TY - JOUR
AU1 - Song, Nan
AU2 - Cai, Wanzhi
AU3 - Li, Hu
AB - Abstract The hemipteran suborder Auchenorrhyncha consists of four superfamily rank taxa (i.e. Cicadoidea, Membracoidea, Cercopoidea and Fulgoroidea), with more than 42000 described species worldwide. The monophyly of Auchenorrhyncha and the higher-level relationships within this group remain questionable, despite recent research using morphological and molecular data. In this study, we sequenced 18 mitogenomes of representatives of Membracoidea, three of Cercopoidea and one of Psylloidea and conducted phylogenetic analyses together with 35 existing mitogenomes of Hemiptera, with special emphasis on the auchenorrhynchan monophyly. The phylogenetic inferences from the mitogenomic data are strongly affected by increased rates of sequence substitution associated with several lineages, which leads to the significant long-branch attraction. Under these conditions, the monophyly of Auchenorrhyncha is not supported even with the comprehensive data coding schemes. This shows the limitations of the mitogenome in resolving the higher-level phylogeny of Auchenorrhyncha. In addition, the removal of fast-evolving sites using a pattern sorting method (OV-sorting) significantly improves phylogenetic estimates under both homogeneous and heterogeneous models. Moreover, data sets with a higher proportion of conserved sites recover the monophyly of Auchenorrhyncha and support a superfamily relationship of [Fulgoroidea + ((Cicadoidea + Cercopoidea) + Membracoidea)]. Auchenorrhyncha, mitochondrial genome, phylogeny INTRODUCTION Auchenorrhyncha is a group within the insect order Hemiptera. Traditionally, this group has been recognized as a suborder with two main clades: Cicadomorpha and Fulgoromorpha (Hennig, 1969, 1981; Carver, Gross & Woodward, 1991). Cicadomorpha comprises three superfamilies: Cicadoidea (cicadas), Membracoidea (leafhoppers and treehoppers) and Cercopoidea (spittlebugs and froghoppers), with 35000 described species belonging to 12 extant families (Dietrich et al., 2001; Cryan, 2005). Fulgoromorpha includes only the superfamily Fulgoroidea (planthoppers) and contains more than 9000 described species across c. 20 extant families (O’Brien & Wilson, 1985; Urban & Cryan, 2007). All species of Auchenorrhyncha are phytophagous insects, many of which are considered agricultural pests. For example, some delphacid planthoppers in Fulgoroidea can reduce crop productivity through feeding damage and by transmitting plant viral diseases (Cryan & Urban, 2012; Song, Liang & Bu, 2012). Despite their economic impact, there is little phylogenetic research focused on Auchenorrhyncha and the group’s phylogeny is debated. Specifically, studies of auchenorrhynchan monophyly vary across different analytical approaches and character sets (Kristensen, 1975; Hennig, 1981; Zrzavý, 1992; Bourgoin, 1993; Campbell et al., 1995; von Dohlen & Moran, 1995; Cryan, 2005; Cryan & Urban, 2012; Song et al., 2012). Understanding the phylogenetic structure of Auchenorrhyncha will help develop a stable, natural classification scheme and serve as a framework for studying the ecological and evolutionary patterns of this insect group. Therefore, we reinvestigated the phylogeny of Auchenorrhyncha, using a new data type and taxon sampling. Detailed reviews of the morphological and molecular evidence for and against the monophyly of Auchenorrhyncha were recently summarized by Cryan & Urban (2012). Proposed characters supporting auchenorrhynchan monophyly include structures of the head, wings and abdomen (Yoshizawa & Saigusa, 2001; Cryan & Urban, 2012). Characters supporting auchenorrhynchan paraphyly include features of the alimentary canal, head capsule and female reproductive system (Forero, 2008; Cryan & Urban, 2012). Therefore, monophyly of Auchenorrhyncha is difficult to resolve with morphological characters alone. According to Bourgoin & Campbell (2002), a consensus of paraphyly was reached by studying 18S rDNA data (Campbell et al., 1995; von Dohlen & Moran, 1995; Sorensen et al., 1995; Bourgoin et al., 1999, 2001; Ouvrard et al., 2000) and fossil records (Shcherbakov, 1984, 1988, 1996; Popov & Shcherbakov, 1996). The paraphyly of Auchenorrhyncha is mainly due to a close affinity between Cicadomorpha or Fulgoromorpha and Heteropterodea (i.e. the clade Heteroptera + Coleorrhyncha). Yet, two recent studies provide new molecular evidence supporting auchenorrhynchan monophyly (Urban & Cryan, 2007; Cryan & Urban, 2012). Specifically, Cryan & Urban (2012) attempted to address the issue of auchenorrhynchan monophyly using multilocus molecular data from nuclear and mitochondrial gene fragments, although their preferred tree topology had only moderate statistical support for this group. The issue of auchenorrhynchan monophyly also pertains to the phylogenetic placement of this suborder within Hemiptera (Hennig, 1981; Zrzavý, 1992; Cryan & Urban, 2012). Evans (1963) treated Auchenorrhyncha as the sister group of a clade composed of two hemipteran suborders: Coleorrhyncha plus Sternorrhyncha. Hennig (1981) placed Auchenorrhyncha as the putative sister group of Sternorrhyncha, which was largely consistent with the hypothesis of a ‘Homoptera long regarded as a separate order’. However, a cladistic analysis of morphological characters by Zrzavý (1992) recovered Auchenorrhyncha as the sister group to Heteropterodea, while Sternorrhyncha was found to be sister to the remaining hemipteran lineages. Thus, Zrzavý (1992) refuted the monophyly of Homoptera. The majority of morphological evidence (e.g. Goodchild, 1966; Hamilton, 1981), palaeontological information and the early 18S rDNA only data sets recover Cicadomorpha and Fulgoromorpha as two independent, non-sister lineages of Hemiptera, rendering a paraphyletic Auchenorrhyncha. However, other morphology-based studies (e.g. D’Urso, 2002) and recent multilocus molecular phylogenetic analyses recover the monophyly of Auchenorrhyncha. Thus, the phylogeny of Auchenorrhyncha remains a problem for hemipterists. There have been no phylogenetic studies that examined the auchenorrhynchan monophyly with only mitogenomic data. Mitogenome sequences have been used for inferring phylogenetic relationships within Insecta at all taxonomic levels (see reviews of Simon et al., 2006; Simon & Hadrys, 2013; Cameron, 2014a, b). mtDNA has several characteristic properties, including maternal inheritance, relatively rapid rates of evolution and high genomic copy numbers (Boore, 1999), making mtDNA easy to amplify. Therefore, it has been an important marker for molecular phylogenetic analysis in insects (Timmermans et al., 2010; Cameron, 2014a, b). Mitogenome sequencing allows for much larger-scale sampling, as genome-scale data sequencing is still costly. Furthermore, the high-throughput sequencing technologies used have revolutionized approaches for obtaining complete insect mitogenomes (Timmermans et al., 2010; Gillett et al., 2014; Tang et al., 2014; Crampton-Platt et al., 2015; Timmermans et al., 2016; Nie et al., 2017). These studies significantly increase the scope of insect phylogenetic studies based on mitogenomic data. Compared to Sanger-based approaches, high-throughput sequencing can overcome the difficulty of designing species-specific primers, long-PCR amplifications and the high cost of obtaining de novo full mitogenomes (Cameron, 2014a; Gillett et al., 2014). Despite the wide use of the mitogenome in phylogenetics, it is contentious because it is a rapidly evolving organelle genome. Phylogenetic studies are limited when using only a small fraction of the total genome (Rubinoff & Holland, 2005). Indeed, phylogenetic inference within several paraneopteran taxa (i.e. Thysanoptera, Phthiraptera and Hemiptera) appears to be influenced by the accelerated substitution rates of mitogenomes (Talavera & Vila, 2011; Simon & Hadrys, 2013). Lineage-specific substitution rates have the potential to cause long-branch artefacts (LBA) (Sheffield et al., 2009; Simon & Hadrys, 2013; Li et al., 2015). Removal of problematic long-branching taxa has been applied to avoid LBA problems (Bergsten, 2005), but often the phylogenetic position of the omitted taxa is of special interest. Therefore, better data matrix treatments and increased taxon sampling are needed to improve insect mitogenomic phylogenies. Common data treatment methods include removing third codon positions (Fenn et al., 2008; Ma et al., 2009), RY coding for the first and/or third codon positions (Phillips, Delsuc & Penny, 2004; Pagès et al., 2010) and degen-coding (Regier et al., 2010; Zwick, Regier & Zwickl, 2012). The OV-sorting method recently developed by Goremykin, Nikiforova & Bininda-Emonds (2010) can sort and remove highly variable sites to generate a series of increasingly conserved alignments. This method is useful for evaluating and reducing the impact of systematic error in large-scale phylogenomic analyses (Goremykin et al., 2010). By November 2016, complete or nearly complete mitogenomes for 33 species of Auchenorrhyncha were available in GenBank. However, the most diverse superfamily Membracoidea with c. 25000 described species (Cryan, 2005) was represented by only nine. To achieve a more balanced taxon sampling and a better-resolved phylogeny of Auchenorrhyncha, we sequenced additional mitogenomes from the superfamilies Membracoidea (18 species) and Cercopoidea (three species) in Auchenorrhyncha, plus additional outgroup taxa from the family Psylloidea (Sternorrhyncha). The newly generated data were combined with the publicly available hemipteran mitogenomes to test the monophyly of Auchenorrhyncha. Maximum likelihood (ML) and Bayesian methods were used to analyse nucleotide and amino acid sequences under various coding schemes. METHODS Taxon sampling A total of 49 ingroup taxa represented all auchenorrhynchan superfamilies: 24 Membracoidea, ten Cercopoidea, five Cicadoidea and ten Fulgoromorpha (Table 1). The outgroup included three species of Psocoptera, three Heteroptera, two Sternorrhyncha and three Coleorrhyncha. The primary specimen materials used for DNA extraction were from the Entomological Museum of Henan Agricultural University (voucher numbers in Table 1). Table 1. Taxonomic information and GenBank accession numbers for the taxa included in this study Superfamily Species Accession number Voucher numbers for newly sequenced species Collecting information Membracoidea Darthula hardwickii NC_026699 – – Membracoidea Leptobelus gazella NC 023219 – – Membracoidea Centrotus cornutus KX437728 MT-2015 isolate Zz041601 Vietnam, Pingxiang, 22°11′N, 106°70′E, July 2012 Membracoidea Empoasca vitis NC 024838 – – Membracoidea Drabescoides nuchalis NC_028154 – – Membracoidea Homalodisca vitripennis NC_006899 – – Membracoidea Nephotettix cincticeps NC_026977 – – Membracoidea Balclutha sp. KX437738 MT-2015 isolate Zz052711 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus sp. KX437735 MT-2015 isolate Zz052706 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cicadula sp. KX437724 MT-2015 isolate Zz052713 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cuerna sp. KX437741 MT-2015 isolate Zz052311 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Dryadomorpha sp. KX437736 MT-2015 isolate Zz060407 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Empoasca sp. KX437737 MT-2015 isolate Zz031203 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Exitianus indicus KX437722 MT-2015 isolate Zz052320 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Graphocephala sp. KX437740 MT-2015 isolate Zz052315 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus phycitis KX437727 MT-2015 isolate Zz052707 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Nephotettix sp. KX437725 MT-2015 isolate Zz053036 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Osbornellus sp. KX437739 MT-2015 isolate Zz052506 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Petalocephala ochracea KX437734 MT-2015 isolate 15052715 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Phlogotettix sp. KX437721 MT-2015 isolate Zz052705 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Psammotettix sp. KX437742 MT-2015 isolate Zz060503 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Sophonia linealis KX437723 MT-2015 isolate Zz052717 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Atkinsoniclla sp. KX437743 MT-2015 isolate Zz060410 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Athysanopsis sp. KX437726 MT-2015 isolate Zz052318 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Philaenus spumarius AY630340 – – Cercopoidea Callitettix versicolor EU725832 – – Cercopoidea Callitettix braconoides NC_025497 – – Cercopoidea Callitettix biformis NC_025496 – – Cercopoidea Abidama producta NC 015799 – – Cercopoidea Aeneolamia contigua NC_025495 – – Cercopoidea Cosmoscarta bispecularis NC_026289 – – Cercopoidea Paphnutius ruficeps KX437731 MT-2015 isolate Zz052908 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Aphrophora intermedia KX437729 MT-2015 isolate Zz062722 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Clovia sp. KX437730 MT-2015 isolate Zz052918 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cicadoidea Magicicada tredecim KM000130 – – Cicadoidea Gaeana maculata KM244671 – – Cicadoidea Tettigades auropilosa KM000129 – – Cicadoidea Tettigades ulnaria KM000128 – – Cicadoidea Diceroprocta semicincta KM000131 – – Fulgoroidea Laodelphax striatellus FJ360695 – – Fulgoroidea Laodelphax striatella JX880068 – – Fulgoroidea Nilaparvata lugens JN563995 – – Fulgoroidea Sogatella furcifera NC_021417 – – Fulgoroidea Nilaparvata muiri NC_024627 – – Fulgoroidea Lycorma delicatula EU909203 – – Fulgoroidea Pyrops candelaria FJ006724 – – Fulgoroidea Ricania marginalis JN242415 – – Fulgoroidea Geisha distinctissima FJ230961 – – Fulgoroidea Sivaloka damnosus FJ360694 – – Peloridioidea Xenophyes cascus JF323862 – – Peloridioidea Hemiodoecus leai NC_025329 – – Peloridioidea Hackeriella veitchi NC_020309 – – Psylloidea Pachypsylla venusta AY263317 – – Psylloidea Cacopsylla chinensis KX437732 MT-2015 isolate Zz060501 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cimicoidea Riptortus pedestris EU427344 – – Cimicoidea Hydaropsis longirostris EU427337 – – Lygaeoidea Geocoris pallidipennis EU427336 – – Atropetae Lepidopsocidae sp. AF335994 – – Psocetae Longivalvus hyalospilus JQ910986 – – Psocetae Psococerastis albimaculata NC_021400 – – Superfamily Species Accession number Voucher numbers for newly sequenced species Collecting information Membracoidea Darthula hardwickii NC_026699 – – Membracoidea Leptobelus gazella NC 023219 – – Membracoidea Centrotus cornutus KX437728 MT-2015 isolate Zz041601 Vietnam, Pingxiang, 22°11′N, 106°70′E, July 2012 Membracoidea Empoasca vitis NC 024838 – – Membracoidea Drabescoides nuchalis NC_028154 – – Membracoidea Homalodisca vitripennis NC_006899 – – Membracoidea Nephotettix cincticeps NC_026977 – – Membracoidea Balclutha sp. KX437738 MT-2015 isolate Zz052711 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus sp. KX437735 MT-2015 isolate Zz052706 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cicadula sp. KX437724 MT-2015 isolate Zz052713 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cuerna sp. KX437741 MT-2015 isolate Zz052311 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Dryadomorpha sp. KX437736 MT-2015 isolate Zz060407 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Empoasca sp. KX437737 MT-2015 isolate Zz031203 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Exitianus indicus KX437722 MT-2015 isolate Zz052320 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Graphocephala sp. KX437740 MT-2015 isolate Zz052315 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus phycitis KX437727 MT-2015 isolate Zz052707 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Nephotettix sp. KX437725 MT-2015 isolate Zz053036 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Osbornellus sp. KX437739 MT-2015 isolate Zz052506 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Petalocephala ochracea KX437734 MT-2015 isolate 15052715 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Phlogotettix sp. KX437721 MT-2015 isolate Zz052705 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Psammotettix sp. KX437742 MT-2015 isolate Zz060503 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Sophonia linealis KX437723 MT-2015 isolate Zz052717 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Atkinsoniclla sp. KX437743 MT-2015 isolate Zz060410 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Athysanopsis sp. KX437726 MT-2015 isolate Zz052318 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Philaenus spumarius AY630340 – – Cercopoidea Callitettix versicolor EU725832 – – Cercopoidea Callitettix braconoides NC_025497 – – Cercopoidea Callitettix biformis NC_025496 – – Cercopoidea Abidama producta NC 015799 – – Cercopoidea Aeneolamia contigua NC_025495 – – Cercopoidea Cosmoscarta bispecularis NC_026289 – – Cercopoidea Paphnutius ruficeps KX437731 MT-2015 isolate Zz052908 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Aphrophora intermedia KX437729 MT-2015 isolate Zz062722 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Clovia sp. KX437730 MT-2015 isolate Zz052918 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cicadoidea Magicicada tredecim KM000130 – – Cicadoidea Gaeana maculata KM244671 – – Cicadoidea Tettigades auropilosa KM000129 – – Cicadoidea Tettigades ulnaria KM000128 – – Cicadoidea Diceroprocta semicincta KM000131 – – Fulgoroidea Laodelphax striatellus FJ360695 – – Fulgoroidea Laodelphax striatella JX880068 – – Fulgoroidea Nilaparvata lugens JN563995 – – Fulgoroidea Sogatella furcifera NC_021417 – – Fulgoroidea Nilaparvata muiri NC_024627 – – Fulgoroidea Lycorma delicatula EU909203 – – Fulgoroidea Pyrops candelaria FJ006724 – – Fulgoroidea Ricania marginalis JN242415 – – Fulgoroidea Geisha distinctissima FJ230961 – – Fulgoroidea Sivaloka damnosus FJ360694 – – Peloridioidea Xenophyes cascus JF323862 – – Peloridioidea Hemiodoecus leai NC_025329 – – Peloridioidea Hackeriella veitchi NC_020309 – – Psylloidea Pachypsylla venusta AY263317 – – Psylloidea Cacopsylla chinensis KX437732 MT-2015 isolate Zz060501 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cimicoidea Riptortus pedestris EU427344 – – Cimicoidea Hydaropsis longirostris EU427337 – – Lygaeoidea Geocoris pallidipennis EU427336 – – Atropetae Lepidopsocidae sp. AF335994 – – Psocetae Longivalvus hyalospilus JQ910986 – – Psocetae Psococerastis albimaculata NC_021400 – – Mitogenomes newly sequenced in this study are indicated in bold. View Large Table 1. Taxonomic information and GenBank accession numbers for the taxa included in this study Superfamily Species Accession number Voucher numbers for newly sequenced species Collecting information Membracoidea Darthula hardwickii NC_026699 – – Membracoidea Leptobelus gazella NC 023219 – – Membracoidea Centrotus cornutus KX437728 MT-2015 isolate Zz041601 Vietnam, Pingxiang, 22°11′N, 106°70′E, July 2012 Membracoidea Empoasca vitis NC 024838 – – Membracoidea Drabescoides nuchalis NC_028154 – – Membracoidea Homalodisca vitripennis NC_006899 – – Membracoidea Nephotettix cincticeps NC_026977 – – Membracoidea Balclutha sp. KX437738 MT-2015 isolate Zz052711 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus sp. KX437735 MT-2015 isolate Zz052706 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cicadula sp. KX437724 MT-2015 isolate Zz052713 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cuerna sp. KX437741 MT-2015 isolate Zz052311 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Dryadomorpha sp. KX437736 MT-2015 isolate Zz060407 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Empoasca sp. KX437737 MT-2015 isolate Zz031203 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Exitianus indicus KX437722 MT-2015 isolate Zz052320 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Graphocephala sp. KX437740 MT-2015 isolate Zz052315 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus phycitis KX437727 MT-2015 isolate Zz052707 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Nephotettix sp. KX437725 MT-2015 isolate Zz053036 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Osbornellus sp. KX437739 MT-2015 isolate Zz052506 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Petalocephala ochracea KX437734 MT-2015 isolate 15052715 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Phlogotettix sp. KX437721 MT-2015 isolate Zz052705 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Psammotettix sp. KX437742 MT-2015 isolate Zz060503 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Sophonia linealis KX437723 MT-2015 isolate Zz052717 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Atkinsoniclla sp. KX437743 MT-2015 isolate Zz060410 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Athysanopsis sp. KX437726 MT-2015 isolate Zz052318 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Philaenus spumarius AY630340 – – Cercopoidea Callitettix versicolor EU725832 – – Cercopoidea Callitettix braconoides NC_025497 – – Cercopoidea Callitettix biformis NC_025496 – – Cercopoidea Abidama producta NC 015799 – – Cercopoidea Aeneolamia contigua NC_025495 – – Cercopoidea Cosmoscarta bispecularis NC_026289 – – Cercopoidea Paphnutius ruficeps KX437731 MT-2015 isolate Zz052908 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Aphrophora intermedia KX437729 MT-2015 isolate Zz062722 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Clovia sp. KX437730 MT-2015 isolate Zz052918 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cicadoidea Magicicada tredecim KM000130 – – Cicadoidea Gaeana maculata KM244671 – – Cicadoidea Tettigades auropilosa KM000129 – – Cicadoidea Tettigades ulnaria KM000128 – – Cicadoidea Diceroprocta semicincta KM000131 – – Fulgoroidea Laodelphax striatellus FJ360695 – – Fulgoroidea Laodelphax striatella JX880068 – – Fulgoroidea Nilaparvata lugens JN563995 – – Fulgoroidea Sogatella furcifera NC_021417 – – Fulgoroidea Nilaparvata muiri NC_024627 – – Fulgoroidea Lycorma delicatula EU909203 – – Fulgoroidea Pyrops candelaria FJ006724 – – Fulgoroidea Ricania marginalis JN242415 – – Fulgoroidea Geisha distinctissima FJ230961 – – Fulgoroidea Sivaloka damnosus FJ360694 – – Peloridioidea Xenophyes cascus JF323862 – – Peloridioidea Hemiodoecus leai NC_025329 – – Peloridioidea Hackeriella veitchi NC_020309 – – Psylloidea Pachypsylla venusta AY263317 – – Psylloidea Cacopsylla chinensis KX437732 MT-2015 isolate Zz060501 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cimicoidea Riptortus pedestris EU427344 – – Cimicoidea Hydaropsis longirostris EU427337 – – Lygaeoidea Geocoris pallidipennis EU427336 – – Atropetae Lepidopsocidae sp. AF335994 – – Psocetae Longivalvus hyalospilus JQ910986 – – Psocetae Psococerastis albimaculata NC_021400 – – Superfamily Species Accession number Voucher numbers for newly sequenced species Collecting information Membracoidea Darthula hardwickii NC_026699 – – Membracoidea Leptobelus gazella NC 023219 – – Membracoidea Centrotus cornutus KX437728 MT-2015 isolate Zz041601 Vietnam, Pingxiang, 22°11′N, 106°70′E, July 2012 Membracoidea Empoasca vitis NC 024838 – – Membracoidea Drabescoides nuchalis NC_028154 – – Membracoidea Homalodisca vitripennis NC_006899 – – Membracoidea Nephotettix cincticeps NC_026977 – – Membracoidea Balclutha sp. KX437738 MT-2015 isolate Zz052711 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus sp. KX437735 MT-2015 isolate Zz052706 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cicadula sp. KX437724 MT-2015 isolate Zz052713 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Cuerna sp. KX437741 MT-2015 isolate Zz052311 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Dryadomorpha sp. KX437736 MT-2015 isolate Zz060407 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Empoasca sp. KX437737 MT-2015 isolate Zz031203 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Exitianus indicus KX437722 MT-2015 isolate Zz052320 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Graphocephala sp. KX437740 MT-2015 isolate Zz052315 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Hishimonus phycitis KX437727 MT-2015 isolate Zz052707 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Nephotettix sp. KX437725 MT-2015 isolate Zz053036 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Osbornellus sp. KX437739 MT-2015 isolate Zz052506 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Petalocephala ochracea KX437734 MT-2015 isolate 15052715 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Phlogotettix sp. KX437721 MT-2015 isolate Zz052705 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Psammotettix sp. KX437742 MT-2015 isolate Zz060503 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Sophonia linealis KX437723 MT-2015 isolate Zz052717 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Atkinsoniclla sp. KX437743 MT-2015 isolate Zz060410 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Membracoidea Athysanopsis sp. KX437726 MT-2015 isolate Zz052318 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Philaenus spumarius AY630340 – – Cercopoidea Callitettix versicolor EU725832 – – Cercopoidea Callitettix braconoides NC_025497 – – Cercopoidea Callitettix biformis NC_025496 – – Cercopoidea Abidama producta NC 015799 – – Cercopoidea Aeneolamia contigua NC_025495 – – Cercopoidea Cosmoscarta bispecularis NC_026289 – – Cercopoidea Paphnutius ruficeps KX437731 MT-2015 isolate Zz052908 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Aphrophora intermedia KX437729 MT-2015 isolate Zz062722 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cercopoidea Clovia sp. KX437730 MT-2015 isolate Zz052918 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cicadoidea Magicicada tredecim KM000130 – – Cicadoidea Gaeana maculata KM244671 – – Cicadoidea Tettigades auropilosa KM000129 – – Cicadoidea Tettigades ulnaria KM000128 – – Cicadoidea Diceroprocta semicincta KM000131 – – Fulgoroidea Laodelphax striatellus FJ360695 – – Fulgoroidea Laodelphax striatella JX880068 – – Fulgoroidea Nilaparvata lugens JN563995 – – Fulgoroidea Sogatella furcifera NC_021417 – – Fulgoroidea Nilaparvata muiri NC_024627 – – Fulgoroidea Lycorma delicatula EU909203 – – Fulgoroidea Pyrops candelaria FJ006724 – – Fulgoroidea Ricania marginalis JN242415 – – Fulgoroidea Geisha distinctissima FJ230961 – – Fulgoroidea Sivaloka damnosus FJ360694 – – Peloridioidea Xenophyes cascus JF323862 – – Peloridioidea Hemiodoecus leai NC_025329 – – Peloridioidea Hackeriella veitchi NC_020309 – – Psylloidea Pachypsylla venusta AY263317 – – Psylloidea Cacopsylla chinensis KX437732 MT-2015 isolate Zz060501 China, Xinyang, Mt. Jigong, 31°50′N, 114°05′E, July 2015 Cimicoidea Riptortus pedestris EU427344 – – Cimicoidea Hydaropsis longirostris EU427337 – – Lygaeoidea Geocoris pallidipennis EU427336 – – Atropetae Lepidopsocidae sp. AF335994 – – Psocetae Longivalvus hyalospilus JQ910986 – – Psocetae Psococerastis albimaculata NC_021400 – – Mitogenomes newly sequenced in this study are indicated in bold. View Large Dna extraction Genomic DNA was individually extracted from each specimen preserved in 95–100% ethanol using the TIANamp Micro DNA Kit (Tiangen Biotech Co., Ltd) following the manufacturer’s instructions. Mitogenome assembly from next-generation sequencing data The process for assembling mitogenomes from next-generation sequencing data followed the protocol by Gillett et al. (2014). This approach was designed for mitogenome reconstruction from pooled DNA samples, with the following steps: sample and library preparation, genome sequencing with Illumina HiSeq Sequencer (Supporting Information, Table S1 and Fig. S1), searching for target mitogenome (Supporting Information, Tables S2, S3) and mitogenome annotation. The detailed protocols are provided in Supporting Information. Sequence alignment and data set concatenation Each of the 37 mitochondrial genes was aligned separately for further analysis. For protein-coding genes, stop codons were removed and aligned based on codons using the invertebrate mitochondrial genetic code in the Perl script transAlign (Bininda-Emonds, 2005). Both the mitochondrial tRNA and rRNA genes were aligned using MAFFT (version 7) under the iterative refinement method, incorporating the most accurate local (E-INS-i) pairwise alignment information (Katoh & Standley, 2013). Alignments were checked in MEGA 6 (Tamura et al., 2013). Gaps were striped with Gap Strip/Squeeze v2.1.0, with a 40% gap tolerance (http://www.hiv.lanl.gov/content/sequence/GAPSTREEZE/gap.html). All alignments were concatenated into two matrices using FASconCAT_v1.0 (Kück & Meusemann, 2010), one including RNA genes and one excluding. In addition, to alleviate the effect of substitution saturation of codon positions on phylogenetic estimation, protein-coding genes were recoded or masked to construct the following data sets: (1) PCG_AA, (2) PCG3RY, (3) PCG13RY, (4) PCGDegen, (5) Alicut_PCG and (6) Alicut_PCGRNA. The detailed interpretation of each data recoding scheme are given in Supporting Information. Sequence characteristic analyses Nucleotide composition was calculated using MEGA 6 (Supporting Information, Table S4). Sequence saturation was assessed using the index of substitution saturation (Iss) of Xia et al. (2003) as implemented in DAMBE 5 (Xia et al., 2003; Xia, 2013) (Supporting Information, Table S5). Nucleotide homogeneity across taxa was assessed for alignments of PCG, PCG3RY, PCGDegen and PCG13RY using a chi-square test (Farris et al., 1994) implemented in PAUP*4.0b10 (Swofford, 2002) (Supporting Information, Table S6-A). To explore the source of the heterogeneity of base frequencies, a chi-square test was performed based on the nucleotide data sets (PCG, PCG3RY, PCGDegen and PCG13RY) and the amino acid data set (PCG_AA) using TREE-PUZZLE ver. 5.2 (Schmidt et al., 2002) under the GTR model for nucleotides and the mtREVE model for amino acids (Supporting Information, Table S6-B). Estimates of nonsynonymous (dN) and synonymous (dS) substitution rates of concatenated protein-coding genes were obtained using the method by Yang & Nielsen (2000) and the program yn00 as implemented in PAML 4.9 (Yang, 2007) (Supporting Information, Table S7). Phylogenetic analyses Phylogenetic analyses were performed under ML and Bayesian inferences. Partitioned ML searches were carried out using IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) (Nguyen et al., 2015; Trifinopoulos et al., 2016). PartitionFinder (Lanfear et al., 2012) was used to infer optimal partitioning strategy and best-fit models (Supporting Information, Table S8). The Bayesian analyses were conducted using PhyloBayes (Lartillot & Philippe, 2004; Lartillot, Lepage & Blanquart, 2009) as implemented in the CIPRES Portal (Miller, Pfeiffer & Schwartz, 2010). The detailed parameter settings of ML and the Bayesian analyses are provided in Supporting Information. For each tree reconstruction, we used FigTree v1.4.3 (Rambaut, 2009) to visualize the topology and to calculate the corresponding branch lengths. One-way analyses of variance (ANOVAs) for branch lengths of major groups were performed in Excel 2016 (Table 2). Table 2. Nodal supports and branch lengths for major lineages in each tree Data set Heteroptera Psylloidea Peloridioidea Fulgoroidea Cicadoidea Cercopoidea Membracoidea NS BL NS BL NS BL NS BL NS BL NS BL NS BL Maximum likelihood analyses using IQ-TREE CG 100 2.79 100 7.07 100 5.53 100 5.75 100 3.79 100 3.67 100 6.1 PCG3RY 100 0.77 100 2.04 100 1.6 100 1.73 100 1.06 100 1.03 100 1.76 PCGDegen 100 0.52 100 1.15 100 0.88 100 1 100 0.62 100 0.61 100 0.99 PCG13RY 100 0.77 100 1.64 100 1.25 100 1.41 100 0.9 100 0.91 100 1.56 Alicut-PCG 100 0.36 100 0.84 100 0.78 100 0.72 100 0.54 100 0.52 100 0.88 PCG_AA 100 0.85 100 2.05 100 1.7 100 1.79 100 1.09 100 1.19 100 1.53 PCGRNA 100 1.84 100 4.59 100 3.5 100 3.86 100 2.63 100 2.55 100 3.99 PCGDegenRNA 100 0.41 100 1.02 100 0.8 100 0.92 100 0.59 100 0.53 100 0.92 Alicut-PCGRNA 100 0.31 100 0.72 100 0.65 100 0.64 100 0.41 100 0.42 100 0.71 Bayesian analyses using PhyloBayes PCG 1 1.51 1 4.87 1 3.13 1 4.84 1 2.01 1 1.89 1 3.88 PCG3RY 1 0.45 1 1.6 1 0.96 1 1.29 1 0.53 1 0.61 1 1.03 PCGDegen 1 0.68 1 2.38 1 1.44 1 2.27 1 0.81 1 0.85 1 1.69 PCG13RY 1 0.45 1 1.78 1 1.12 1 1.77 1 0.55 1 0.61 1 1.04 Alicut-PCG 1 1.56 1 6.15 1 3.86 1 6.16 1 2.02 1 2.13 1 4.23 PCG_AA 1 1.35 1 4.22 1 3.27 1 3.44 1 1.89 1 1.99 1 3.14 PCGRNA 1 1.29 1 3.99 1 2.58 1 3.4 1 1.99 1 1.79 1 3.32 PCGDegenRNA 1 0.69 1 2.49 1 2.02 1 2.02 1 0.85 1 0.83 1 1.76 Alicut-PCGRNA 1 1.23 1 4.69 1 2.94 1 4.22 1 1.61 1 1.56 1 3.24 Data set Heteroptera Psylloidea Peloridioidea Fulgoroidea Cicadoidea Cercopoidea Membracoidea NS BL NS BL NS BL NS BL NS BL NS BL NS BL Maximum likelihood analyses using IQ-TREE CG 100 2.79 100 7.07 100 5.53 100 5.75 100 3.79 100 3.67 100 6.1 PCG3RY 100 0.77 100 2.04 100 1.6 100 1.73 100 1.06 100 1.03 100 1.76 PCGDegen 100 0.52 100 1.15 100 0.88 100 1 100 0.62 100 0.61 100 0.99 PCG13RY 100 0.77 100 1.64 100 1.25 100 1.41 100 0.9 100 0.91 100 1.56 Alicut-PCG 100 0.36 100 0.84 100 0.78 100 0.72 100 0.54 100 0.52 100 0.88 PCG_AA 100 0.85 100 2.05 100 1.7 100 1.79 100 1.09 100 1.19 100 1.53 PCGRNA 100 1.84 100 4.59 100 3.5 100 3.86 100 2.63 100 2.55 100 3.99 PCGDegenRNA 100 0.41 100 1.02 100 0.8 100 0.92 100 0.59 100 0.53 100 0.92 Alicut-PCGRNA 100 0.31 100 0.72 100 0.65 100 0.64 100 0.41 100 0.42 100 0.71 Bayesian analyses using PhyloBayes PCG 1 1.51 1 4.87 1 3.13 1 4.84 1 2.01 1 1.89 1 3.88 PCG3RY 1 0.45 1 1.6 1 0.96 1 1.29 1 0.53 1 0.61 1 1.03 PCGDegen 1 0.68 1 2.38 1 1.44 1 2.27 1 0.81 1 0.85 1 1.69 PCG13RY 1 0.45 1 1.78 1 1.12 1 1.77 1 0.55 1 0.61 1 1.04 Alicut-PCG 1 1.56 1 6.15 1 3.86 1 6.16 1 2.02 1 2.13 1 4.23 PCG_AA 1 1.35 1 4.22 1 3.27 1 3.44 1 1.89 1 1.99 1 3.14 PCGRNA 1 1.29 1 3.99 1 2.58 1 3.4 1 1.99 1 1.79 1 3.32 PCGDegenRNA 1 0.69 1 2.49 1 2.02 1 2.02 1 0.85 1 0.83 1 1.76 Alicut-PCGRNA 1 1.23 1 4.69 1 2.94 1 4.22 1 1.61 1 1.56 1 3.24 The branch lengths were calculated from the longest terminal taxon of each lineage to the common ancestor to the Psocoptera. NS, nodal support; BL, branch length. View Large Table 2. Nodal supports and branch lengths for major lineages in each tree Data set Heteroptera Psylloidea Peloridioidea Fulgoroidea Cicadoidea Cercopoidea Membracoidea NS BL NS BL NS BL NS BL NS BL NS BL NS BL Maximum likelihood analyses using IQ-TREE CG 100 2.79 100 7.07 100 5.53 100 5.75 100 3.79 100 3.67 100 6.1 PCG3RY 100 0.77 100 2.04 100 1.6 100 1.73 100 1.06 100 1.03 100 1.76 PCGDegen 100 0.52 100 1.15 100 0.88 100 1 100 0.62 100 0.61 100 0.99 PCG13RY 100 0.77 100 1.64 100 1.25 100 1.41 100 0.9 100 0.91 100 1.56 Alicut-PCG 100 0.36 100 0.84 100 0.78 100 0.72 100 0.54 100 0.52 100 0.88 PCG_AA 100 0.85 100 2.05 100 1.7 100 1.79 100 1.09 100 1.19 100 1.53 PCGRNA 100 1.84 100 4.59 100 3.5 100 3.86 100 2.63 100 2.55 100 3.99 PCGDegenRNA 100 0.41 100 1.02 100 0.8 100 0.92 100 0.59 100 0.53 100 0.92 Alicut-PCGRNA 100 0.31 100 0.72 100 0.65 100 0.64 100 0.41 100 0.42 100 0.71 Bayesian analyses using PhyloBayes PCG 1 1.51 1 4.87 1 3.13 1 4.84 1 2.01 1 1.89 1 3.88 PCG3RY 1 0.45 1 1.6 1 0.96 1 1.29 1 0.53 1 0.61 1 1.03 PCGDegen 1 0.68 1 2.38 1 1.44 1 2.27 1 0.81 1 0.85 1 1.69 PCG13RY 1 0.45 1 1.78 1 1.12 1 1.77 1 0.55 1 0.61 1 1.04 Alicut-PCG 1 1.56 1 6.15 1 3.86 1 6.16 1 2.02 1 2.13 1 4.23 PCG_AA 1 1.35 1 4.22 1 3.27 1 3.44 1 1.89 1 1.99 1 3.14 PCGRNA 1 1.29 1 3.99 1 2.58 1 3.4 1 1.99 1 1.79 1 3.32 PCGDegenRNA 1 0.69 1 2.49 1 2.02 1 2.02 1 0.85 1 0.83 1 1.76 Alicut-PCGRNA 1 1.23 1 4.69 1 2.94 1 4.22 1 1.61 1 1.56 1 3.24 Data set Heteroptera Psylloidea Peloridioidea Fulgoroidea Cicadoidea Cercopoidea Membracoidea NS BL NS BL NS BL NS BL NS BL NS BL NS BL Maximum likelihood analyses using IQ-TREE CG 100 2.79 100 7.07 100 5.53 100 5.75 100 3.79 100 3.67 100 6.1 PCG3RY 100 0.77 100 2.04 100 1.6 100 1.73 100 1.06 100 1.03 100 1.76 PCGDegen 100 0.52 100 1.15 100 0.88 100 1 100 0.62 100 0.61 100 0.99 PCG13RY 100 0.77 100 1.64 100 1.25 100 1.41 100 0.9 100 0.91 100 1.56 Alicut-PCG 100 0.36 100 0.84 100 0.78 100 0.72 100 0.54 100 0.52 100 0.88 PCG_AA 100 0.85 100 2.05 100 1.7 100 1.79 100 1.09 100 1.19 100 1.53 PCGRNA 100 1.84 100 4.59 100 3.5 100 3.86 100 2.63 100 2.55 100 3.99 PCGDegenRNA 100 0.41 100 1.02 100 0.8 100 0.92 100 0.59 100 0.53 100 0.92 Alicut-PCGRNA 100 0.31 100 0.72 100 0.65 100 0.64 100 0.41 100 0.42 100 0.71 Bayesian analyses using PhyloBayes PCG 1 1.51 1 4.87 1 3.13 1 4.84 1 2.01 1 1.89 1 3.88 PCG3RY 1 0.45 1 1.6 1 0.96 1 1.29 1 0.53 1 0.61 1 1.03 PCGDegen 1 0.68 1 2.38 1 1.44 1 2.27 1 0.81 1 0.85 1 1.69 PCG13RY 1 0.45 1 1.78 1 1.12 1 1.77 1 0.55 1 0.61 1 1.04 Alicut-PCG 1 1.56 1 6.15 1 3.86 1 6.16 1 2.02 1 2.13 1 4.23 PCG_AA 1 1.35 1 4.22 1 3.27 1 3.44 1 1.89 1 1.99 1 3.14 PCGRNA 1 1.29 1 3.99 1 2.58 1 3.4 1 1.99 1 1.79 1 3.32 PCGDegenRNA 1 0.69 1 2.49 1 2.02 1 2.02 1 0.85 1 0.83 1 1.76 Alicut-PCGRNA 1 1.23 1 4.69 1 2.94 1 4.22 1 1.61 1 1.56 1 3.24 The branch lengths were calculated from the longest terminal taxon of each lineage to the common ancestor to the Psocoptera. NS, nodal support; BL, branch length. View Large To evaluate the impact of character state variation on tree topology, we ran the Goremykin et al. (2010) NoiseReductor script (sorter.pl) on the full data set (i.e. PCGRNA) and performed both ML and Bayesian inferences. In addition, the nucleotide compositional heterogeneity of the data set PCG was tested through pairwise Euclidean distances (Regier & Zwick, 2011; Zwick et al., 2012; Regier et al., 2013). The detailed protocols of both analyses are provided in Supporting Information. RESULTS The complete or nearly complete mitogenomes were recovered for ten species with the same gene organization as the ancestral insect (Cameron, 2014b). The other 12 species had partial mitogenomes and the missing genes were mainly located adjacent to the putative control region. A detailed description of mitogenome assembly and the characteristics of the data matrix are provided in Supporting Information. The monophyly of Hemiptera received strong support from all analyses (bootstrap support value [BP] = 100, Bayesian posterior probability value [PP] = 1.0). All superfamilies represented by more than two species were monophyletic. However, the concatenated alignments with or without RNA genes, different data recoding strategies for protein-coding genes and tree construction methods resulted in the incongruent relationships among superfamilies. ML analyses Most of the ML analyses under site-homogeneous models yielded the same tree topology (Fig. 1), except for those from Degen-coding and alignment masking. Heteroptera was well supported as the sister group to all other hemipterans (BP = 100). The remaining hemipteran taxa were divided into two main clades. The first clade comprised three superfamilies: Peloridioidea, Psylloidea and Fulgoroidea. Most ML analyses recovered Peloridioidea and Psylloidea as a clade (BP > 70), collectively sister to the superfamily Fulgoroidea. The second clade was composed of the three Cicadomorpha superfamilies: Membracoidea, Cicadoidea and Cercopoidea. In all ML analyses, relationships within Cicadomorpha were consistent [Membracoidea + (Cicadoidea + Cercopoidea)], with strong nodal supports for the major nodes (BP ≥ 90). In the ML analyses, only the data set PCGDegen embedded Heteroptera within an ingroup and as sister to Cicadomorpha. For the analyses with alignment masking (Alicut-PCG and Alicut-PCGRNA), the only difference occurred in the first major clade. The topological pattern [Peloridioidea + (Psylloidea + Fulgoroidea)] was retrieved by Alicut-PCG and Alicut-PCGRNA. Overall, Auchenorrhyncha was rendered paraphyletic, due to the Fulgoroidea grouping with the clade (Psylloidea + Peloridioidea) or with Psylloidea alone. Visual inspection of the ML trees revealed that two representatives from outgroup Psylloidea (Sternorrhyncha), five representatives from ingroup Fulgoroidea (all sample in Delphacidae) and two representatives from ingroup Membracoidea (i.e. Darthula hardwickii and Petalocephala ochracea) exhibited the conspicuously long branches. The one-way ANOVA revealed no difference in the branch lengths between major groups (P > 0.05) (Table 2). Figure 1. View largeDownload slide Maximum likelihood tree estimated with IQ-TREE from the data set of PCGRNA, with the partition schemes and best-fit models selected by PartitionFinder. Node numbers show bootstrap support values (> 50). Scale bar represents substitutions/site. Asterisks designate the species newly sequenced in this study. Figure 1. View largeDownload slide Maximum likelihood tree estimated with IQ-TREE from the data set of PCGRNA, with the partition schemes and best-fit models selected by PartitionFinder. Node numbers show bootstrap support values (> 50). Scale bar represents substitutions/site. Asterisks designate the species newly sequenced in this study. Bayesian analyses In most of the PhyloBayes analyses, the site- heterogeneous CAT model had no important effect on deep relationships. The Bayesian trees recovered a paraphyletic Auchenorrhyncha, quite similar to the trees recovered by ML analysis under the site-homogeneous model. The differences observed between ML and Bayesian trees were centred on the following aspects. (1) Within the clade containing Peloridioidea, Psylloidea and Fulgoroidea, PCG, Alicut_PCG, PCGRNA and Alicut_PCGRNA resulted in [Peloridioidea + (Psylloidea + Fulgoroidea)] (as in ML Alicut-PCG and in ML Alicut-PCGRNA), whereas PCGDegen, PCG13RY, PCG_AA and PCGDegenRNA recovered [Fulgoroidea + (Psylloidea + Peloridioidea)] (as in all other ML trees). In the PhyloBayes analysis of PCG3RY, the relationship between Peloridioidea, Psylloidea and Fulgoroidea was unresolved. (2) For Cicadomorpha, the Bayesian analyses of PCG3RY, PCG13RY, PCGDegen and PCGDegenRNA recovered the sister relationship of [Cicadoidea + (Cercopoidea + Membracoidea)]. (3) For the position of Heteroptera, Bayesian analyses revealed three possibilities: sister group to all other Hemiptera in most cases, sister group to Cicadomorpha in PCG3RY tree (but without significant statistical support, PP = 0.63), clustered with the grouping [Peloridioidea + (Psylloidea + Fulgoroidea)] in the trees from Alicut-PCG and Alicut-PCGRNA. The branch-length heterogeneity is more obvious in the Bayesian trees due to longer branch lengths leading to Psylloidea, Peloridioidea and Fulgoroidea. The one-way ANOVA showed significant differences in the branch lengths between major groups (P = 0.0000 < 0.05). Results from the OV-sorted alignments For the ML analyses of OV-sorted alignments, Auchenorrhyncha was paraphyletic until the 5500 most variable sites were deleted (data set 8765 bp): [Fulgoroidea + ((Cicadoidea + Cercopoidea) + Membracoidea)] (Supporting Information, Fig. S2). In the subsequent three sorting analyses (i.e. the data sets 7265, 6765 and 6265 bp), a monophyletic Auchenorrhyncha was still retrieved, but the inter-superfamily branches became too short to be distinguished. After data set 5765 bp, relationships within Hemiptera became unresolved, but Heteroptera remained sister to the rest. In the PhyloBayes analyses of OV-sorted alignments (CAT-GTR model), the monophyly of Auchenorrhyncha was recovered after the 5500 most variable sites were deleted (data set 8765 bp) (Fig. 2). An identical topology was recovered at the next shortening step (data set 8265 bp). In both trees, Sternorrhyncha was sister to the remaining Hemiptera; Heteropterodea was not supported as Heteroptera was sister to a monophyletic Auchenorrhyncha. Within Auchenorrhyncha, superfamily relationships were concordant with the ML analyses of the OV-sorted data set 8765 bp: [Fulgoroidea + ((Cicadoidea + Cercopoidea) + Membracoidea)]. Although Auchenorrhyncha was still supported in the next two shorter data sets (7765 and 7265 bp), an unexpected superfamily relationship [(Cicadoidea + Cercopoidea) + (Fulgoroidea + Membracoidea)] was retrieved (Supporting Information, Fig. S3). These two sister group relationships were weakly supported. In subsequent analyses with further removal of variable positions, relationships within Hemiptera collapsed due to information loss. Figure 2. View largeDownload slide Bayesian tree inferred from the OV-sorted PCGRNA data set (8765 bp) using PhyloBayes with the CAT-GTR model. Numbers on the tree indicate the Bayesian posterior probability values (> 0.90). Scale bar represents substitutions/site. Figure 2. View largeDownload slide Bayesian tree inferred from the OV-sorted PCGRNA data set (8765 bp) using PhyloBayes with the CAT-GTR model. Numbers on the tree indicate the Bayesian posterior probability values (> 0.90). Scale bar represents substitutions/site. Assessment of compositional heterogeneity A Euclidean distance analysis of the nucleotide composition of PCG showed evidence of strong compositional heterogeneity, especially for Coleorrhyncha with long subtending branches (Supporting Information, Fig. S4). This indicated that the three species of Coleorrhyncha shared a similar sequence composition to the remaining Hemiptera. Compositional heterogeneity is likely to have a distorting effect on analyses that included Coleorrhyncha. Unfortunately, removal of Coleorrhyncha did not significantly alter phylogenetic inference; paraphyletic Auchenorrhyncha was still found in the analyses under both homogeneous and heterogeneous models without Coleorrhyncha (Supporting Information, Fig. S5). Comparisons across compositional distance trees indicated that neither coding scheme nor the OV-sorting method effectively removed compositional heterogeneity. A long-branch Coleorrhyncha was consistently found in all compositional distance trees (Supporting Information, Fig. S6). DISCUSSION Limits of mitogenomes in resolving higher-level auchenorrhynchan phylogeny With continued technological development, the availability of insect mitogenomes has increased dramatically. Using the mitogenome as a molecular marker has generally been reserved for resolving intra-ordinal phylogenetic relationships of insects, such as in Coleoptera (Gillett et al., 2014; Timmermans et al., 2016; Nie et al., 2017), Diptera (Cameron et al., 2007) and Orthoptera (Ma et al., 2012). The mitogenome has been shown to be insufficient for recovering deeper relationships (Talavera & Vila, 2011; Song et al., 2016a), and its use often resulted in a conclusion incongruent with other phylogenetic evidence. Talavera & Vila (2011) revealed that fast sequence evolution of some insect groups compromised the utility of the mitogenome in solving higher-level relationships. Attempts to test the monophyly of Auchenorrhyncha using mitogenomic data initially included almost all available paraneopteran mitogenomes. However, several lineages with accelerated sequence evolutionary rates exhibited significant long-branch attraction, where Phthiraptera and Thysanoptera were nested within Hemiptera, with Sternorrhyncha (in particular with aphid and white flies). Therefore, we selected relatively slow-evolving mitogenomes from Psocoptera and Psylloidea (Sternorrhyncha) for this study. However, removing fasting-evolving outgroups did not overcome the long-branch effect. Within Hemiptera, long-branched Sternorrhyncha and Fulgoromorpha were still clustered in an assemblage. In the mitogenomic study by Simon & Hadrys (2013), the sister group relationship between Sternorrhyncha and Fulgoromorpha was identified as systematically erroneous. Thus, the paraphyly of Auchenorrhyncha, attributed to attraction between Sternorrhyncha and Fulgoromorpha, is not an accepted hypothesis. Long branches are often correlated with accelerated rates of substitution (Talavera & Vila, 2011; Bernt et al., 2013; Simon & Hadrys, 2013) and/or composition heterogeneity (Regier & Zwick, 2011; Zwick et al., 2012; Regier et al., 2013). Our substitution rate analysis indicated that accelerated substitution rates were shared by Peloridioidea, Psylloidea and Fulgoroidea (Supporting Information, Table S7). Furthermore, Euclidean distance analyses showed significant compositional heterogeneity in the data (Supporting Information, Figs S4, S6). To avoid saturation and composition heterogeneity of the mitogenomic data, we used comprehensive data recoding strategies and reran the phylogenetic analyses. The long-branched assemblage (i.e. Peloridioidea, Psylloidea and Fulgoroidea) could not be separated. Comparison of the compositional distance trees from the recoded data sets demonstrated that no data treatment removed all compositional heterogeneity (Supporting Information, Fig. S6). This also suggests why most analyses failed to break up long branches: the compositional signal is stronger than the phylogenetic signal. Several recent studies have shown that the use of the Bayesian mixture model, CAT, can effectively avoid or partly overcome long-branch attraction (Lartillot, Brinkmann & Philippe, 2007; Lartillot et al., 2009; Talavera & Vila, 2011; Song et al., 2016b). However, tree searches using the CAT-GTR or CAT model implemented in the software PhyloBayes produced similar topologies to ML analyses under the site-homogeneous model. The Bayesian mixture model could not substantially avoid long-branch attraction artefacts for the current mitogenomic data sets with full taxa, even when combined with recoding schemes. This again demonstrated the difficulty in resolving the ‘Auchenorrhyncha question’ with mitogenomes. Taken together, the complicating factors, including biased base composition, substitution saturation, compositional heterogeneity and lineage-specific rate, limit the phylogenetic utility of the mitogenome in recovering deeper relationships in Auchenorrhyncha. In further research, using genomic data to uncover new nuclear genes with relatively slow-evolving rates may be more effective. Higher-level phylogeny of Auchenorrhyncha Highly variable positions can increase long-branch attraction effects. Using OV-sorting (Goremykin et al., 2010), the proportion of noise in the data set decreased as highly variable positions were successively removed. The effect of removing saturated sites was identified by comparing topologies from a series of data sets with different proportions of conserved and variable sites. In the OV-sorting analyses, alignments with reduced variability supported auchenorrhynchan monophyly (Fig. 2 and Supporting Information, Figs S2, S3). At the same time, there was a decrease in nodal support for [Fulgoroidea + (Psylloidea + Peloridioidea)] as the proportion of highly variable sites reduced. The results of the OV-sorting analyses also indicated that auchenorrhynchan paraphyly is probably an artefact caused by the evolutionary characteristics of mitogenome sequences, for example high substitution saturation and higher overall substitution rates. Therefore, inference based on noise-reduced alignments may be more reliable. At the superfamily level, the relationship [Membracoidea + (Cicadoidea + Cercopoidea)] was well supported by all analyses under ML criteria regardless of data treatments, and in five of nine Bayesian analyses (i.e. PCG, PCGAA, Alicut-PCG, PCGRNA and Alicut-PCGRNA). This arrangement is concordant with previous molecular studies based on nuclear and/or mitochondrial genes (Cryan, 2005; Cryan & Urban, 2012). In addition, analyses based on more conserved data sets with the OV-sorting treatment tended to also support [Membracoidea + (Cicadoidea + Cercopoidea)]. Therefore, we conclude that the mitogenome may be useful for relationships below the infraorder of Auchenorrhyncha. The placement of Heteroptera Although the placement of Heteroptera is beyond the scope of this study, this clade is one of the outgroups closely related to the recovered ingroup Auchenorrhyncha. In most previous phylogenetic studies, Heteroptera was suggested to be derived from within ‘Homoptera’ (Campbell et al., 1995; von Dohlen & Moran, 1995; Sorensen et al., 1995; Bourgoin & Campbell, 2002). Based on the comprehensive fossil, molecular and morphological interpretations, Bourgoin & Campbell (2002) proposed a sister group relationship between Heteropterodea (comprising Heteroptera and Coleorrhyncha) and Cicadomorpha. In this study, the position of Heteroptera was variable: sister to the remaining Hemiptera, sister to Cicadomorpha, clustered with the grouping [Peloridioidea + (Psylloidea + Fulgoroidea)] or sister to a monophyletic Auchenorrhyncha. Although the cause of these conflicting results between methods is complicated, long-branch attraction may be one of the factors. That Heteroptera was retrieved as a sister group to all other Hemitpera may result from clustering caused by attraction to all other long-branch taxa. In the existing mitogenomic data, long-branch attraction occurs not only between the outgroup and the ingroup but also between the taxa of the ingroup. To avoid the possible long-branch attraction between the outgroup and ingroup, we removed all long-branch outgroup taxa (i.e. Coleorrhyncha and Sternorrhyncha) and compiled the reduced data set with only Psocoptera and Heterotera as outgroups. The results revealed the possibility of long-branch attraction between Fulgoroidea and Membracoidea (Supporting Information, Fig. S7). Thus, elucidating the placement of Heteroptera relative to taxa of Auchenorrhyncha was difficult with the available mitogenomic data. CONCLUSION The anomalous characteristics in the Hemiptera mitogenomes, such as biased base composition, substitution saturation, compositional heterogeneity and clade-specific rate, lead to the striking differences in branch length between groups. These introduce the possibility of long-branch attraction, thus limiting the applicability of mitogenomic data in phylogenetic reconstructions of Hemiptera or Auchenorrhyncha. This study presents a series of exploratory analyses required to obtain ‘reasonable’ phylogenetic results from insect mitogenome data sets. Although newly sequenced data do not contribute substantially towards resolving the monophyly of Auchenorrhyncha, this article serves as a cautionary tale for future researchers attempting phylogenomic analyses to obtain plausible results for this insect group. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Statistics associated with the sequencing of mitogenomes using NGS-Illumina technology in 22 hemipteran species. Table S2. Primers designed for amplifying ‘Bait’ sequences. Table S3. Local blast for the ‘Bait’ sequences of each species newly determined in this study. Table S4. The mitochondrial genome nucleotide composition for the major lineage included in this study. Table S5. Saturation test for protein-coding genes, RNA genes and the reduced data sets with OV-sorting. Table S6. Chi-square test of homogeneity of base frequencies across taxa performed by (A) PAUP and (B) TREE-PUZZLE. Table S7. Estimation of synonymous and nonsynonymous substitution rates by yn00 implemented in PAML. Table S8. Partition schemes and best-fitting models selected by PartitionFinder for (A) PCG, (B) PCG_AA, and (C) PCGRNA. Figure S1. Mean sequencing coverage vs. concentration of genomic DNA in the sample pool for 22 identified mitogenomic assemblies. Figure S2. Maximum likelihood tree inferred from the OV-sorted PCGRNA data set (8765 bp) using IQ-TREE under the GTR model. Node numbers show bootstrap support values (> 50). Scale bar represents substitutions/site. Figure S3. Bayesian tree inferred from the OV-sorted PCGRNA data set (7765 bp) using PhyloBayes with the CAT-GTR model. Numbers on the tree indicate the Bayesian posterior probability values (> 0.90). Scale bar represents substitutions/site. Figure S4. Euclidean distance tree for the data set of PCG. Bootstrap percentages are displayed for the significantly long branches and indicate the strength of the compositional signal at particular nodes. The branch lengths represent the compositional heterogeneity in the data set: the longer a branch is, the stronger is the compositional signal. Figure S5. Maximum likelihood tree inferred from the PCG data set without three species from Coleorrhyncha using IQ-TREE under the GTR model. The PhyloBayes analysis based on the same data set provided largely identical tree topology. Node numbers show bootstrap support values (> 50, left) and Bayesian posterior probability values (> 0.90, right). Dashes denote the relationships not being retrieved or BP < 50 or PP < 0.9. Scale bar represents substitutions/site. Figure S6. Compositional distance trees for eight data sets – PCG3RY, PCGDegen, Alicut_PCG, PCGRNA, PCGDegenRNA, Alicut_PCGRNA, OV-sorting 8765bp, and OV-sorting 7765 bp. Red lines highlight three taxa from Coleorrhyncha. The branch length sum illustrates the total amount of compositional heterogeneity in the data set. Figure S7. (A) Maximum likelihood tree inferred from the PCG data set without Coleorrhyncha and Sternorrhyncha using IQ-TREE under the GTR model. Node numbers show bootstrap support values (> 50). Scale bar represents substitutions/site. The Cicadomorpha was non-monophyletic with respect to the nested position of Fulgoroidea. (B) The PhyloBayes tree under the CAT-GTR model based on the same data set. Node numbers show Bayesian posterior probability values (> 0.90). Scale bar represents substitutions/site. In this analysis, the CAT-GTR model suppressed the effect of long-branch attraction between Fulgoroidea and Membracoidea and recovered a monophyletic Cicadomorpha. ACKNOWLEDGEMENTS The detailed information for some sections of this article are tabulated in Supporting Information. We thank Doctor Peng Liu for advice in analysing data and writing this article. We are grateful to Dr Andreas Zwick for his kindly help in the analyses of compositional heterogeneity and for his helpful comments and suggestions on the manuscript. We acknowledge Professor Bojian Zhong for offering the Perl script for OV-sorting analyses. This research is supported by grants from the National Natural Science Foundation of China (No. 31402002), Key Scientific Research Projects of Henan Province (Grant Nos 14B210036 and 16A210029) and Henan Academician Workstation of Pest Green Prevention and Control for Plants in Southern Henan (YZ201601). REFERENCES Bergsten J . 2005 . A review of long-branch attraction . Cladistics 21 : 163 – 193 . Google Scholar CrossRef Search ADS Bernt M , Bleidorn C , Braband A , Dambach J , Donath A , Fritzsch G , Golombek A , Hadrys H , Jühling F , Meusemann K , Middendorf M , Misof B , Perseke M , Podsiadlowski L , von Reumont B , Schierwater B , Schlegel M , Schrödl M , Simon S , Stadler PF , Stöger I , Struck TH . 2013 . 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TI - Insufficient power of mitogenomic data in resolving the auchenorrhynchan monophyly
JF - Zoological Journal of the Linnean Society
DO - 10.1093/zoolinnean/zlx096
DA - 2017-12-26
UR - https://www.deepdyve.com/lp/oxford-university-press/insufficient-power-of-mitogenomic-data-in-resolving-the-KdvtBTs8Px
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -