ~445 Gb plusmn 045) A further consider-ation for the implementation of our approach is taxon sam-pling and the mitogenomic assembly pipeline Our samplingfor the higher level taxonomic relationships within theCurculionoidea provides little challenge for the pipeline asmtDNA genomes sampled from different genera exhibit highDNA sequence divergence Genome divergence facilitatesgenome reassembly from a mixed pool of genome fragmentsand the pipeline efficiency will eventually be compromised asmtDNA genome relatedness increases Our data suggest thatthis limit lies somewhere below an uncorrected divergence of10 for cox1 and cytB that characterizes the two species ofCionus (C olens and C griseus) included in our sampling Toascertain genome relatedness thresholds for the reassemblypipeline simulation analyses can be employed However it isimportant to point out that as NGS technology and readlengths improve relatedness thresholds will also becomemore favorable

Materials and Methods

Taxon Sampling DNA Extraction and Quantification

Throughout this study the most recent higher level classifi-cation of Curculionoidea proposed by Bouchard et al (2011)is adhered to whereas the assignment of genera to higher taxafollows the catalog of Alonso-Zarazaga and Lyal (1999) DNAwas extracted from each ethanol-preserved specimen individ-ually using DNeasy blood and tissue extraction kits (Qiagen)The concentration of double-stranded DNA (dsDNA) in mostextractions (139 of 173) was assayed on a Qubit fluorometerusing a dsDNA high-sensitivity kit (Invitrogen)

ldquoBaitrdquo Sequence PCR

Standard PCR reactions to amplify four different fragments ofmtDNA (cox1 50 ldquobarcode regionrdquo cox1 30 region rrnL andcytb) were undertaken for each of the 173 samples Primersand reaction conditions are listed in supplementary table S10Supplementary Material online PCR products were firstcleaned with a size-exclusion filter (Merck Millipore) andthen Sanger sequenced the resulting bait sequences weresubsequently employed to identify mitogenomic assembliesin the manner detailed below

2233


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Sample Pooling and Sequencing

To minimize the effects of DNA concentration on assemblysuccess across all samples approximately equimolar quanti-ties of genomic DNA from each of the samples were pooledaiming for 10 ng of dsDNA per sample resulting in a DNApool of approximately 15mg This calculation did not con-sider 31 samples which were not quantified because of limitedsample volume For each of these a fixed volume of either 5 or8ml was added to the pool Based on the findings ofCrampton-Platt AL Timmermans MJTN Gimmel ML KuttySN Cockerill TD Khen CV and Vogler AP (unpublished data)where longer insert size was found to result in longer mito-chondrial contigs a TruSeq library was prepared from thepool aiming for an insert size of 800 bp Quantification ofthe final library indicated that the average insert size was790 bp and this was sequenced on a single Illumina MiSeqrun (500-cycle 250 bp paired-end reads version 2 reagent kit)

Mitogenomic Assembly Pipeline

The bioinformatics assembly pipeline used in this study wasdeveloped by Crampton-Platt AL Timmermans MJTNGimmel ML Kutty SN Cockerill TD Khen CV and VoglerAP (unpublished data) and is followed here with minor mod-ifications A list of the software required (most freely available)is given in table 3 and a schematic overview of the principalsteps is presented in figure 6 In brief the raw data weretrimmed of adapters using Trimmomatic (Bolger et al2014) and putative mitochondrial reads were identified in aBLAST search (Altschul et al 1990) against a custom referencedatabase of 258 Coleoptera mitogenomes (E = 1e5 no re-striction in length overlap) (Timmermans MJTN Barton CDodsworth S Haran J Ahrens D Foster PG Bocak L andVogler AP unpublished data) The extracted mtDNA readswere subjected to whole-genome shot-gun assembly usingCelera Assembler (Myers et al 2000) and IDBA-UD (Penget al 2012) and the resulting contigs were filtered again formtDNA hits against the Coleoptera reference library for

sequences of more than 1000 bp overlap at E = 1e5 Bothassemblies were merged using Minimus2 (Sommer et al2007) to combine overlapping sequences from both assem-blers into longer scaffolds

To investigate the relationship between the number ofgenerated sequencing reads and assembly success all readswere mapped onto the obtained contigs using Geneious al-lowing for 2 mismatches a maximum gap size of 3 bp andrequiring a minimum overlap of 100 bp Annotations of eachassembly were conducted by first mapping tRNA genes withCOVE (Eddy and Durbin 1994) after which the intervening

Table 3 List of Software Used for the De Novo Assembly and Analysis of Mitogenomes with their Main Function and Source URL

Software Function URLa

FastQC NGS quality assessment httpwwwbioinformaticsbabrahamacukprojectsfastqc

Trimmomatic Adapter trimming httpwwwusadellaborgcmsindexphppage=trimmomatic

Celera Genome assembly httpsourceforgenetappsmediawikiwgs-assemblerindexphptitle=Main_Page

IDBA-UD Genome assembly httpicshkuhk~alsehkubrgprojectsidba_ud

Minimus2 Merging sequence sets httpsourceforgenetappsmediawikiamosindexphptitle=Minimus2

Prinseq Sequence quality control httpedwardssdsueducgi-binprinseqprinseqcgi

COVE tRNA annotation httpselabjaneliaorgsoftwarehtml

FeatureExtract Gene extraction httpwwwcbsdtudkservicesFeatureExtract

Geneious Gene annotationsequence editing httpwwwgeneiouscom

MAFFT Sequence alignment httpmafftcbrcjpalignmentsoftware

BLAST Local alignment search httpblastncbinlmnihgovBlastcgi

PartitionFinder Partitioning scheme selection httpwwwrobertlanfearcompartitionfinder

CIPRES Phylogenetic analysis server httpwwwphyloorg

RAxML Maximum-likelihood phylogenetic analysis httpscoh-itsorgexelixissoftwarehtml

ldquoAPErdquo package in R Phylogenetic analysis httpape-packageirdfr

aAll URLs were last accessed on May 10 2014

DNA extracon

lsquoBaitrsquo PCR(cox1 cytB rrnL)

Sanger sequencing

Idenfied lsquobaitsrsquo

dsDNA concentraon assay

Equimolar sample pooling

NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon

BLAST idenficaonof mitogenomes

with lsquobaitsrsquo

Phylogeny reconstrucon

FIG 6 Schematic flowchart of the principal steps for the bulk de novoassembly of mitogenomes and identification with PCR-amplified ldquobaitrdquosequences

2234


niversity of East A



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protein and rRNA coding genes were extracted withFeatureExtract 12 (Wernersson 2005) To identify thesegenes the resulting sequences were mapped to theTribolium castaneum mitogenome (GenBank accessionnumber NC_003081) using Geneious and were afterward ex-ported by gene into separate FASTA files Sequences of lessthan one-third of total gene length were discarded


To identify the mitogenomic assemblies by association withtheir respective originating specimen BLAST searches wereconducted for each bait sequence reference against all corre-sponding gene sequences extracted from the mitogenomeassemblies (separately for cox1-50 and 30 regions cytB andrrnL) Only hits with 100 pairwise identity and more than100 bp overlap were considered a successful identificationWhere multiple bait sequences from a single specimenwere available each bait was checked to have hit the samelong assembly unequivocally to test for possible chimeras Ifbaits from a single specimen matched multiple nonoverlap-ping assemblies they presumably correspond to the sameincompletely assembled mitogenome These assemblieswere combined and retained if they included eight or moregenes in total Once mitogenomic assemblies were identifiedthe tRNA gene order in the cluster of six tRNA genes locatedbetween nad3 and nad5 was visually recorded

Sequence Alignment and Data Set Concatenation

The sequences for the genes nad5 nad4 nad4L and nad1which are transcribed on the reverse strand of the mitochon-drial genome were reverse complemented prior to alignmentTwenty-eight additional curculionoid mitogenome sequenceswere obtained from GenBank (primarily those generated byHaran et al [2013] supplementary table S1 SupplementaryMaterial online) to maximize taxon sampling Two membersof Chrysomeloidea were included as outgroups followingHaran et al (2013) The combined sequences from each ofthe separated 13 protein-coding and two rRNA genes wereindividually aligned using the MAFFT 70 online server underthe FFT-NS-I slow iterative refinement strategy (Katoh et al2002) Alignments were thereafter checked manually inGeneious for quality and to ensure that protein-codinggenes were in the correct reading frame Genes were conca-tenated together to make six different data matrices as fol-lows All genes (A) only protein-coding genes (B) all geneswith third codon positions removed from protein-codinggenes (C) protein-coding genes only with third codon posi-tions removed (D) all genes with third codon positions re-moved from protein-coding genes and first codon positionsR-Y coded (E) and only protein-coding genes with thirdcodon positions removed and first codon positions R-Ycoded (F)


Each of the six data sets was analyzed under the ML optimal-ity criterion using RAxML 766 (Stamatakis 2006) run on the

CIPRES web-based server (Miller et al 2010) To assess nodalsupport a rapid BS with 1000 iterations was run in parallelwith tree-building The data sets were each analyzed bothpartitioned by gene and unpartitioned (ie a single partition)Additionally three of the data sets (A B and E) were firsttested using PartitionFinder (Lanfear et al 2012) to objectivelyselect the best-fitting partitioning scheme and model of mo-lecular evolution for each alignment This was performedusing the Bayesian Information Criterion from an initialpartitioning of each of the three codon positions for eachamino acid-coding sequence and each rRNA gene beingseparate partitions The resulting ML trees were made ultra-metric using the chronos function of the APE package in R(Paradis et al 2004) which uses penalized likelihood to fit achronogram to a phylogenetic tree (Paradis 2013) To obtaina measure of the suitability of the mitogenomic data torobustly support relationships across different nodal ages(putative taxonomic ranks) we investigated the distributionof nodal support across trees by calculating the branch lengthfrom the root for each node using a custom R script andplotting this against its respective RAxML BS support Wealso constructed a strict consensus tree from the 15 MLtrees to visualize the distribution of consistent nodesacross all our analyses We performed additional RAxML anal-yses on data sets A and B partitioned by gene and separatecodon positions for each protein-coding gene (41 and 39partitions respectively) and various RAxML analyseson these two data sets with different combinations ofpartitioning schemes and topological constraints assummarized in table 2 in order to calculate the AIC as ameans for preferred model selection (Posada and Buckley2004)

To investigate how successfully subsets of the full-datamatrix were able to reconstruct the phylogeny we also ana-lyzed (using RAxML with data partitioned by gene and byPartitionFinder-derived partitions) three additional data setscomposed of 1) only the reverse-transcribed protein-codinggenes (nad5 nad4 nad4L and nad1) 2) the remaining nineforward-transcribed protein-coding genes and 3) only theavailable ldquobaitrdquo sequences The latter analysis was undertakento ascertain whether there is any benefit in assembling themitogenomes for phylogeny reconstruction over the PCRsequences alone

Compositional heterogeneity was assessed on theprotein-coding genes using the w2 statistics (Swofford 2002)The resulting P value is the probability that the data are ho-mogeneous and is considered significant when less than 5This test suffers from a high probability of Type II error be-cause the test assumes independence of the data which theyare not We therefore also used the test of Foster (2004)which uses simulations based on the ML tree model anddata size to generate a valid null distribution of a w2 valuefrom the original data The ML tree for the concatenated datawas used in all cases when assessing heterogeneity in eachgene with any missing taxa pruned off Model parametersand branch lengths were reoptimized under a GTR + Gmodel

2235


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Supplementary MaterialSupplementary tables S1 S2 S4 S7 and S10 and figures S3 S5S6 S8 and S9 are available at Molecular Biology and Evolutiononline (httpwwwmbeoxfordjournalsorg)

Acknowledgments

The authors are indebted to the following individuals whohave lent or donated weevil specimens used in this studyMax Barclay Roberto Caldara Christiana Faria MichaelGillett Levent Gultekin James Kitson Christopher LyalMassimo Meregalli Rolf Oberprieler Charles OrsquoBrienPedro Oromı Li Ren and Clive Turner They also thankPeter Foster (NHM) for his analytical assistance and twoanonymous reviewers for their constructive commentsThis work was supported by a Natural EnvironmentResearch Council CASE PhD studentship (NEG01194X1)to CPDTG the University of East Anglia and the NaturalHistory Museum the NHM Biodiversity Initiative theLeverhulme Trust (F00696P) and a NERC PostdoctoralFellowship (NEI0215781) to MJTNT

ReferencesAlonso-Zarazaga MA Lyal CHC 1999 A world catalogue of families and

genera of Curculionoidea (Insecta Coleoptera) (excepting Scolytidaeand Platypodidae) Barcelona Entomopraxis

Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic localalignment search tool J Mol Biol 215403ndash410

Bolger AM Lohse M Usadel B 2014 Trimmomatic a flexible trimmerfor Illumina sequence data Bioinformatics Advance Access pub-lished April 12014 doi 101093bioinformaticsbtu170

Botero-Castro F Tilak MK Justy F Catzeflis F Delsuc F Douzery EJP2013 Next-generation sequencing and phylogenetic signal of com-plete mitochondrial genomes for resolving the evolutionary historyof leaf-nosed bats (Phyllostomidae) Mol Phylogenet Evol 69728ndash739

Bouchard P Bousquet Y Davies AE Alonso-Zarazaga MA Lawrence JFLyal CHC Newton AF Reid CAM Schmitt M Slipinski SA et al2011 Family-group names in Coleoptera (Insecta) Zookeys 881ndash972

Bernt M Bleidorn C Braband A Dambach J Donath A Fritzsch GGolombek A Hadrys H Juhling F Meusemann K et al 2013 Acomprehensive analysis of bilateran mitochondrial genomes andphylogeny Mol Phylogenet Evol 69352ndash364

Cameron SL 2014 Insect mitochondrial genomics implications for evo-lution and phylogeny Annu Rev Entomol 5995ndash117

Cameron SL Lo N Bourguignon T Svenson GJ Evans TA 2012 Amitochondrial genome phylogeny of termites (BlattodeaTermitoidae) robust support for interfamilial relationships and mo-lecular synapomorphies define major clades Mol Phylogenet Evol 65163ndash173

Castoe TA Sasa MM Parkinson C 2005 Modeling nucleotide evolutionat the mesoscale the phylogeny of the neotropical pitvipers of thePorthidium group (Viperidae Crotalinae) Mol Phylogenet Evol 37881ndash898

Crowson RA 1955 The natural classification of the families ofColeoptera London Nathaniel Lloyd amp Co

Curole JP Kocher TD 1999 Mitogenomics digging deeper with com-plete mitochondrial genomes Trends Ecol Evol 14394ndash398

Eddy S Durbin R 1994 RNA sequence analysis using covariance modelsNucleic Acids Res 222079ndash2088

Finstermeier K Zinner D Brameier M Meyer M Kreuz E Hofreiter MRoos C 2013 A mitogenomic phylogeny of living primates PLoSOne 8e69504

Foster PG 2004 Modeling compositional heterogeneity Syst Biol 53485ndash495

Franz NM Engel MS 2010 Can higher-level phylogenies of weevils ex-plain their evolutionary success A critical review Syst Entomol 35597ndash606

Funk DJ Omland KE 2003 Species-level paraphyly and polyphyly fre-quency causes and consequences with insights from animal mito-chondrial DNA Annu Rev Ecol Evol Syst 34397ndash423

Haran J Timmermans MJTN Vogler AP 2013 Mitogenome se-quences stabilize the phylogenetics of weevils (Curculionoidea)and establish the monophyly of larval ectophagy Mol PhylogenetEvol 67156ndash166

Hundsdoerfer AK Rheinheimer J Wink M 2009 Towards the phylogenyof the Curculionoidea (Coleoptera) reconstructions from mito-chondrial and nuclear ribosomal DNA sequences Zool Anz 2489ndash31

Jordal BH Sequeira AS Cognato AI 2011 The age and phylogeny ofwood boring weevils and the origin of subsociality Mol PhylogenetEvol 59708ndash724

Katoh K Misawa K Kuma K Miyata T 2002 MAFFT a novel methodfor rapid multiple sequence alignment based on fast Fourier trans-form Nucleic Acids Res 303059ndash3066

Kayal E Roure B Philippe H Collins AG Lavrov DV 2013 Cnidarianphylogenetic relationships as revealed by mitogenomics BMC EvolBiol 135

Kuschel G 1995 A phylogenetic classification of Curculionoideato families and subfamilies Mem Entomol Soc Washington 145ndash33

Lanfear R Calcott B Ho SYW Guindon S 2012 PartitionFinder com-bined selection of partitioning schemes and substitution models forphylogenetic analyses Mol Biol Evol 291695ndash1701

Langley CH Crepeau M Cerdeno C Corbett-Detig R Stevens K 2011Circumventing heterozygosity sequencing the amplified genome ofa single haploid Drosophila melanogaster embryo Genetics 188239ndash246

Marvaldi AE 1997 Higher level phylogeny of Curculionidae (Coleoptera Curculionoidea) based mainly on larval characters with special ref-erence to broad-nosed weevils Cladistics 13285ndash312

McKenna DD Sequeira AS Marvaldi AE Farrell BD 2009 Temporal lagsand overlap in the diversification of weevils and flowering plantsProc Natl Acad Sci U S A 1067083ndash7088

Meregalli M Osella G 2007 Dichotrachelus kahleni sp n a new weevilspecies from the Carnian Alps north-eastern Italy (ColeopteraCurculionidae Entiminae) Deut Entomol Z 54169ndash177

Miller MA Pfeiffer W Schwartz T 2010 Creating the CIPRES ScienceGateway for inference of large phylogenetic trees In Proceedings ofthe Gateway Computing Environments Workshop (GCE) 2010 Nov14 New Orleans (LA) IEEE p 1ndash8

Myers EW Sutton CG Delcher AL Dew IM Fasulo DP Flanigan MJKravitz SA Mobarry CM Reinert KH Remington KA et al 2000 Awhole-genome assembly of Drosophila Science 2872196ndash2204

Oberprieler RG Marvaldi AE Anderson RS 2007 Weevils weevils wee-vils everywhere Zootaxa 1668491ndash520

Osigus H-J Eitel M Bernt M Donath A Schierwater B 2013 Mitoge-nomics at the base of Metazoa Mol Phylogenet Evol 69339ndash351

Pacheco MA Battistuzzi FU Lentino M Aguilar RF Kumar S EscalanteAA 2011 Evolution of modern birds revealed by mitogenomicstiming the radiation and origin of major orders Mol Biol Evol 281927ndash1942

Papadopoulou A Anastasiou I Vogler AP 2010 Revisiting the insectmitochondrial molecular clock the mid-Aegean trench calibrationMol Biol Evol 271659ndash1672

Paradis E 2013 Molecular dating of phylogenies by likelihood methodsa comparison of models and a new information criterion MolPhylogenet Evol 67436ndash444

Paradis E Claude J Strimmer K 2004 APE analyses of phylogenetics andevolution in R language Bioinformatics 20289ndash290

Peng Y Leung HCM Yiu SM Chin FYL 2012 IBDA-UD a de novoassembler for single-cell and metagenomic sequencing data wothhighly uneven depth Bioinformatics 281420ndash1428

2236


niversity of East A



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Pons J Ribera I Bertranpetit J Balke M 2010 Nucleotide substitutionrates for the full set of mitochondrial protein-coding genes inColeoptera Mol Phylogenet Evol 56796ndash807

Posada D Buckley T 2004 Model selection and model averaging inphylogenetics advantages of Akaike information criterion andBayesian approaches over likelihood ratio tests Syst Biol 53793ndash808

Roos J Aggarwal RK Janke A 2007 Extended mitogenomic phylogeneticanalyses yield new insight into crocodylian evolution and their sur-vival of the Cretaceous-Tertiary boundary Mol Phylogenet Evol 45663ndash673

Rubinstein ND Feldstein T Shenkar N Botero-Castro F Griggio FMastrototaro F Delsuc F Douzery EJP Gissi C Huchon D 2013Deep sequencing of mixed total DNA without barcodes allows ef-ficient assembly of highly plastic Ascidian mitochondrial genomesGenome Biol Evol 51185ndash1199

Sheffield NC Song H Cameron SL Whiting MF 2008 A comparativeanalysis of mitochondrial genomes in Coleoptera (ArthropodaInsecta) and genome descriptions of six new beetles Mol BiolEvol 252499ndash2509

Sheffield NC Song H Cameron SL Whiting MF 2009 Nonstationaryevolution and compositional heterogeneity in beetle mitochondrialphylogenetics Syst Biol 58381ndash394

Sommer DD Delcher AL Salzberg SL Pop M 2007 Minimus a fastlightweight genome assembler BMC Bioinformatics 864

Song HJ Sheffield NC Cameron SL Miller KB Whiting MF 2010 Whenphylogenetic assumptions are violated base compositional hetero-geneity and among-site rate variation in beetle mitochondrial phy-logenomics Syst Entomol 35429ndash448

Stamatakis A 2006 RAxML-VI-HPC maximum likelihood-based phylo-genetic analyses with thousands of taxa and mixed modelsBioinformatics 222688ndash2690

Swofford DL 2002 PAUP phylogenetic analysis using parsimony(and other methods) Version 4 Sunderland (MA) SinauerAssociates

Talavera G Vila R 2011 What is the phylogenetic signal limitfrom mitogenomes The reconciliation between mitochondrialand nuclear data in the Insecta class phylogeny BMC Evol Biol 11315

Thompson RT 1992 Observations on the morphology and classificationof weevils (Coleoptera Curculionoidea) with a key to major groups JNat Hist 26835ndash891

Timmermans MJTN Dodsworth S Culverwell CL Bocak L Ahrens DLittlewood DTJ Pons J Vogler AP 2010 Why barcode High-throughput multiplex sequencing of mitochondrial genomes formolecular systematics Nucleic Acids Res 381ndash14

Wei S-j Shi M Sharkey MJ van Achterberg C Chen X-X 2010Comparative mitogenomics of Braconidae (InsectaHymenoptera) and the phylogenetic utility of mitochondrial ge-nomes with special reference to holometabolous insects BMCGenomics 11371

Wernersson R 2005 FeatureExtractmdashextraction of sequence annotationmade easy Nucleic Acids Res 33W567ndashW569

Williams S Foster PG Littlewood DTJ 2014 The complete mitochon-drial genome of a turbinid vetigastropod from MiSeq Illumina se-quencing of genomic DNA and steps towards a resolved gastropodphylogeny Gene 53338ndash47

Winkelmann I Campos PF Strugnell J Cherel Y Smith PJKubodera T Allcock L Kampmann M-L Schroeder H GuerraA et al 2013 Mitochondrial genome diversity and populationstructure of the giant squid Architeuthis genetics sheds newlight on one of the most enigmatic marine species Proc R SocB 2801759

2237


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1 cockroach and 13 termite mitogenomes in Cameron et al[2012]) Techniques have almost always included either shot-gun sequencing of expensive multiple-indexed libraries(Botero-Castro et al 2013) or a target-enrichment stepsuch as primer walking using standard polymerase chain re-action (PCR) amplification of overlapping fragments (Botero-Castro et al 2013) long-range PCR followed by eithersequencing-primer walking (Roos et al 2007) or shot-gunsequencing (Timmermans et al 2010) and hybrid-captureusing sheared long-range PCR products as ldquobaitsrdquo immobi-lized on magnetic beads (Winkelmann et al 2013) Althoughthese techniques can generate full mitochondrial genomeseach of them has limitations that generally restrain thenumber of taxa or samples that can be incorporated econom-ically within a study

This study aims to address this sampling bottleneck bytesting the possibility of parallel de novo mitogenome assem-bly from a single library of pooled genomic DNA from a bulksample consisting of many species This method has recentlybeen applied to sequencing of environmental samples of ar-thropods from a rainforest canopy (Crampton-Platt ALTimmermans MJTN Gimmel ML Kutty SN Cockerill TDKhen CV Vogler AP unpublished data) Here we apply thistechnique to investigate the higher level phylogeny of an ex-tremely diverse superfamily of insects the weevils(Coleoptera Curculionoidea) Mitogenome sequences inthe Coleoptera have to date been accumulated graduallyfor major lineages including the four suborders mostlyusing Sanger sequencing (Sheffield et al 2008 2009 Ponset al 2010 Song et al 2010 Timmermans et al 2010)These studies consistently encountered difficulties in resolv-ing basal relationships in Coleoptera due to apparent com-positional heterogeneity (Sheffield et al 2009 Song et al 2010)and markedly different rates of molecular evolution (Ponset al 2010) However it is not known whether heterogeneitythat confounds deep-level divergences also affects subcladesfor example at the level of superfamilies and families(Cameron 2014) In addition the effect of different data par-titioning schemes remains to be investigated across taxo-nomic levels (Cameron 2014)

The Curculionoidea are composed of no fewer than 62000described species distributed wherever terrestrial plants grow(Oberprieler et al 2007) The current higher level classificationproposed by Bouchard et al (2011) recognizes nine extantfamilies among which the Curculionidae s str is by far thelargest containing at least 51000 species in 17 subfamilies and292 tribes and subtribes The phylogenetic classification of theweevils was recognized by the eminent beetle taxonomistCrowson (1955) as ldquo probably the largest and most impor-tant problem in the higher classification of Coleoptera rdquoSince that time there have been considerable advances in ourunderstanding of the phylogeny of this group with significantmorphological analyses by Kuschel (1995) and Marvaldi(1997) More recently molecular data have contributed to-ward reconstructing weevil higher level relationships includ-ing studies by McKenna et al (2009) Hundsdoerfer et al(2009) and Jordal et al (2011) which each incorporated be-tween two and six gene markers A recent analysis of 27 weevil

mitogenomes using 12 protein-coding genes (Haran et al2013) supported the paraphyly of Curculionoidea s str ascurrently defined because the subfamily Platypodinae wasrecovered in a distant position in a clade with the familiesDryophthoridae and Brachyceridae that together were sisterto all other Curculionoidea Although undertaken with lim-ited taxon sampling within the Curculionoidea s str (18tribes) this last study also supported the division of thefamily into two large clades One comprising the ldquobroad-nosedrdquo weevils (subfamilies Entiminae Cyclominae andHyperinae) and another containing the remaining subfamilies(except for Platypodinae) In the same study a tRNAAla totRNAArg gene order rearrangement was identified in a clusterof six tRNA genes located between nad3 and nad5 whichappears to be a synapomorphy for the ldquobroad-nosedrdquo weevilsubfamilies further supporting their monophyly This topol-ogy was consistent with that proposed by McKenna et al(2009) who concluded that the initial diversification of wee-vils occurred on gymnosperm plants during the Early to earlyMiddle Jurassic

The Platypodinae is one of several weevil subfamilies thatare specialist wood-borers together with the bark-beetles(Scolytinae) and the subfamily Cossoninae although othersubfamilies also contain xylophagous members (egMolytinae Cryptorhynchinae and Conoderinae) The evolu-tion of wood-boring behavior was investigated in detail byJordal et al (2011) whose analyses incorporated morpholog-ical characters together with molecular data concluding thatboth Scolytinae and Platypodinae are derived lineages withinthe Curculionoidea sensu Oberprieler et al (2007) Howeverseveral important head characters that underpin this relation-ship are likely to be homoplasious and associated with tun-neling habit (Jordal et al 2011) Thompson (1992) identifieddistinct characters of the platypodine eighth abdominal ster-nite and male genitalia which indicated a distant relationshipto Scolytinae and a possible justification for their inclusion ina separate curculionoid family Therefore the question aboutthe polyphyly of wood-boring lineages remains open and thefailure of previous mitogenome studies to recover the platy-podine and scolytine lineages as monophyletic (Haran et al2013) may be due to limited taxon sampling The issue there-fore may only be resolved if Jordal et alrsquos (2011) comprehen-sive taxon sampling of wood-boring lineages could bematched using mitochondrial genomes

Results

Mitogenomic Assembly

Specimens were selected to represent a wide taxonomic cov-erage and included 173 species from six different families ofCurculionoidea and 16 subfamilies and 104 tribes ofCurculionidae They were acquired from various sourcesand in different stages of preservation leading to variableDNA quality as is common in phylogenetic studies that in-volve lineages for which DNA-ready material is difficult toobtain Individual DNA extracts were not characterized ingreat detail but based on bait PCR success they are likelyto differ in the degree of degradation and purity All DNA

2224


niversity of East A



ownloaded from







0

5000

10000

15000

20000

25000

Asse

mbl

y le

ngth

(bp)


IDBA-UD

Celera

Minimus2


2225


niversity of East A



ownloaded from


IDBAminusUD


Freq

uenc

y

1000 5000 9000 13000 17000

020

4060

8011

0

Celera


Freq

uenc

y

0 3000 7000 12000 17000

020

4060

8011

0

Minimus2


Freq

uenc

y

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

020

4060

8011

0


0

5000

10000

15000

20000

25000

0 50 100 150 200 250 300

Con

g le

ngth

Coverage

0

25

5

75

10

1 10 100 1000

ng g

DNA

Coverage

A

B


0

20

40

60

80

100

120

140


Total baits

Total bait hits



2226


niversity of East A



ownloaded from









1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

All genes

P1 X X X X X X



P4 X X X X X

P5 X X X X

P6 X X X X





P4 X X X X

P5 X X X X


2227


niversity of East A



ownloaded from










Number ofPartitions

SubstitutionModel

Number ofParameters

Ln L AIC AIC


Protein-codinggenes



2228


niversity of East A



ownloaded from

01

SC

OL

Sco

lytin

i S

coly

tus

sp (F

ranc

e)S

CO

L S

coly

tini

Sco

lytu

s sc

olyt

us (D

enm

ark)

SC

OL

Dia

mer

ini

Dia

mer

us in

erm

is (T

anza

nia)

SC

OL

Hex

acol

ini

Sco

lyto

des

caud

atus

(Cos

ta R

ica)

SC

OL

Phl

oeot

ribin

i P

hloe

otrib

us s

pinu

losu

s (N

orw

ay)

SC

OL

Hyl

esin

i H

yles

inus

var

ius

(Sw

eden

)S

CO

L H

ylas

tini

Hyl

aste

s op

acus

(Sw

eden

)S

CO

L To

mic

ini

Tom

icus

pin

iper

da (N

orw

ay)

SC

OL

Poly

grap

hini

Pol

ygra

phus

pol

igra

phus

(Sw

eden

)S

CO

L C

rypt

urgi

ni C

rypt

urgu

s pu

sillu

s (N

orw

ay)

SC

OL

Cor

thyl

ini

Pity

opht

horu

s m

icro

grap

hus

(Sw

eden

)S

CO

L C

orth

ylin

i C

orth

ylus

rubr

icol

lis (C

osta

Ric

a)S

CO

L C

ryph

alin

i C

ryph

alus

sal

tuar

ius

(Nor

way

)S

CO

L X

yloc

toni

ni X

yloc

tonu

s m

acul

atus

(RS

A)

SC

OL

Hyp

obor

ini

Hyp

obor

us fi

cus

(Mor

occo

)S

CO

L P

rem

nobi

ini

Pre

mno

bius

cav

ipen

nis

(RS

A)

SC

OL

Ipin

i Ip

s ac

umin

atus

(Nor

way

)S

CO

L Ip

ini

Ips

cem

brae

(Fra

nce)

SC

OL

Dry

ocoe

tini

Dry

ocoe

tes

auto

grap

hus

(Nor

way

)S

CO

L sp

2 (C

hina

)S

CO

L X

yleb

orin

i A

nisa

ndru

s di

spar

(Nor

way

)S

CO

L sp

1 (C

hina

)C

OS

S P

enta

rthrin

i Pe

ntar

thru

s el

umbe

(Eng

land

)CO

SS

sp1

(Chi

na)

CO

NO

Mec

opin

i M

ecop

us s

p (A

ustra

lia)

MO

LY s

p3 (C

hina

)M

OLY

sp1

(Chi

na)

CR

YP

Cry

ptor

hync

hini

sp

(Cam

eroo

n)C

ON

O Z

ygop

ini

Pelto

phor

us s

p (U

SA

)C

RY

P C

rypt

orhy

nchi

ni P

seud

omop

sis

(Sab

a)C

RY

P C

rypt

orhy

nchi

ni A

calle

s au

bei (

Fran

ce)

CR

YP

Cry

ptor

hync

hini

Per

isso

ps s

p (A

ustra

lia)

CR

YP

Cry

ptor

hync

hini

Our

opor

opte

rus

sp (A

ustra

lia)

CU

RC

Cio

nini

Cio

nus

gris

eus

(Can

arie

s)C

UR

C C

ioni

ni C

ionu

s ol

ens

(Fra

nce)

CE

UT

Phy

tobi

ni R

hino

ncus

sp

(Tur

key)

CE

UT

Mon

onyc

hini

Mon

onyc

hus

punc

tum

albu

m (I

taly

)C

EU

T C

euto

rhyn

chin

i C

euto

rhyn

chus

ass

imili

s (F

ranc

e)C

UR

C S

tore

ini

Mel

ante

rius

sp (A

ustra

lia)

CU

RC

Mec

inin

i M

iaru

s sp

(RS

A)

ME

SO

Mag

dalin

ini

Mag

dalis

sp

(Ital

y)M

ES

O L

aem

osac

cini

Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

hini

ni C

ampt

orhi

nus

sp (A

ustra

lia)

BA

RI B

arid

ini

Mel

anob

aris

latic

ollis

(Fra

nce)

CU

RC

Tyc

hiin

i S

ibin

ia fu

lva

(US

A)

SC

OL

Cop

tono

tini

Cop

tono

tus

cycl

opus

(Cos

ta R

ica)

CO

NO

Lob

otra

chel

ini

sp1

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp2

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp3

(Chi

na)

MO

LY s

p4 (C

hina

)C

UR

C A

caly

ptin

i A

caly

ptus

sp

(Ital

y)M

OLY

Pis

sodi

ni P

isso

des

sp (I

taly

)M

OLY

Lep

yrin

i Le

pyru

s sp

(Chi

na)

MO

LY H

ylob

ini

Hyl

obiu

s ab

ietis

(Fra

nce)

MO

LY s

p2 (C

hina

)LI

XI R

hino

cylli

ni B

anga

ster

nus

sp (T

urke

y)LI

XI L

ixin

i La

rinus

turb

inat

us (F

ranc

e)C

UR

C E

ugno

min

i A

ncyt

talia

sp

(Aus

tralia

)C

UR

C C

rypt

oplin

i H

aplo

nyx

sp (A

ustra

lia)

CO

SS

Neu

mat

orin

i B

rach

ytem

nus

porc

atus

(Fra

nce)

CU

RC

Ant

hono

min

i A

ntho

nom

us p

omor

um (F

ranc

e)C

UR

C C

erat

opin

i C

erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

0

2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

ilyO

ther

curc

ulio

noi

dfa

mili

esha

veth

eir

nam

ela

bels

colo

red

byfa

mily

Num

bers

adja

cen

tto

nod

esar

eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

mor

eth

an80

hi

ghlig

hted

inre

dT

heth

ree

pri

nci

pal

woo

d-bo

rin

gsu

bfam

ilies

are

rep

rese

nte

dby

dash

edbr

anch

esan

dth

en

odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

culio

nid

aere

ferr

edto

inth

ete

xtN

odes

indi

cate

din

gree

nco

rres

pon

dto

nod

esp

rese

nt

inth

est

rict

con

sen

sus

tree

and

nod

esin

dica

ted

inbl

uear

eco

nsi

sten

tw

ith

itT

hep

osit

ion

sof

the

thre

etR

NA

rear

ran

gem

ents

are

indi

cate

dSc

ale

bar

rep

rese

nts

subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from







0

5000

10000

15000

20000

25000

Asse

mbl

y le

ngth

(bp)


IDBA-UD

Celera

Minimus2


2225


niversity of East A



ownloaded from


IDBAminusUD


Freq

uenc

y

1000 5000 9000 13000 17000

020

4060

8011

0

Celera


Freq

uenc

y

0 3000 7000 12000 17000

020

4060

8011

0

Minimus2


Freq

uenc

y

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

020

4060

8011

0


0

5000

10000

15000

20000

25000

0 50 100 150 200 250 300

Con

g le

ngth

Coverage

0

25

5

75

10

1 10 100 1000

ng g

DNA

Coverage

A

B


0

20

40

60

80

100

120

140


Total baits

Total bait hits



2226


niversity of East A



ownloaded from









1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

All genes

P1 X X X X X X



P4 X X X X X

P5 X X X X

P6 X X X X





P4 X X X X

P5 X X X X


2227


niversity of East A



ownloaded from










Number ofPartitions

SubstitutionModel

Number ofParameters

Ln L AIC AIC


Protein-codinggenes



2228


niversity of East A



ownloaded from

01

SC

OL

Sco

lytin

i S

coly

tus

sp (F

ranc

e)S

CO

L S

coly

tini

Sco

lytu

s sc

olyt

us (D

enm

ark)

SC

OL

Dia

mer

ini

Dia

mer

us in

erm

is (T

anza

nia)

SC

OL

Hex

acol

ini

Sco

lyto

des

caud

atus

(Cos

ta R

ica)

SC

OL

Phl

oeot

ribin

i P

hloe

otrib

us s

pinu

losu

s (N

orw

ay)

SC

OL

Hyl

esin

i H

yles

inus

var

ius

(Sw

eden

)S

CO

L H

ylas

tini

Hyl

aste

s op

acus

(Sw

eden

)S

CO

L To

mic

ini

Tom

icus

pin

iper

da (N

orw

ay)

SC

OL

Poly

grap

hini

Pol

ygra

phus

pol

igra

phus

(Sw

eden

)S

CO

L C

rypt

urgi

ni C

rypt

urgu

s pu

sillu

s (N

orw

ay)

SC

OL

Cor

thyl

ini

Pity

opht

horu

s m

icro

grap

hus

(Sw

eden

)S

CO

L C

orth

ylin

i C

orth

ylus

rubr

icol

lis (C

osta

Ric

a)S

CO

L C

ryph

alin

i C

ryph

alus

sal

tuar

ius

(Nor

way

)S

CO

L X

yloc

toni

ni X

yloc

tonu

s m

acul

atus

(RS

A)

SC

OL

Hyp

obor

ini

Hyp

obor

us fi

cus

(Mor

occo

)S

CO

L P

rem

nobi

ini

Pre

mno

bius

cav

ipen

nis

(RS

A)

SC

OL

Ipin

i Ip

s ac

umin

atus

(Nor

way

)S

CO

L Ip

ini

Ips

cem

brae

(Fra

nce)

SC

OL

Dry

ocoe

tini

Dry

ocoe

tes

auto

grap

hus

(Nor

way

)S

CO

L sp

2 (C

hina

)S

CO

L X

yleb

orin

i A

nisa

ndru

s di

spar

(Nor

way

)S

CO

L sp

1 (C

hina

)C

OS

S P

enta

rthrin

i Pe

ntar

thru

s el

umbe

(Eng

land

)CO

SS

sp1

(Chi

na)

CO

NO

Mec

opin

i M

ecop

us s

p (A

ustra

lia)

MO

LY s

p3 (C

hina

)M

OLY

sp1

(Chi

na)

CR

YP

Cry

ptor

hync

hini

sp

(Cam

eroo

n)C

ON

O Z

ygop

ini

Pelto

phor

us s

p (U

SA

)C

RY

P C

rypt

orhy

nchi

ni P

seud

omop

sis

(Sab

a)C

RY

P C

rypt

orhy

nchi

ni A

calle

s au

bei (

Fran

ce)

CR

YP

Cry

ptor

hync

hini

Per

isso

ps s

p (A

ustra

lia)

CR

YP

Cry

ptor

hync

hini

Our

opor

opte

rus

sp (A

ustra

lia)

CU

RC

Cio

nini

Cio

nus

gris

eus

(Can

arie

s)C

UR

C C

ioni

ni C

ionu

s ol

ens

(Fra

nce)

CE

UT

Phy

tobi

ni R

hino

ncus

sp

(Tur

key)

CE

UT

Mon

onyc

hini

Mon

onyc

hus

punc

tum

albu

m (I

taly

)C

EU

T C

euto

rhyn

chin

i C

euto

rhyn

chus

ass

imili

s (F

ranc

e)C

UR

C S

tore

ini

Mel

ante

rius

sp (A

ustra

lia)

CU

RC

Mec

inin

i M

iaru

s sp

(RS

A)

ME

SO

Mag

dalin

ini

Mag

dalis

sp

(Ital

y)M

ES

O L

aem

osac

cini

Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

hini

ni C

ampt

orhi

nus

sp (A

ustra

lia)

BA

RI B

arid

ini

Mel

anob

aris

latic

ollis

(Fra

nce)

CU

RC

Tyc

hiin

i S

ibin

ia fu

lva

(US

A)

SC

OL

Cop

tono

tini

Cop

tono

tus

cycl

opus

(Cos

ta R

ica)

CO

NO

Lob

otra

chel

ini

sp1

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp2

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp3

(Chi

na)

MO

LY s

p4 (C

hina

)C

UR

C A

caly

ptin

i A

caly

ptus

sp

(Ital

y)M

OLY

Pis

sodi

ni P

isso

des

sp (I

taly

)M

OLY

Lep

yrin

i Le

pyru

s sp

(Chi

na)

MO

LY H

ylob

ini

Hyl

obiu

s ab

ietis

(Fra

nce)

MO

LY s

p2 (C

hina

)LI

XI R

hino

cylli

ni B

anga

ster

nus

sp (T

urke

y)LI

XI L

ixin

i La

rinus

turb

inat

us (F

ranc

e)C

UR

C E

ugno

min

i A

ncyt

talia

sp

(Aus

tralia

)C

UR

C C

rypt

oplin

i H

aplo

nyx

sp (A

ustra

lia)

CO

SS

Neu

mat

orin

i B

rach

ytem

nus

porc

atus

(Fra

nce)

CU

RC

Ant

hono

min

i A

ntho

nom

us p

omor

um (F

ranc

e)C

UR

C C

erat

opin

i C

erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

0

2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

ilyO

ther

curc

ulio

noi

dfa

mili

esha

veth

eir

nam

ela

bels

colo

red

byfa

mily

Num

bers

adja

cen

tto

nod

esar

eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

mor

eth

an80

hi

ghlig

hted

inre

dT

heth

ree

pri

nci

pal

woo

d-bo

rin

gsu

bfam

ilies

are

rep

rese

nte

dby

dash

edbr

anch

esan

dth

en

odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

culio

nid

aere

ferr

edto

inth

ete

xtN

odes

indi

cate

din

gree

nco

rres

pon

dto

nod

esp

rese

nt

inth

est

rict

con

sen

sus

tree

and

nod

esin

dica

ted

inbl

uear

eco

nsi

sten

tw

ith

itT

hep

osit

ion

sof

the

thre

etR

NA

rear

ran

gem

ents

are

indi

cate

dSc

ale

bar

rep

rese

nts

subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from


IDBAminusUD


Freq

uenc

y

1000 5000 9000 13000 17000

020

4060

8011

0

Celera


Freq

uenc

y

0 3000 7000 12000 17000

020

4060

8011

0

Minimus2


Freq

uenc

y

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

020

4060

8011

0


0

5000

10000

15000

20000

25000

0 50 100 150 200 250 300

Con

g le

ngth

Coverage

0

25

5

75

10

1 10 100 1000

ng g

DNA

Coverage

A

B


0

20

40

60

80

100

120

140


Total baits

Total bait hits



2226


niversity of East A



ownloaded from









1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

All genes

P1 X X X X X X



P4 X X X X X

P5 X X X X

P6 X X X X





P4 X X X X

P5 X X X X


2227


niversity of East A



ownloaded from










Number ofPartitions

SubstitutionModel

Number ofParameters

Ln L AIC AIC


Protein-codinggenes



2228


niversity of East A



ownloaded from

01

SC

OL

Sco

lytin

i S

coly

tus

sp (F

ranc

e)S

CO

L S

coly

tini

Sco

lytu

s sc

olyt

us (D

enm

ark)

SC

OL

Dia

mer

ini

Dia

mer

us in

erm

is (T

anza

nia)

SC

OL

Hex

acol

ini

Sco

lyto

des

caud

atus

(Cos

ta R

ica)

SC

OL

Phl

oeot

ribin

i P

hloe

otrib

us s

pinu

losu

s (N

orw

ay)

SC

OL

Hyl

esin

i H

yles

inus

var

ius

(Sw

eden

)S

CO

L H

ylas

tini

Hyl

aste

s op

acus

(Sw

eden

)S

CO

L To

mic

ini

Tom

icus

pin

iper

da (N

orw

ay)

SC

OL

Poly

grap

hini

Pol

ygra

phus

pol

igra

phus

(Sw

eden

)S

CO

L C

rypt

urgi

ni C

rypt

urgu

s pu

sillu

s (N

orw

ay)

SC

OL

Cor

thyl

ini

Pity

opht

horu

s m

icro

grap

hus

(Sw

eden

)S

CO

L C

orth

ylin

i C

orth

ylus

rubr

icol

lis (C

osta

Ric

a)S

CO

L C

ryph

alin

i C

ryph

alus

sal

tuar

ius

(Nor

way

)S

CO

L X

yloc

toni

ni X

yloc

tonu

s m

acul

atus

(RS

A)

SC

OL

Hyp

obor

ini

Hyp

obor

us fi

cus

(Mor

occo

)S

CO

L P

rem

nobi

ini

Pre

mno

bius

cav

ipen

nis

(RS

A)

SC

OL

Ipin

i Ip

s ac

umin

atus

(Nor

way

)S

CO

L Ip

ini

Ips

cem

brae

(Fra

nce)

SC

OL

Dry

ocoe

tini

Dry

ocoe

tes

auto

grap

hus

(Nor

way

)S

CO

L sp

2 (C

hina

)S

CO

L X

yleb

orin

i A

nisa

ndru

s di

spar

(Nor

way

)S

CO

L sp

1 (C

hina

)C

OS

S P

enta

rthrin

i Pe

ntar

thru

s el

umbe

(Eng

land

)CO

SS

sp1

(Chi

na)

CO

NO

Mec

opin

i M

ecop

us s

p (A

ustra

lia)

MO

LY s

p3 (C

hina

)M

OLY

sp1

(Chi

na)

CR

YP

Cry

ptor

hync

hini

sp

(Cam

eroo

n)C

ON

O Z

ygop

ini

Pelto

phor

us s

p (U

SA

)C

RY

P C

rypt

orhy

nchi

ni P

seud

omop

sis

(Sab

a)C

RY

P C

rypt

orhy

nchi

ni A

calle

s au

bei (

Fran

ce)

CR

YP

Cry

ptor

hync

hini

Per

isso

ps s

p (A

ustra

lia)

CR

YP

Cry

ptor

hync

hini

Our

opor

opte

rus

sp (A

ustra

lia)

CU

RC

Cio

nini

Cio

nus

gris

eus

(Can

arie

s)C

UR

C C

ioni

ni C

ionu

s ol

ens

(Fra

nce)

CE

UT

Phy

tobi

ni R

hino

ncus

sp

(Tur

key)

CE

UT

Mon

onyc

hini

Mon

onyc

hus

punc

tum

albu

m (I

taly

)C

EU

T C

euto

rhyn

chin

i C

euto

rhyn

chus

ass

imili

s (F

ranc

e)C

UR

C S

tore

ini

Mel

ante

rius

sp (A

ustra

lia)

CU

RC

Mec

inin

i M

iaru

s sp

(RS

A)

ME

SO

Mag

dalin

ini

Mag

dalis

sp

(Ital

y)M

ES

O L

aem

osac

cini

Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

hini

ni C

ampt

orhi

nus

sp (A

ustra

lia)

BA

RI B

arid

ini

Mel

anob

aris

latic

ollis

(Fra

nce)

CU

RC

Tyc

hiin

i S

ibin

ia fu

lva

(US

A)

SC

OL

Cop

tono

tini

Cop

tono

tus

cycl

opus

(Cos

ta R

ica)

CO

NO

Lob

otra

chel

ini

sp1

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp2

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp3

(Chi

na)

MO

LY s

p4 (C

hina

)C

UR

C A

caly

ptin

i A

caly

ptus

sp

(Ital

y)M

OLY

Pis

sodi

ni P

isso

des

sp (I

taly

)M

OLY

Lep

yrin

i Le

pyru

s sp

(Chi

na)

MO

LY H

ylob

ini

Hyl

obiu

s ab

ietis

(Fra

nce)

MO

LY s

p2 (C

hina

)LI

XI R

hino

cylli

ni B

anga

ster

nus

sp (T

urke

y)LI

XI L

ixin

i La

rinus

turb

inat

us (F

ranc

e)C

UR

C E

ugno

min

i A

ncyt

talia

sp

(Aus

tralia

)C

UR

C C

rypt

oplin

i H

aplo

nyx

sp (A

ustra

lia)

CO

SS

Neu

mat

orin

i B

rach

ytem

nus

porc

atus

(Fra

nce)

CU

RC

Ant

hono

min

i A

ntho

nom

us p

omor

um (F

ranc

e)C

UR

C C

erat

opin

i C

erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

0

2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

ilyO

ther

curc

ulio

noi

dfa

mili

esha

veth

eir

nam

ela

bels

colo

red

byfa

mily

Num

bers

adja

cen

tto

nod

esar

eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

mor

eth

an80

hi

ghlig

hted

inre

dT

heth

ree

pri

nci

pal

woo

d-bo

rin

gsu

bfam

ilies

are

rep

rese

nte

dby

dash

edbr

anch

esan

dth

en

odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

culio

nid

aere

ferr

edto

inth

ete

xtN

odes

indi

cate

din

gree

nco

rres

pon

dto

nod

esp

rese

nt

inth

est

rict

con

sen

sus

tree

and

nod

esin

dica

ted

inbl

uear

eco

nsi

sten

tw

ith

itT

hep

osit

ion

sof

the

thre

etR

NA

rear

ran

gem

ents

are

indi

cate

dSc

ale

bar

rep

rese

nts

subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from









1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

All genes

P1 X X X X X X



P4 X X X X X

P5 X X X X

P6 X X X X





P4 X X X X

P5 X X X X


2227


niversity of East A



ownloaded from










Number ofPartitions

SubstitutionModel

Number ofParameters

Ln L AIC AIC


Protein-codinggenes



2228


niversity of East A



ownloaded from

01

SC

OL

Sco

lytin

i S

coly

tus

sp (F

ranc

e)S

CO

L S

coly

tini

Sco

lytu

s sc

olyt

us (D

enm

ark)

SC

OL

Dia

mer

ini

Dia

mer

us in

erm

is (T

anza

nia)

SC

OL

Hex

acol

ini

Sco

lyto

des

caud

atus

(Cos

ta R

ica)

SC

OL

Phl

oeot

ribin

i P

hloe

otrib

us s

pinu

losu

s (N

orw

ay)

SC

OL

Hyl

esin

i H

yles

inus

var

ius

(Sw

eden

)S

CO

L H

ylas

tini

Hyl

aste

s op

acus

(Sw

eden

)S

CO

L To

mic

ini

Tom

icus

pin

iper

da (N

orw

ay)

SC

OL

Poly

grap

hini

Pol

ygra

phus

pol

igra

phus

(Sw

eden

)S

CO

L C

rypt

urgi

ni C

rypt

urgu

s pu

sillu

s (N

orw

ay)

SC

OL

Cor

thyl

ini

Pity

opht

horu

s m

icro

grap

hus

(Sw

eden

)S

CO

L C

orth

ylin

i C

orth

ylus

rubr

icol

lis (C

osta

Ric

a)S

CO

L C

ryph

alin

i C

ryph

alus

sal

tuar

ius

(Nor

way

)S

CO

L X

yloc

toni

ni X

yloc

tonu

s m

acul

atus

(RS

A)

SC

OL

Hyp

obor

ini

Hyp

obor

us fi

cus

(Mor

occo

)S

CO

L P

rem

nobi

ini

Pre

mno

bius

cav

ipen

nis

(RS

A)

SC

OL

Ipin

i Ip

s ac

umin

atus

(Nor

way

)S

CO

L Ip

ini

Ips

cem

brae

(Fra

nce)

SC

OL

Dry

ocoe

tini

Dry

ocoe

tes

auto

grap

hus

(Nor

way

)S

CO

L sp

2 (C

hina

)S

CO

L X

yleb

orin

i A

nisa

ndru

s di

spar

(Nor

way

)S

CO

L sp

1 (C

hina

)C

OS

S P

enta

rthrin

i Pe

ntar

thru

s el

umbe

(Eng

land

)CO

SS

sp1

(Chi

na)

CO

NO

Mec

opin

i M

ecop

us s

p (A

ustra

lia)

MO

LY s

p3 (C

hina

)M

OLY

sp1

(Chi

na)

CR

YP

Cry

ptor

hync

hini

sp

(Cam

eroo

n)C

ON

O Z

ygop

ini

Pelto

phor

us s

p (U

SA

)C

RY

P C

rypt

orhy

nchi

ni P

seud

omop

sis

(Sab

a)C

RY

P C

rypt

orhy

nchi

ni A

calle

s au

bei (

Fran

ce)

CR

YP

Cry

ptor

hync

hini

Per

isso

ps s

p (A

ustra

lia)

CR

YP

Cry

ptor

hync

hini

Our

opor

opte

rus

sp (A

ustra

lia)

CU

RC

Cio

nini

Cio

nus

gris

eus

(Can

arie

s)C

UR

C C

ioni

ni C

ionu

s ol

ens

(Fra

nce)

CE

UT

Phy

tobi

ni R

hino

ncus

sp

(Tur

key)

CE

UT

Mon

onyc

hini

Mon

onyc

hus

punc

tum

albu

m (I

taly

)C

EU

T C

euto

rhyn

chin

i C

euto

rhyn

chus

ass

imili

s (F

ranc

e)C

UR

C S

tore

ini

Mel

ante

rius

sp (A

ustra

lia)

CU

RC

Mec

inin

i M

iaru

s sp

(RS

A)

ME

SO

Mag

dalin

ini

Mag

dalis

sp

(Ital

y)M

ES

O L

aem

osac

cini

Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

hini

ni C

ampt

orhi

nus

sp (A

ustra

lia)

BA

RI B

arid

ini

Mel

anob

aris

latic

ollis

(Fra

nce)

CU

RC

Tyc

hiin

i S

ibin

ia fu

lva

(US

A)

SC

OL

Cop

tono

tini

Cop

tono

tus

cycl

opus

(Cos

ta R

ica)

CO

NO

Lob

otra

chel

ini

sp1

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp2

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp3

(Chi

na)

MO

LY s

p4 (C

hina

)C

UR

C A

caly

ptin

i A

caly

ptus

sp

(Ital

y)M

OLY

Pis

sodi

ni P

isso

des

sp (I

taly

)M

OLY

Lep

yrin

i Le

pyru

s sp

(Chi

na)

MO

LY H

ylob

ini

Hyl

obiu

s ab

ietis

(Fra

nce)

MO

LY s

p2 (C

hina

)LI

XI R

hino

cylli

ni B

anga

ster

nus

sp (T

urke

y)LI

XI L

ixin

i La

rinus

turb

inat

us (F

ranc

e)C

UR

C E

ugno

min

i A

ncyt

talia

sp

(Aus

tralia

)C

UR

C C

rypt

oplin

i H

aplo

nyx

sp (A

ustra

lia)

CO

SS

Neu

mat

orin

i B

rach

ytem

nus

porc

atus

(Fra

nce)

CU

RC

Ant

hono

min

i A

ntho

nom

us p

omor

um (F

ranc

e)C

UR

C C

erat

opin

i C

erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

0

2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

ilyO

ther

curc

ulio

noi

dfa

mili

esha

veth

eir

nam

ela

bels

colo

red

byfa

mily

Num

bers

adja

cen

tto

nod

esar

eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

mor

eth

an80

hi

ghlig

hted

inre

dT

heth

ree

pri

nci

pal

woo

d-bo

rin

gsu

bfam

ilies

are

rep

rese

nte

dby

dash

edbr

anch

esan

dth

en

odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

culio

nid

aere

ferr

edto

inth

ete

xtN

odes

indi

cate

din

gree

nco

rres

pon

dto

nod

esp

rese

nt

inth

est

rict

con

sen

sus

tree

and

nod

esin

dica

ted

inbl

uear

eco

nsi

sten

tw

ith

itT

hep

osit

ion

sof

the

thre

etR

NA

rear

ran

gem

ents

are

indi

cate

dSc

ale

bar

rep

rese

nts

subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from










Number ofPartitions

SubstitutionModel

Number ofParameters

Ln L AIC AIC


Protein-codinggenes



2228


niversity of East A



ownloaded from

01

SC

OL

Sco

lytin

i S

coly

tus

sp (F

ranc

e)S

CO

L S

coly

tini

Sco

lytu

s sc

olyt

us (D

enm

ark)

SC

OL

Dia

mer

ini

Dia

mer

us in

erm

is (T

anza

nia)

SC

OL

Hex

acol

ini

Sco

lyto

des

caud

atus

(Cos

ta R

ica)

SC

OL

Phl

oeot

ribin

i P

hloe

otrib

us s

pinu

losu

s (N

orw

ay)

SC

OL

Hyl

esin

i H

yles

inus

var

ius

(Sw

eden

)S

CO

L H

ylas

tini

Hyl

aste

s op

acus

(Sw

eden

)S

CO

L To

mic

ini

Tom

icus

pin

iper

da (N

orw

ay)

SC

OL

Poly

grap

hini

Pol

ygra

phus

pol

igra

phus

(Sw

eden

)S

CO

L C

rypt

urgi

ni C

rypt

urgu

s pu

sillu

s (N

orw

ay)

SC

OL

Cor

thyl

ini

Pity

opht

horu

s m

icro

grap

hus

(Sw

eden

)S

CO

L C

orth

ylin

i C

orth

ylus

rubr

icol

lis (C

osta

Ric

a)S

CO

L C

ryph

alin

i C

ryph

alus

sal

tuar

ius

(Nor

way

)S

CO

L X

yloc

toni

ni X

yloc

tonu

s m

acul

atus

(RS

A)

SC

OL

Hyp

obor

ini

Hyp

obor

us fi

cus

(Mor

occo

)S

CO

L P

rem

nobi

ini

Pre

mno

bius

cav

ipen

nis

(RS

A)

SC

OL

Ipin

i Ip

s ac

umin

atus

(Nor

way

)S

CO

L Ip

ini

Ips

cem

brae

(Fra

nce)

SC

OL

Dry

ocoe

tini

Dry

ocoe

tes

auto

grap

hus

(Nor

way

)S

CO

L sp

2 (C

hina

)S

CO

L X

yleb

orin

i A

nisa

ndru

s di

spar

(Nor

way

)S

CO

L sp

1 (C

hina

)C

OS

S P

enta

rthrin

i Pe

ntar

thru

s el

umbe

(Eng

land

)CO

SS

sp1

(Chi

na)

CO

NO

Mec

opin

i M

ecop

us s

p (A

ustra

lia)

MO

LY s

p3 (C

hina

)M

OLY

sp1

(Chi

na)

CR

YP

Cry

ptor

hync

hini

sp

(Cam

eroo

n)C

ON

O Z

ygop

ini

Pelto

phor

us s

p (U

SA

)C

RY

P C

rypt

orhy

nchi

ni P

seud

omop

sis

(Sab

a)C

RY

P C

rypt

orhy

nchi

ni A

calle

s au

bei (

Fran

ce)

CR

YP

Cry

ptor

hync

hini

Per

isso

ps s

p (A

ustra

lia)

CR

YP

Cry

ptor

hync

hini

Our

opor

opte

rus

sp (A

ustra

lia)

CU

RC

Cio

nini

Cio

nus

gris

eus

(Can

arie

s)C

UR

C C

ioni

ni C

ionu

s ol

ens

(Fra

nce)

CE

UT

Phy

tobi

ni R

hino

ncus

sp

(Tur

key)

CE

UT

Mon

onyc

hini

Mon

onyc

hus

punc

tum

albu

m (I

taly

)C

EU

T C

euto

rhyn

chin

i C

euto

rhyn

chus

ass

imili

s (F

ranc

e)C

UR

C S

tore

ini

Mel

ante

rius

sp (A

ustra

lia)

CU

RC

Mec

inin

i M

iaru

s sp

(RS

A)

ME

SO

Mag

dalin

ini

Mag

dalis

sp

(Ital

y)M

ES

O L

aem

osac

cini

Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

hini

ni C

ampt

orhi

nus

sp (A

ustra

lia)

BA

RI B

arid

ini

Mel

anob

aris

latic

ollis

(Fra

nce)

CU

RC

Tyc

hiin

i S

ibin

ia fu

lva

(US

A)

SC

OL

Cop

tono

tini

Cop

tono

tus

cycl

opus

(Cos

ta R

ica)

CO

NO

Lob

otra

chel

ini

sp1

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp2

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp3

(Chi

na)

MO

LY s

p4 (C

hina

)C

UR

C A

caly

ptin

i A

caly

ptus

sp

(Ital

y)M

OLY

Pis

sodi

ni P

isso

des

sp (I

taly

)M

OLY

Lep

yrin

i Le

pyru

s sp

(Chi

na)

MO

LY H

ylob

ini

Hyl

obiu

s ab

ietis

(Fra

nce)

MO

LY s

p2 (C

hina

)LI

XI R

hino

cylli

ni B

anga

ster

nus

sp (T

urke

y)LI

XI L

ixin

i La

rinus

turb

inat

us (F

ranc

e)C

UR

C E

ugno

min

i A

ncyt

talia

sp

(Aus

tralia

)C

UR

C C

rypt

oplin

i H

aplo

nyx

sp (A

ustra

lia)

CO

SS

Neu

mat

orin

i B

rach

ytem

nus

porc

atus

(Fra

nce)

CU

RC

Ant

hono

min

i A

ntho

nom

us p

omor

um (F

ranc

e)C

UR

C C

erat

opin

i C

erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

0

2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

ilyO

ther

curc

ulio

noi

dfa

mili

esha

veth

eir

nam

ela

bels

colo

red

byfa

mily

Num

bers

adja

cen

tto

nod

esar

eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

mor

eth

an80

hi

ghlig

hted

inre

dT

heth

ree

pri

nci

pal

woo

d-bo

rin

gsu

bfam

ilies

are

rep

rese

nte

dby

dash

edbr

anch

esan

dth

en

odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

culio

nid

aere

ferr

edto

inth

ete

xtN

odes

indi

cate

din

gree

nco

rres

pon

dto

nod

esp

rese

nt

inth

est

rict

con

sen

sus

tree

and

nod

esin

dica

ted

inbl

uear

eco

nsi

sten

tw

ith

itT

hep

osit

ion

sof

the

thre

etR

NA

rear

ran

gem

ents

are

indi

cate

dSc

ale

bar

rep

rese

nts

subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from

01

SC

OL

Sco

lytin

i S

coly

tus

sp (F

ranc

e)S

CO

L S

coly

tini

Sco

lytu

s sc

olyt

us (D

enm

ark)

SC

OL

Dia

mer

ini

Dia

mer

us in

erm

is (T

anza

nia)

SC

OL

Hex

acol

ini

Sco

lyto

des

caud

atus

(Cos

ta R

ica)

SC

OL

Phl

oeot

ribin

i P

hloe

otrib

us s

pinu

losu

s (N

orw

ay)

SC

OL

Hyl

esin

i H

yles

inus

var

ius

(Sw

eden

)S

CO

L H

ylas

tini

Hyl

aste

s op

acus

(Sw

eden

)S

CO

L To

mic

ini

Tom

icus

pin

iper

da (N

orw

ay)

SC

OL

Poly

grap

hini

Pol

ygra

phus

pol

igra

phus

(Sw

eden

)S

CO

L C

rypt

urgi

ni C

rypt

urgu

s pu

sillu

s (N

orw

ay)

SC

OL

Cor

thyl

ini

Pity

opht

horu

s m

icro

grap

hus

(Sw

eden

)S

CO

L C

orth

ylin

i C

orth

ylus

rubr

icol

lis (C

osta

Ric

a)S

CO

L C

ryph

alin

i C

ryph

alus

sal

tuar

ius

(Nor

way

)S

CO

L X

yloc

toni

ni X

yloc

tonu

s m

acul

atus

(RS

A)

SC

OL

Hyp

obor

ini

Hyp

obor

us fi

cus

(Mor

occo

)S

CO

L P

rem

nobi

ini

Pre

mno

bius

cav

ipen

nis

(RS

A)

SC

OL

Ipin

i Ip

s ac

umin

atus

(Nor

way

)S

CO

L Ip

ini

Ips

cem

brae

(Fra

nce)

SC

OL

Dry

ocoe

tini

Dry

ocoe

tes

auto

grap

hus

(Nor

way

)S

CO

L sp

2 (C

hina

)S

CO

L X

yleb

orin

i A

nisa

ndru

s di

spar

(Nor

way

)S

CO

L sp

1 (C

hina

)C

OS

S P

enta

rthrin

i Pe

ntar

thru

s el

umbe

(Eng

land

)CO

SS

sp1

(Chi

na)

CO

NO

Mec

opin

i M

ecop

us s

p (A

ustra

lia)

MO

LY s

p3 (C

hina

)M

OLY

sp1

(Chi

na)

CR

YP

Cry

ptor

hync

hini

sp

(Cam

eroo

n)C

ON

O Z

ygop

ini

Pelto

phor

us s

p (U

SA

)C

RY

P C

rypt

orhy

nchi

ni P

seud

omop

sis

(Sab

a)C

RY

P C

rypt

orhy

nchi

ni A

calle

s au

bei (

Fran

ce)

CR

YP

Cry

ptor

hync

hini

Per

isso

ps s

p (A

ustra

lia)

CR

YP

Cry

ptor

hync

hini

Our

opor

opte

rus

sp (A

ustra

lia)

CU

RC

Cio

nini

Cio

nus

gris

eus

(Can

arie

s)C

UR

C C

ioni

ni C

ionu

s ol

ens

(Fra

nce)

CE

UT

Phy

tobi

ni R

hino

ncus

sp

(Tur

key)

CE

UT

Mon

onyc

hini

Mon

onyc

hus

punc

tum

albu

m (I

taly

)C

EU

T C

euto

rhyn

chin

i C

euto

rhyn

chus

ass

imili

s (F

ranc

e)C

UR

C S

tore

ini

Mel

ante

rius

sp (A

ustra

lia)

CU

RC

Mec

inin

i M

iaru

s sp

(RS

A)

ME

SO

Mag

dalin

ini

Mag

dalis

sp

(Ital

y)M

ES

O L

aem

osac

cini

Lae

mos

accu

s sp

(US

A)

CR

YP

Cam

ptor

hini

ni C

ampt

orhi

nus

sp (A

ustra

lia)

BA

RI B

arid

ini

Mel

anob

aris

latic

ollis

(Fra

nce)

CU

RC

Tyc

hiin

i S

ibin

ia fu

lva

(US

A)

SC

OL

Cop

tono

tini

Cop

tono

tus

cycl

opus

(Cos

ta R

ica)

CO

NO

Lob

otra

chel

ini

sp1

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp2

(Chi

na)

CO

NO

Lob

otra

chel

ini

sp3

(Chi

na)

MO

LY s

p4 (C

hina

)C

UR

C A

caly

ptin

i A

caly

ptus

sp

(Ital

y)M

OLY

Pis

sodi

ni P

isso

des

sp (I

taly

)M

OLY

Lep

yrin

i Le

pyru

s sp

(Chi

na)

MO

LY H

ylob

ini

Hyl

obiu

s ab

ietis

(Fra

nce)

MO

LY s

p2 (C

hina

)LI

XI R

hino

cylli

ni B

anga

ster

nus

sp (T

urke

y)LI

XI L

ixin

i La

rinus

turb

inat

us (F

ranc

e)C

UR

C E

ugno

min

i A

ncyt

talia

sp

(Aus

tralia

)C

UR

C C

rypt

oplin

i H

aplo

nyx

sp (A

ustra

lia)

CO

SS

Neu

mat

orin

i B

rach

ytem

nus

porc

atus

(Fra

nce)

CU

RC

Ant

hono

min

i A

ntho

nom

us p

omor

um (F

ranc

e)C

UR

C C

erat

opin

i C

erat

opus

sp

(Sab

a)

22

2180

7

3

2

31

1

11160 35

3210

0

1676 36

7

2

1810

010

0

2424

333

40

1

8110

079

100

12

9810

0

76

16

510

0

96

96

54

11

100

100

100

76

100

100

46

100

66

100

9910

0

7272

100

Curculionidae sstr

B

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

Part

1

FIG

5

(Par

ts1

and

2)M

Ltr

eere

sult

ing

from

the

anal

ysis

ofth

eldquoa

llge

nesrdquo

data

set

par

titi

oned

acco

rdin

gto

the

six

Part

itio

nFi

nde

rp

arti

tion

s(s

eeta

ble

1)W

ithi

nC

urcu

lion

idae

sst

r(s

ensu

Bouc

hard

etal

201

1)br

anch

esar

eco

lore

dac

cord

ing

tosu

bfam

ilyO

ther

curc

ulio

noi

dfa

mili

esha

veth

eir

nam

ela

bels

colo

red

byfa

mily

Num

bers

adja

cen

tto

nod

esar

eR

AxM

Lra

pid

boot

stra

psc

ores

wit

hva

lues

mor

eth

an80

hi

ghlig

hted

inre

dT

heth

ree

pri

nci

pal

woo

d-bo

rin

gsu

bfam

ilies

are

rep

rese

nte

dby

dash

edbr

anch

esan

dth

en

odes

labe

led

Aan

dB

indi

cate

the

two

larg

edi

visi

ons

wit

hin

Cur

culio

nid

aere

ferr

edto

inth

ete

xtN

odes

indi

cate

din

gree

nco

rres

pon

dto

nod

esp

rese

nt

inth

est

rict

con

sen

sus

tree

and

nod

esin

dica

ted

inbl

uear

eco

nsi

sten

tw

ith

itT

hep

osit

ion

sof

the

thre

etR

NA

rear

ran

gem

ents

are

indi

cate

dSc

ale

bar

rep

rese

nts

subs

titu

tion

rate

Fam

ilyan

dsu

bfam

ilyco

des

pre

cede

taxa

nam

esas

follo

ws

An

thri

bida

e(A

NT

H)

Att

elab

idae

(AT

TE)

Bra

chyc

erid

ae(B

RA

C)

Bren

tida

e(B

REN

)D

ryop

htho

ridae

(DR

YO

)N

emon

ychi

dae

(NEM

O)

Bago

inae

(BA

GO

)Ba

ridi

nae

(BA

RI)

C

euto

rhyn

chin

ae(C

EUT

)C

onod

erin

ae(C

ON

O)

Cos

son

inae

(CO

SS)

Cry

pto

rhyn

chin

ae(C

RY

P)

Cur

culio

nin

ae(C

UR

C)

Lixi

nae

(LIX

I)

Mes

opti

llin

ae(M

ESO

)M

olyt

inae

(MO

LY)

Plat

ypod

inae

(PLA

T)

and

Scol

ytin

ae(S

CO

L)

2229


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from

CE

RA

MB

YC

IDA

E

Ano

plop

hora

gla

brip

enni

sC

HR

YS

OM

ELI

DA

E

Crio

ceris

duo

deci

mpu

ncta

taA

NTH

Ant

hrib

inae

Pla

tyst

omos

alb

inus

(Fra

nce)

AN

TH s

p1 (C

hina

)N

EM

O C

imbe

ridin

ae D

oydi

rhyn

chus

aus

triac

us (F

ranc

e)AT

TE R

hync

hitin

ae B

yctis

cus

popu

li (F

ranc

e)AT

TE R

hync

hitin

ae D

epor

aus

betu

lae

(Eng

land

)AT

TE A

ttela

bina

e s

p (C

hina

)AT

TE A

pode

rinae

sp

(Chi

na)

ATTE

Apo

derin

ae A

pode

rus

cory

li (F

ranc

e)B

RE

N s

p1 (C

hina

)B

RE

N A

pion

inae

Rho

pala

pion

long

irost

re (F

ranc

e)B

RE

N N

anop

hyin

ae N

anop

hyes

sp

(Tur

key)

BR

EN

Nan

ophy

inae

Nan

ophy

es m

arm

orat

us (F

ranc

e)B

RAC

Erir

hirin

ini

Ech

inoc

nem

is s

p (A

ustra

lia)

BR

AC B

rach

ycer

inae

Bra

chyc

erus

mur

icat

us (F

ranc

e)B

RAC

Ocl

adiin

ae O

clad

ius

sp (R

SA

)D

RYO

Rhy

ncho

phor

inae

Cos

mop

olite

s so

rdid

us (C

hina

)D

RYO

sp1

(Chi

na)

DR

YO R

hync

hoph

orin

ae S

itoph

ilus

gran

ariu

s (F

ranc

e)D

RYO

Orth

ogna

thin

ae R

hino

stom

us b

arbi

rost

ris (B

eliz

e)P

LAT

Tess

eroc

erin

i D

iapu

s un

ispi

neus

(PN

G)

PLA

T P

laty

podi

ni P

laty

pus

cylin

dric

us (F

ranc

e)B

AGO

Bag

oina

e B

agou

s sp

(Eng

land

)H

YP

E H

yper

ini

Hyp

era

post

ica

(Fra

nce)

EN

TI S

itoni

ni S

itona

line

atus

(Fra

nce)

CY

CL

Dic

hotra

chel

ini

Dic

hotra

chel

us m

anue

li (It

aly)

CY

CL

Rhy

thirr

inin

i R

hyth

irrin

us s

p (R

SA

)C

YC

L R

hyth

irrin

ini

Cis

olea

sp

(Aus

tralia

)C

YC

L A

terp

ini

Rha

dino

som

us s

p (A

ustra

lia)

CY

CL

Ate

rpin

i Pe

lolo

rhin

us s

p (A

ustra

lia)

EN

TI T

ropi

phor

ini

Lept

opiu

s sp

(Aus

tralia

)E

NTI

Tro

piph

orin

i C

atas

arcu

s sp

(Aus

tralia

)E

NTI

Nau

pact

ini

Lito

styl

us p

uden

s (S

aba)

EN

TI N

aupa

ctin

i N

aupa

ctus

xan

thog

raph

us (R

SA

)E

NTI

Oph

ryas

tini

Oph

ryas

tes

sp (U

SA

)E

NTI

Geo

nem

ini

Lach

nopu

s cu

rvip

es (S

aba)

EN

TI G

eone

min

i B

aryn

otus

obs

curu

s (F

ranc

e)E

NTI

Psa

llidi

ini

Psa

llidi

um s

p (T

urke

y)E

NTI

Cra

topi

ni C

rato

pus

sum

ptuo

sus

(La

Reu

nion

)E

NTI

Tan

ymec

ini

Geo

tragu

s sp

(Chi

na)

EN

TI s

p4 (C

hina

)E

NTI

sp2

(Chi

na)

EN

TI s

p1 (C

hina

)E

NTI

Oos

omin

i B

aria

nus

sp (J

uan

de N

ova)

EN

TI s

p3 (C

hina

)E

NTI

Bra

chyd

erin

i S

troph

osom

a sp

(Eng

land

)E

NTI

Bra

chyd

erin

i S

troph

osom

a m

elan

ogra

mm

um (F

ranc

e)E

NTI

Bra

chyd

erin

i B

rach

yder

es ru

gatu

s (C

anar

ies)

EN

TI s

p5 (C

hina

)E

NTI

Lap

aroc

erin

i La

paro

ceru

s fre

yi (C

anar

ies)

EN

TI P

olyd

rusi

ni P

olyd

rusu

s m

argi

natu

s (F

ranc

e)E

NTI

Pol

ydru

sini

Lio

phlo

eus

tess

ulat

us (F

ranc

e)E

NTI

Tra

chyp

hloe

ini

Trac

hyph

loeu

s sp

(Eng

land

)E

NTI

Myo

rhin

ini

sp (R

SA

)E

NTI

Tro

piph

orin

i Tr

opip

horu

s be

rtolin

i (Ita

ly)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

rugo

sost

riatu

s (F

ranc

e)E

NTI

Otio

rhyn

chin

i O

tiorh

ynch

us s

p (E

ngla

nd)

EN

TI O

tiorh

ynch

ini

Otio

rhyn

chus

glo

bulu

s (It

aly)

28

99

100

100

6

4

90

70

100

100

44

4978

28

1810

010

050

36 995

54

40

2410

082

73

100

62

39

613

100

6 21

9610

0

95

4147

55

53

45

34

4210

010

0

100

100

5

100

9310

0

100

100

100

97

100

640

1

Curculionidae sstr

A

Other families

PLA

T P

laty

podi

ni P

laty

pus

sp (E

ngla

nd)

T

TT T

Part

2

TA

RN

SE

F to

RA

NS

EF

tRN

A tr

ansl

ocat

ion

Nod

e pr

esen

t in

stric

t con

sens

us tr

ee

Nod

e co

nsis

tent

with

stri

ct c

onse

nsus

tree

Woo

d-bo

ring

beha

viou

r

AR

NS

EF

to R

NS

AE

F tR

NA

tran

sloc

atio

n

AR

NS

EF

to R

EA

NS

F tR

NA

tran

sloc

atio

n

T T

FIG

5

Con

tin

ued

2230


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from



Discussion







2231


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from









2232


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from












2233


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from

























DNA extracon


Sanger sequencing




NGS

Mitogenome Assembly

BLAST for mtDNA

Gene annotaon





2234


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from











2235


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from


Acknowledgments







































2236


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from


















2237


niversity of East A



ownloaded from

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