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Phylogenomics of Hemiptera (Insecta: Paraneoptera) based on mitochondrial genomes

ARTICLE in SYSTEMATIC ENTOMOLOGY · JANUARY 2013 Impact Factor: 2.55 · DOI: 10.1111/j.1365-3113.2012.00660.x

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Qiang Xie Jianfu Zhou Nankai University Dartmouth College

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Xiaoguang Liu Wenjun Bu Nankai Univrsity Nankai University

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Available from: Kai Dang Retrieved on: 17 August 2015 Systematic Entomology (2013), 38, 233–245 DOI: 10.1111/j.1365-3113.2012.00660.x

Phylogenomics of Hemiptera (Insecta: Paraneoptera) based on mitochondrial genomes

∗ ∗ ∗ YING CUI1 , QIANG XIE1 ,JIMENGHUA1 , KAI DANG1, JIANFU ZHOU2, XIAOGUANG LIU2, GANG WANG2,XINYU1 and W E N J U N B U1

1Institute of Entomology, College of Life Sciences, Nankai University, Tianjin, China and 2College of Information Technical Science, Nankai University, Tianjin, China

Abstract. Hemiptera is the largest order in Paraneoptera and the ﬁfth largest in Insecta. Disputes about hemipteran phylogeny have concerned the monophyly of Auchenorrhyncha and relationships between the suborders Fulgoromorpha, Cicado- morpha, Coleorrhyncha and Heteroptera. In a phylogenomic study of Hemiptera, we add two new mitochondrial genomes of Peloridiidae (Coleorrhyncha) to those reported in GenBank, to complete the taxon sampling of all suborders. We used two types of data – amino acid sequences and nucleotides of various combinations between protein coding genes, tRNAs and rRNAs – to infer the phylogeny of Hemiptera. In total 27 taxa of Paraneoptera were sampled, 24 of them being hemipterans. Bayesian inference, maximum likelihood and maximum parsimony analyses were employed. The relationship of Cicadomorpha + Heteroptera is always stable in the results with different combinations between data types and phylogenetic methods, but our results challenge the monophyly of ‘Homoptera’ and Auchenorrhyncha. In evaluating the relative contribution of each gene, the phylograms generated by single genes CO1, ND1, ND2, ND4 and ND5, respectively, closely matched the tree yielded by the combined datasets. In light of the taxon-sampling sensitivity of trees based on mitochondrial genomes, the results need to be tested with further data from nuclear genes.

Introduction accepted since the 1960s to the present (Hennig, 1969; Carver et al., 1991; Kristensen, 1991; Yoshizawa & Saigusa, 2001). Hemiptera (true bugs) and Homoptera (planthoppers, leafhop- In this study, Hemiptera is used to mean Hemiptera (s.l.). pers, treehoppers, cicadas, spittlebugs, aphids, psyllids, scales, Different views of hemipteran phylogeny are summarized whiteﬂies, etc.) were established originally by Linnaeus (1758) in Fig. 1. Traditionally, no matter whether ‘Homoptera’ is according to features of the wing. Although merged into Ryn- regarded as a distinct order or as a suborder of Hemiptera, it gota in 1775 by Fabricius (modiﬁed to Rhynchota by Burmeis- consists of Sternorrhyncha and Auchenorrhyncha. Sternorrhyn- ter in 1835) based on the mouthpart structure, they were still cha was supported as a sister group to all the other hemipterans, treated as two separate orders until the 1930s (e.g. Brues & and named Euhemiptera (Zrzavy,´ 1990), by numerous phy- Melander, 1932), except by Latreille (1810) who recognized logenetic studies based on morphological evidence (Hennig, Heteroptera and ‘Homoptera’ as two sections of his order 1969, 1981; Kristensen, 1975, 1991; Schuh, 1979; Popov, Hemiptera (s.l.). The concept of Hemiptera (s.l.)hasbeen 1981; Minet & Bourgoin, 1986; Wootton & Betts, 1986; Correspondence: Wenjun Bu, Institute of Entomology, College of Zrzavy,´ 1990, 1992) and molecular evidence (Wheeler et al., Life Sciences, Nankai University, Weijin Road No. 94, Tianjin 300071, 1993; Campbell et al., 1994, 1995; Sorensen et al., 1995; China. E-mail: [email protected] von Dohlen & Moran, 1995; Ouvrard et al., 2000; Cryan & Urban, 2012). However, Homoptera has been argued to ∗These authors contributed equally to this work. be monophyletic based on evidence from the mitochondrial

© 2012 The Royal Entomological Society 233 234 Y. Cui et al. genomes (mitogenomes), and thus the controversy over the characteristics (Shcherbakov, 1988; Zrzavy,´ 1990, 1992; Popov monophyly of Homoptera was revived (Song et al., 2010). & Shcherbakov, 1991, 1996; D’Urso, 1993; Grimaldi & Engel, Auchenorrhyncha traditionally has been divided into Fulgo- 2005). Although Shcherbakov (1988) regarded the extant romorpha and Cicadomorpha (Grimaldi & Engel, 2005). As to cicadomorphs and heteropterans as descendants of a common the monophyly of Auchenorrhyncha, both the molecular evi- ancestor, Popov & Shcherbakov (1991, 1996) argued that dence and the morphological evidence has been controversial Coleorrhyncha and Heteroptera did not have an immediate (Fig. 1). In fact, the nonmonophyletic view of Auchenorrhyn- common ancestor and were descended independently from cha includes different opinions. According to one hypothe- separate lineages. Hence, the phylogenetic relationships among sis, Fulgoromorpha rather than Cicadomorpha is more closely the higher-level hemipteran lineages remain unclear (Fig. 1). related to Heteroptera (Goodchild, 1966; Bourgoin, 1988, Mitogenomes have been used successfully to reconstruct 1993; Campbell et al., 1994; von Dohlen & Moran, 1995) or the phylogenetic relationships within some orders of Insecta Heteropterodea (Campbell et al., 1995; Sorensen et al., 1995). (Cameron et al., 2007, 2008; Fenn et al., 2008; Hua et al., In an alternative hypothesis, Cicadomorpha is placed as a sis- 2008, 2009; Wei et al., 2010). Until now, the mitogenomes ter group to Heteropterodea based on 18S rDNA (Ouvrard of four of the five hemipteran suborders – Sternorrhyncha, et al., 2000; Xie et al., 2008). Hamilton (1981) proposed that Cicadomorpha, Fulgoromorpha and Heteroptera – have been Cicadomorpha was sister group to Sternorrhyncha. reported in GenBank, which leaves the corresponding data for Coleorrhyncha, proposed originally by Myers & China Coleorrhyncha blank. Restricted distribution, small individual (1929), are small bugs with a crptic lifestyle. They possess size and secluded lifestyle may all make it hard to provide a mixture of cicadomorphan and bug-like characters (Bechly complete mitogenomic sequences for Coleorrhyncha. How- & Szwedo, 2007), and represent a separate suborder within ever, without the mitogenomic sequences of Coleorrhyncha, Hemiptera. This suborder includes a single extant family, phylogenomic sampling of Hemiptera are incomplete. Here we Peloridiidae, which is distributed now only in Patagonia included two mitogenomes of Coleorrhyncha and reconstructed and the Australian continent (Burckhardt et al., 2011). Its the higher-level phylogenomic relationships of Hemiptera with complete subordinal sampling for the first time. phylogenetic position within Hemiptera has long fascinated hemipterists. Peloridiids were placed first in Heteroptera (Breddin, 1897), but transferred to a suborder of their own Materials and methods (China, 1924) or included in the ‘Homoptera’ (Myers & China, 1929; China, 1962; Evans, 1963). Peloridiidae and Heteroptera Taxon sampling were treated as sister groups by Schlee (1969) but Cobben (1978) considered Schlee’s synapomorphies as superficial. Twenty-seven taxa were sampled in this study. Twenty- Although the close relationship between Peloridiidae and four of these were ingroups, which included all five suborders Heteroptera has been supported consistently by nuclear rDNAs of Hemiptera. Among them, the mitogenomes of Xenophyes (Campbell et al., 1995; Sorensen et al., 1995; Ouvrard et al., cascus Bergroth and Hackeriella veitchi (Hacker) are reported 2000; Xie et al., 2008; Cryan & Urban, 2012), controversies here for the first time. The remaining outgroups were sampled remain from the view of morphological and paleontological from Phthiraptera and Thysanoptera (Table 1).

Fig. 1. The summarized phylogenetic relationships among the higher-level hemipteran lineages.

◦ Table 1. Hemipteran taxon sampling and the accession numbers of initial denaturation at 94 C, 32 cycles of 20 s denaturation at ◦ ◦ the corresponding mitogenomes. 94 C, 1 min annealing at 47–60 C and 1–10 mins elongation ◦ at 72 C depending on the size of products, and a final Taxa Accession number ◦ elongation for 10 min at 72 C. The primers used in this study Phthiraptera — are listed in File S1 and the specific primers named with the Bothriometopus macrocnemis NC_009983 first three letters ‘Xen’ were used for amplifying the sequences Campanulotes bidentatus compar NC_007884 of Xenophyes cascus and ‘Hac’ for Hackeriella veitchi. Thysanoptera — The PCR products were electrophoresed in 0.7–1% agarose Thrips imaginis NC_004371 gel and then purified. All the products were sequenced directly Hemiptera — Sternorrhyncha — in double directions by primer walking. Those fragments which Aleurochiton aceris NC_006160 failed direct sequencing were cloned into TA-cloning vector Aleurodicus dugesii NC_005939 pMD-18T (TaKaRa) and transformed into competent E. coli Acyrthosiphon pisum NC_011594 DH5α. Putative clones containing the PCR fragments were Bemisia tabaci NC_006279 selected and sequenced by the universal primers M13F (-47)/ Neomaskellia andropogonis NC_006159 M13R (-48) (File S1). Pachypsylla venusta NC_006157 Schizaphis graminum NC_006158 Tetraleurodes acaciae NC_006292 Fulgoromorpha — Analysis of sequence data Geisha distinctissima NC_012617 Laodelphax striatellus NC_013706 Sequences were edited and assembled primarily by BioEdit Lycorma delicatula NC_012835 v7.0 (Hall, 1999). Protein-coding genes (PCGs) were identified Sivaloka damnosa NC_014286 by Open Reading Frame Finder (ORF Finder) implemented Cicadomorpha — in the NCBI website (http://www.ncbi.nlm.nih.gov/gorf/gorf. Abidama producta NC_015799 html) with invertebrate mitochondrial genetic codes. Riboso- Homalodisca vitripennis NC_006899 mal RNA (rRNA) genes, which were assumed to extend to Philaenus spumarius NC_005944 the boundaries of flanking genes, were compared with other Coleorrhyncha — Hackeriella veitchi GQ884145 hemipteran mitochondrial sequences by MUSCLE which was Xenophyes cascus JF323862 implemented in MEGA v5.05 (Tamura et al., 2011). Transfer Heteroptera – RNA (tRNA) analyses were conducted using tRNAscan-SE Laccotrephes robustus NC_012817 v1.21 (Schattner et al., 2005) with the invertebrate mitochon- Hydrometra sp. NKMT020 NC_012842 drial codon predictors. The tRNAs which were not detected by Neuroctenus parus NC_012459 tRNAscan-SE were identified by BLAST search and compar- Nezara viridula NC_011755 ison with the reported mitogenomes of other hemipterans. Saldula arsenjevi NC_012463 Stenopirates sp. HL2011 NC_016017 Valentia hoffmanni NC_012823 Phylogenetic analyses

Material preparation Each of the 13 PCGs was aligned individually using the ‘align translated mt Proteins’ option of the PRANK program Adult specimens of Xenophyes cascus and Hackeriella web server (http://www.ebi.ac.uk/goldman-srv/webPRANK) veitchi (Peloridiidae) were collected by J. Damgaard and with default parameters. Stop codons were all removed from G. W. Gibbs from New Zealand in 2008 and by Geoff Mon- those sequences before alignments. The processes of align- teith in Springbrook National Park, Australia, in 2006, respec- ment were repeated several times until the results became tively. All specimens were preserved in 95% ethanol and stored stable. Subsequently, each aligned gene was translated to pro- ◦ at −20 C until DNA extraction. The voucher specimens were tein according to the Invertebrate Mitochondrial Genetic Code deposited in the Laboratory of Insect Molecular Systematics, implemented in MEGA v5.05. The sequences of tRNA and Institute of Entomology, Nankai University, China. rRNA were aligned respectively using MXSCARNA (Tabei et al., 2008), which is a computer-based alignment approach to consider the predicted secondary structure of noncoding Mitochondrial DNA preparation and amplification RNA (ncRNA). According to the substitution saturation test implemented in the program DAMBE (Xia & Xie, 2001), Total genomic DNA was extracted from muscle tissue the third codon position is saturated. It will mask a major of the thorax by a CTAB-based method (Reineke et al., part of the phylogenetic signal and will plague the phyloge- 1998). The mitogenome was amplified by polymerase chain netic analysis of deep branches (Lopez et al., 1999; Philippe reaction (PCR) and the reaction systems were performed with & Forterre, 1999; Xia et al., 2003). Thus, the aligned DNA TaKaRa LA PCR Kit v2.1 according to the manufacturer’s of the 13 PCGs for the further analysis only included the recommendations. The PCR thermal cycling program is 1 min first and second codon positions. Then the aligned protein

© 2012 The Royal Entomological Society, Systematic Entomology, 38, 233–245 236 Y. Cui et al. and DNA of the 13 PCGs were each concatenated and gen- searches were performed using 1000 random addition repli- erated two datasets (AA and PCG12). Sequences of RNAs cates with the tree bisection-reconnection (TBR) option. Gaps were combined and then concatenated with PCG12 to gener- were treated as missing data. All characters had equal weight. ate the nucleotide datasets PCG12T (PCG12 + tRNAs) and The robustness of each internal branch of the MP tree was PCG12RT (PCG12 + rRNAs + tRNAs). The extra tRNAs, evaluated with 1000 bootstrap replications. which were not amongst the commonly reported 22 tRNAs in most insects, were excluded, i.e. tRNA-Met and tRNA-Ser from Acyrthosiphon pisum and Thrips imagines, and tRNA-Trp Phylogenetic examination of separate genes and tRNA-Val from Bothriometopus macrocnemis. These datasets were subjected to Bayesian inference (BI), In order to evaluate the contribution of different genes to maximum likelihood (ML) and maximum parsimony (MP) the reconstructed hemipteran phylogeny, each of the 13 PCGs analyses. The BI was performed with MrBayes v3.1.2 in and the corresponding amino acid sequences were used to parallel (Zhou et al., 2011). For the AA data, the substitution reconstruct phylogeny independently under the BI analyses. model was set as aamodelpr = mixed. For the nucleotide data, The Ktreedist program (Soria-Carrasco et al., 2007) was used the parameter settings of the substitution model GTR + + I, to evaluate the relative contribution of each tree generated by based on the hierarchical likelihood tests by jModeltest v0.1.1 a single gene. The rooted reference tree, i.e. the tree based on (Posada, 2008). It should be pointed out that, for each PCG, AA or PCG12, and estimated trees were saved with estimated only the ﬁrst and second codon positions were taken into branch lengths, and k-scores were calculated by Ktreedist. account in the test of the substitution model, and this is true also for the ML method. A total of 5 million generations were Results run for the AA data, whereas 10 million generations were run for the nucleotides datasets. The numbers of generations to Mitochondrial genome burn were determined with the help of Tracer v1.4 (Rambaut & Drummond, 2007). The sequenced mitogenomes of Xenophyes cascus and The ML method of phylogenetic reconstruction, includ- Hackeriella veitchi, which are 14 117 and 15 793 bp in ing the model test, was implemented using Treeﬁnder length, respectively are accessed in GenBank as JF323862 and software (Jobb, 2008). For the AA dataset, detailed infor- GQ884145. The organizations of the two genomes are analysed mation on model substitution is attached as Table 2. For in Tables 3 and 4. Within the 13 PCGs, 12 initiate with the the nucleotide data, the substitution model of each gene standard start codons ATN in both mitogenomes, whereas CO1 was GTR[Optimum,Empirical]:G[Optimum] under the crite- starts with CGA in Xenophyes cascus and TCG in Hackeriella rion AICc. The number of bootstrap replications was 100 for veitchi. The secondary structures of the tRNAs are summarized both the AA and nucleotides data. The MP analyses were car- in Files S2 and S3. They could be folded into typical cloverleaf ried out with PAUP* v4.0b10 (Swofford, 2002). Heuristic tree secondary structures, except that the stem of the DHU arm was absent in tRNA-Ser (GCT). Table 2. The substitution models of the AA dataset determined by TreeFinder under the criterion AICc. The mitogenomic phylogeny of Hemiptera AA Model

Atp6 mtArt[,Empirical]:G[Optimum] In the phylogenetic results, those produced by the BI (Fig. 2) Atp8 MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: and ML (Fig. 3) analyses were largely the same, but the MP G[Optimum] analyses (Fig. 4) failed to recover many consistently accepted CO1 mtArt[,]:G[Optimum] monophyletic groups, such as the clade of Hemiptera. In all CO2 mtArt[,Empirical]:G[Optimum] phylograms of various combinations between datasets and phy- CO3 mtArt[,]:G[Optimum] logenetic algorithms, neither Homoptera nor Auchenorrhyncha Cytb MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: was monophyletic. However, in BI and ML results, the close G[Optimum] relationship between Cicadomorpha and Heteroptera was sta- ND1 mtArt[,Empirical]:G[Optimum] ND2 MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: bly supported by all the data types except the PCG12RT G[Optimum] (Figs 2, 3; File S4). ND3 MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: G[Optimum] ND4 MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: Phylogenetic examination of separated genes G[Optimum] ND4L mtArt[,]:G[Optimum] In the results of BI analyses (File S5), the clade Cicadomor- ND5 MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: pha + Heteroptera was supported by the genes Atp6, CO1, GI[Optimum] CO3, Cytb, ND1 and ND5 independently. Furthermore, both ND6 MIX[mtREV,mtMam,mtArt][Optimum,Empirical]: the ND2 and ND4 genes suggested a close relationship G[Optimum] between Fulgoromorpha and Coleorrhyncha.

Table 3. Organization of the mitochondrial genome of Xenophyes cascus.

Intergenic Gene Strand Start End Anticodon Length (bp) Start codon Stop codon nucleotidesa (bp) tRNA-Gln — 101 33 TTG 69 — — — tRNA-Met + 112 180 CAT 69 — — 10 ND2 + 181 1171 — 991 ATT T-tRNA 0 tRNA-Trp + 1172 1239 TCA 68 — — 0 tRNA-Cys — 1293 1232 GCA 62 — — −8 tRNA-Tyr — 1360 1295 GTA 66 — — 1 CO1 + 1362 2892 — 1531 CGA T-tRNA 1 tRNA-Leu(TAA) + 2893 2956 TAA 64 — — 0 CO2 + 2957 3641 — 685 ATA T-tRNA 0 tRNA-Lys + 3642 3707 CTT 66 — — 0 tRNA-Asp + 3706 3769 GTC — — — −2 ATPase8 + 3770 3922 — — ATC TAG 0 ATPase6 + 3916 4581 — — ATA TAA −7 CO3 + 4591 5377 — 787 ATG T-tRNA 11 tRNA-Gly + 5378 5440 TCC 63 0 ND3 + 5440 5791 — 352 ATG T-tRNA −1 tRNA-Ala + 5792 5853 TGC 62 — — 0 tRNA-Arg + 5853 5918 TCG 66 — — −1 tRNA-Asn + 5928 5995 GTT 68 — — 9 tRNA-Ser(GCT) + 5995 6056 GCT — — — −1 tRNA-Glu + 6058 6124 TTC 67 — — 1 tRNA-Phe — 6189 6123 GAA 67 — — −2 ND5 — 7888 6190 — 1699 ATT T-tRNA 0 tRNA-His — 7950 7889 GTG 62 — — — ND4 — 9277 7951 — 1327 ATG T-tRNA 0 ND4L — 9561 9271 291 ATT TAA −7 tRNA-Thr + 9564 9625 TGT 62 — — 2 tRNA-Pro — 9692 9626 TGG 67 — — 0 ND6 + 9694 10 209 — 516 ATA TAA 1 CytB + 10 209 11 343 1135 ATG T-tRNA −1 tRNA-Ser(TGA) + 11 344 11 412 TGA 69 — — 0 ND1 — 12 346 11 419 — 928 ATT T-tRNA 7 tRNA-Leu(TAG) — 12 413 12 347 TAG 67 — — 0 lrRNA — 13 712 12 414 — 1300 — — 0 tRNA-Val — 13 775 13 713 TAC 63 — — 0 srRNA — 14 117 13 776 — 342 — — 0 a Numbers correspond to nucleotides separating a gene from an upstream one; the negative numbers refer to overlaps between the adjacent genes.

The results calculated by Ktreedist are given as a set of also in the suborder of Cicadomorpha and Coleorrhyncha. Most k-scores (Fig. 5). Each high score indicates a poor match mitogenomes of Heteroptera and Fulgoromorpha have the uni- between the estimated tree and the reference tree. The ﬁve versal gene order except for some rearrangements occurring genes CO1, ND1, ND2, ND4 and ND5 are matched well with in speciﬁc species (Hua et al., 2009; Song & Liang, 2009; Li the topology of the reference tree. Atp8, CO2, ND3, ND4L and et al., 2012). However, no synapomorphy on the gene order ND6 presented the most deviant topologies. has been found among the suborders.

Discussion Mitogenomic phylogeny of Hemiptera

Mitochondrial genome Compared to the phylogenetic algorithms BI and ML, the algorithm MP obtained inconsistent results in reconstructing In all available mitogenomes of Hemiptera, the extents of the high-level phylogeny of Hemiptera, failing to recover many gene rearrangements vary according to different suborders. widely accepted monophyletic groups, especially the mono- In Sternorrhyncha, Aleyrodoidea may have highly rearranged phyly of Hemiptera and Sternorrhyncha. tRNA, PCGs and rRNA (Thao et al., 2004), whereas Aphi- Although the results of the BI and ML analyses showed doidea and Psylloidea have almost the same gene order to that differences among the different datasets, all the results sup- of Drosophila yakuba (Clary et al., 1985), which was found ported the hypothesis that Sternorrhyncha was the sister group

Table 4. Organization of the mitochondrial genome of Hackeriella veitchi.

Intergenic Gene Strand Start End Anticodon Length (bp) Start codon Stop codon nucleotidesa (bp) tRNA-Ile J 1 66 GAT 66 — — — tRNA-Gln N 65 140 TTG 76 — — −2 tRNA-Met J 146 210 CAT 65 — — 6 ND2 J 210 1202 — 993 ATT T-tRNA −1 tRNA-Trp J 1201 1264 TCA 64 — — −2 tRNA-Cys N 1257 1317 GCA 61 — — −8 tRNA-Tyr N 1318 1382 GTA 65 — — 0 CO1 J 1382 2915 — 1534 TCG T-tRNA −1 tRNA-Leu(TAA) J 2916 2979 TAA 64 — — 0 CO2 J 2980 3664 — 685 ATA T-tRNA 0 tRNA-Lys J 3665 3730 CTT 66 — — 0 tRNA-Asp J 3729 3790 GTC 62 — — −2 ATPase8 J 3800 3937 — 138 ATA TAA 11 ATPase6 J 3931 4596 — 666 ATG TAA −7 CO3 J 4601 5387 — 1453 ATG T-tRNA 5 tRNA-Gly J 5388 5448 TCC 61 — — 0 ND3 J 5449 5800 — 352 ATA T-tRNA 0 tRNA-Ala J 5801 5861 TGC 61 — — 0 tRNA-Arg J 5860 5919 TCG 60 — — −2 tRNA-Asn J 5921 5988 GTT 68 — — 1 tRNA-Ser(GCT) J 5988 6051 GCT 64 — — −1 tRNA-Glu J 6051 6116 TTC 66 — — −1 tRNA-Phe N 6115 6183 GAA 69 — — −2 ND5 N 6184 7886 — 1703 ATG T-tRNA 0 tRNA-His N 7887 7949 GTG 63 — — 0 ND4 N 7950 9273 — 1324 ATG T-tRNA 0 ND4L N 9267 9554 — 288 ATG TAA −7 tRNA-Thr J 9560 9620 TGT 61 — — 5 tRNA-Pro N 9621 9686 TGG 66 — — 0 ND6 J 9690 10 199 — 510 ATA TAA 3 CytB J 10 199 11 335 — 1137 ATG T-tRNA −1 tRNA-Ser(TGA) J 11 334 11 402 TGA 69 — — −2 ND1 N 11 433 12 336 — 904 ATT T-tRNA 30 tRNA-Leu(TAG) N 12 337 12 405 TAG 69 — — 0 lrRNA N 12 406 13 648 — 1243 — — 0 tRNA-Val N 13 649 13 719 TAC 71 — — 0 srRNA N 13 720 14 497 — 778 — — 0 Control — 14 498 15 793 — 1296 — — 0 a Numbers correspond to nucleotides separating a gene from an upstream one; the negative numbers refer to overlaps between the adjacent genes. to the remaining Hemiptera, except for the PCG12RT dataset. of Homoptera was reasserted by Song et al. (2010) based on In comparison to the other three datasets (AA, PCG12 and mitogenomes. It may seem confusing that very similar molec- PCG12T), the inconsistent results of PCG12RT probably are ular markers have produced obviously different results (Song caused by interference from 16S rRNA and 12S rRNA. Even et al., 2010; Talavera & Vila, 2011). In fact, these markers in the phylogeny at the family level, saturation can be detected could be sensitive for completed taxon-sampling in phyloge- in substitutions of 16S rRNA, which thus make mitochon- netic analyses (Heatke et al., 2006; Heath et al., 2008a,b). In drial rRNAs unsuitable for addressing higher level relation- this study, Phthiraptera and Thysanoptera were included in the ships (Tian et al., 2008). Thus, the discussion of hemipteran taxon sampling of outgroups, and each of the ﬁve suborders of phylogeny below is based on the results of the combinations Hemiptera was represented by at least two taxa. This is proba- between datasets AA, PCG12 and PCG12T, and algorithms BI bly the key reason leading to the differences in the phylogenetic and ML. results of Hemiptera based on mitogenomes. The idea that paraphyletic Homoptera and monophyletic The monophyly of Auchenorrhyncha is another focus Sternorrhyncha are sister groups to the remaining hemipter- of debate: here the paraphyletic status of Auchenorrhyn- ans has been widely accepted (Hennig, 1969, 1981; Wheeler cha is supported consistently. Morphological evidence for a et al., 1993; Hoch et al., 2006; Lee et al., 2009; Talavera & monophyletic Auchenorrhyncha has been controversial. The Vila, 2011; Cryan & Urban, 2012). However, the monophyly evidence for the ‘paraphyletic’ view includes morphology

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Fig. 2. The 27-taxon phylograms of Hemiptera with the results of Bayesian analyses based on different datasets: (A) AA, (B) PCG12 and (C) PCG12T. The node support values above the branches are the Bayesian posterior probabilities (BPP).

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Fig. 3. The summarized 27-taxon phylogeny of Hemiptera based on the results of maximum likelihood analyses of different datasets: (A) AA, (B) PCG12 and (C) PCG12T. The node support values above the branches are the bootstrap (BS) values.

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Fig. 4. The summarized 27-taxon phylogeny of Hemiptera based on the results of maximum parsimony analyses of different datasets: (A) AA, (B) PCG12 and (C) PCG12T. The node support values above the branches are the bootstrap (BS) values.

previous molecular studies based on nuclear rDNAs supported a close relationship between Coleorrhyncha and Heteroptera, more nuclear genes should be utilized. It is a universal phenomenon that discrepancies exist between the phylogenetic relationships revealed by nuclear and mitochondrial datasets (Timmermans et al., 2008). Furthermore, the results given by mitochondrial datasets may vary according to different combinations of data type and methods of phylogenetic reconstruction. Each combination deserves to be tried Fig. 5. Hemipteran gene and protein k-scores scaled from Ktreedist to evaluate results based on mitogenomes and also differences which measure overall differences in the relative branch length and between the results from nuclear and mitochondrial markers topology of the phylogenetic trees generated by the single PCG or AA compared to their combined dataset. (Lin & Danforth, 2004; Talavera & Vila, 2011). Based on the results of this study, we recommend combining the three AA, PCG12 and PCG12T datasets and the two BI and ML algo- and histology of the digestive tract (Goodchild, 1966), wing rithms in subsequent phylogenetic analyses. Jones et al. (2007) (Wootton & Betts, 1986) and head morphology (Bourgoin, stated that mitochondrial DNA could be applied in molecu- 1986a,b), and male (Bourgoin & Huang, 1990) and female gen- lar phylogenetics with an appropriate substitution model. For italia (Bourgoin, 1993). Evidence for the ‘monophyletic view’, instance, Kjer & Honeycutt (2007) retrieved a phylogenetic includes the sclerotized forewing base (Yoshizawa & Saigusa, tree of mammals by applying a site-specific rate model suc- 2001) and wing coupling structures (D’Urso, 2002). cessfully. In insects, the mitogenomes have been successfully As to the molecular evidence concerning the phylogeny of used to resolve intra-ordinal relationships, such as in Diptera Auchenorrhyncha, both 18S rDNAs (Campbell et al., 1994, (Cameron et al., 2007), Hymenoptera (Cameron et al., 2008; 1995; Sorensen et al., 1995; von Dohlen & Moran, 1995; Wei et al., 2010), and Orthoptera (Fenn et al., 2008). In this Ouvrard et al., 2000; Xie et al., 2008), and the complete study, with the site-heterogeneous mixture model CAT used mitogenomes (Song et al., 2010; Talavera & Vila, 2011) for the AA dataset, the result from PhyloBayes (File S6) support its paraphyly. A phylogenetic study based on seven still well supports the monophyletic group of (Cicadomor- combined fragments of DNA indicated the monophyly of pha + Heteroptera). Auchenorrhyncha by molecular evidence for the first time (Cryan & Urban, 2012). Their detailed analyses have assessed the relative contribution of different genes respectively, and Conclusion the genes of CO1 and ND4 have obtained higher scores to support the monophyly of Auchenorrhyncha than other genes. This is the first phylogenomic study of hemipterans with com- However, the evaluation of the contribution of CO1 and ND4 plete suborder sampling. In congruence with current widely is in conflict with the results of this study, in which CO1 and accepted views, the results support the paraphyly of Homoptera + ND4 support the clades (Cicadomorpha Heteroptera) and and Auchenorrhyncha. The close relationship between Cicado- + (Fulgoromorpha Coleorrhyncha), respectively (Fig. 5). morpha and Heteroptera proposed in this study is supported In our results, Cicadomorpha rather than Coleorrhyncha also by the fossil evidence but not by rDNAs. More nuclear was shown consistently to be sister group to Heteroptera genes should be explored to elucidate the relationships between (Figs 2, 3). Schlee (1969) proposed Peloridiidae as sister group Coleorrhyncha, Cicadomorpha and Heteroptera. of Heteroptera, and since then this relationship has been supported by morphological studies (Zrzavy,´ 1990, 1992; D’Urso, 1993) and molecular studies mainly based on nuclear rDNAs Supporting Information (Wheeler et al., 1993; Campbell et al., 1995; Sorensen et al., 1995; Ouvrard et al., 2000; Xie et al., 2008; Cryan & Urban, Additional Supporting Information may be found in the online 2012). However, Cobben (1978) considered the synapomor- version of this article under the DOI reference: + phies of Coleorrhyncha Heteroptera raised by Schlee (1969) 10.1111/j.1365-3113.2012.00660.x to be superficial. Furthermore, based on the fossil record, Popov & Shcherbakov (1991, 1996) stated that synapomor- File S1. The modified or specifically designed primers phies shared by Coleorrhyncha and Heteroptera are at least to amplify the mitogenomes of Xenophyes cascus and dubious. According to Popov & Shcherbakov (1991, 1996), Hackeriella veitchi. Heteroptera share the costal fracture and forewing–thoracic File S2. Putative secondary structures of mitochondrial coupling device with, and doubtless descended from, a super- tRNAs of Xenophyes cascus. Lines indicate the classi- family of primitive Cicadomorpha, Scytinopteroidea. Further- cal Watson-Crick pairs, and asterisks indicate the G = U more they pointed out that the two suborders evolved from matches. generalized Cicadomorpha as independent stocks, acquired wing coupling of the same type and dorsoventrally flattened File S3. Putative secondary structures of mitochondrial habitus with forewing overlap in parallel. However, as all tRNAs of Hackeriella veitchi. Lines indicate the classical

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