Journal of Biotechnology and Sericology 80, 79-88 (2011)

The mitochondrial genome of a stick insect () and the phylogeny of polyneopteran

Shuichiro Tomita, Kenji Yukuhiro and Natuo Kômoto＊

National Institute of Agrobiological Sciences; Tsukuba, Ibaraki 305-8634, Japan (Received August 20, 2011; Accepted April 12, 2012)

Polyneoptera is an assemblage of 11 insect orders consisting of lower neopteran insects. The interordinal rela- tionships within Polyneoptera remain highly controversial and ancient rapid radiations are thought to be respon- sible. Phasmatodea, whose phylogenetic position among Polyneoptera is quite unstable, has been thought to be a key taxon to resolve the polyneopteran phylogeny. We determined the full-length sequence of the mitochondrial genome of a stick insect, Extatosoma tiaratum, and explored the phylogeny of polyneopteran insects using nu- cleic acid as well as amino acid sequences from the mitogenome. Our analyses recovered a close relationship between Phasmatodea, Mantophasmatodea and Grylloblattodea. Dictyoptera was placed as a sister to this group. The monophyly of Orthoptera was confirmed and Plecoptera was placed as a sister group of this order. Six clades are recovered within Phasmatodea in this study and most of them challenge the conventional classifi- cation system. Key words: mitogenomics, molecular phylogeny, Polyneoptera

Bradler, 2006), has also been placed as a sister to Plecop- INTRODUCTION tera, Dermaptera, or Zoraptera. Many previous studies Polyneoptera is a large group represented by 11 insect have found a close relationship between Orthoptera and orders including Blattodea (cockroaches), Dermaptera Phasmatodea (Kamp, 1973; Kukalová-Peck, 1991; Flook (earwigs), Embioptera (web-spinners), Grylloblattodea and Rowell, 1998; Wheeler et al., 2001; Terry and Whit- (ice-crawlers), Isoptera (termites), Mantodea (praying ing, 2005). However, the specialized state of sperm ultra- mantises), Mantophasmatodea (heel-walkers), Phasmato- structure is shared with Dermaptera and Phasmatodea, dea (stick insects), Plecoptera (stoneflies), Orthoptera thus suggesting sister-group relationships among these or- (grasshoppers), and Zoraptera (angel insects). Because the ders (Jamieson, 1987). derived states of morphologic characteristics have a mark- Previous studies have demonstrated that the mitochon- edly sporadic distribution among these orders, their rela- drial genome provides useful information when perform- tionships to each other and to the Paraneoptera and ing phylogenetic analyses (Boore and Brown, 1998; Endopterygota remain unclarified. In addition, because the Lavrov and Lang, 2005). Several convenient features of monophyly of Polyneoptera has not been well established, the mitogenome can be used to infer its phylogeny, such Kristensen et al. (1991) avoided the use of the word as its cellular abundance, the absence of recombination, “Polyneoptera” and instead referred to them as “the lower and the unambiguous orthology of genes, although lin- Neoptera”. Nevertheless, many hypotheses have been sug- eage-specific compositional heterogeneity and accelerated gested to explain the interordinal relationships of Polyne- substitution rates are major factors that negatively affect optera. Only the monophyly of Dictyoptera (Mantodea+ mitochondrial phylogenies. Blattodea+Isoptera) (Inward et al., 2007) and the sister re- In this work, we identified the full-length mitochondrial lationship of Grylloblattodea+Mantophasmatodea (Xenon- genome sequence of a stick insect, Extatosoma tiaratum, omia) (Terry and Whiting, 2005; cf. Plazzi et al., 2011) which inhabits in Queensland and New South Wales, Aus- have been repeatedly recovered by various molecular and tralia (Gurney, 1947). Also, we explored phasmatodean morphological studies (Arillo and Engel, 2006; Kjer et al., and polyneopteran phylogenetics using the most thorough 2006; Ishiwata et al., 2011). species sampling of mitochondrial genomes to date. Among polyneopteroid orders, the phylogenetic place- ment of Phasmatodea is extraordinarily unstable; it has MATERIALS AND METHODS been hypothesized to be a sister group to most orders within Polyneoptera. Embioptera, the most favored sister DNA extraction and sequencing group in recent years (Beutel and Gorb, 2001; Klug and Total genomic DNA was extracted from eggs using  common methods (Sambrook et al., 1989). The eggs were *To whom correspondence should be addressed. laid by a female obtained from a pet shop and the speci- Fax: +81-29-838-6263. Tel: +81-29-838-6285. men of the female individual is maintained as the voucher Email: [email protected] in our laboratory. PCR amplification and the mitochondri- 80 Tomita et al. al genome sequencing strategy were performed as de- al., 2011) as previously described (Kômoto et al., 2011). scribed previously (Kômoto et al., 2011). The sequence of In brief, we aligned the nucleotide sequences of mito- the Extatosoma tiaratum mitochondrial genome, which chondrial protein-coding genes (PCGs) based on their was identified in this study, was deposited in DDBJ with translated amino acid sequences, and rRNA genes were the accession number AB642680. aligned using MUSCLE (Edgar, 2004) in MEGA5. All aligned sets of PCGs and rRNA genes were concatenated Data set preparation and model finding for use in subsequent phylogenetic analyses. The list of Gene identification and alignment was performed using the species used in this study is shown in Table 1. Among ClustalW (Thompson et al., 1994) in MEGA5 (Tamura et them, two Odonata species, Davidius lunatus and Euphaea

Table 1. Species used in this study Order Species accession No. Odonata Davidius lunatus* EU591677 Euphaea formosa* HM126547 Plecoptera Pteronarcys princeps AY687866 Mantodea Tamolanica tamolana DQ241797 Blattaria Blattella germanica EU854321 Eupolyphaga sinensis FJ830540 Periplaneta fuliginosa AB126004 Isoptera Reticulitermes flavipes EF206314 Reticulitermes virginicus EF206318 Grylloblattodea Grylloblatta sculleni DQ241796 Mantophasmatodea Sclerophasma paresisense DQ241798 Phasmatodea: Timematodea Timema californicum DQ241799 Phasmatodea: Verophasmatodea Bacillus atticus GU001955 Bacillus rossius GU001956 Entoria okinawaensis AB477459 Extatosoma tiaratum AB642680 Heteropteryx dilatata AB477468 alpheus AB477471 Micadina phluctainoides AB477466 Neohirasea japonica AB477469 Orestes mouhotii AB477462 Phyllium giganteum AB477461 Phraortes illepidus AB477460 Phraortes sp. (Miyako Is.) AB477465 Phraortes sp. (Iriomote Is.) AB477464 AB477467 Ramulus hainanense FJ156750 Ramulus irregulariterdentatus AB477463 Sipyloidea sipylus AB477470 Orthoptera: Ensifera Anabrus simplex EF373911 Deracantha onos EU137664 Elimaea cheni GU323362 Gampsocleis gratiosa EU527333 Gryllotalpa orientalis AY660929 Myrmecophilus manni EU938370 Ruspolia dubia EF583824 Troglophilus neglectus EU938374 Orthoptera: Caelifera Acrida willemsei EU938372 Atractomorpha sinensis EU263919 Ellipes minuta GU945502 Euchorthippus fusigeniculatus HM583652 Gomphocerus licenti GQ180102 Locusta migratoria X80245 Oedaleus decorus EU513374 Schistocerca gregaria GQ491031 * outgroup taxa Polyneopteran phylogeny based on mitogenome 81 formosa, were used as the outgroup taxa. We constructed burn-in and combined the resulting MCMC tree samples nucleotide data sets composed of 11,621 nucleotide sites for subsequent estimation of posteriors. For amino acid from 13 concatenated mitochondrial PCGs and two rRNA data sets, we ran four concurrent analyses with eight genes (nt123). We also made a nucleotide data set exclud- chains each for 1.4 × 107 generations and discarded the ing the third codon position of the PCGs, which was com- first 70,000 samplings as burn-in. We conducted ML anal- posed of 8,162 sites. Amino acid data sets consisting of yses using RAxML PTHREADS 7.2.8 (Stamatakis, 2006). 3,459 sites were derived from the 13 PCGs. We applied ML tree searches and rapid bootstrapping We used Kakusan4 (Tanabe, 2011) to find nucleotide within one step (1,000 bootstrap replicates). RAxML used substitution models and partitioning strategies for the nu- randomized maximum parsimony starting trees for both cleotide data sets. Breaking down the nucleotide data by nucleotide and amino acid data sets. codon positions within each gene resulted in 41 partitions (first, second, and third codon positions for each of the 13 Topology test PCGs, rrnS, and rrnL) for the full nucleotide data set The tree topologies were statistically tested using Tree- (nt123 data set) and 28 partitions for the data set, exclud- Puzzle 5.2 (Schmidt et al., 2002) and CONSEL v. 0.1i, ing the third codon positions (nt12 data set), which were which perform the Kishino-Hasegawa (KH), Shimodaira- chosen for both Bayesian and maximum likelihood (ML) Hasegawa (SH), and approximately unbiased (AU) tests analyses. The likelihood was calculated based on the se- (Kishino and Hasegawa, 1989; Shimodaira and Hasegawa, quences separated into each codon position of each PCG. 1999, 2001; Shimodaira, 2002). Alternative topologies that Finally, a nucleotide substitution model was selected us- were tested were hypothesized based on the unstable ing the number of sites as the sample size based on the branching in the present analyses. Bayesian information criterion (BIC) for Bayesian analy- ses and Akaike’s information criterion (AIC) for ML anal- Substitution saturation test yses. Because Kakusan4 selects a nucleotide substitution We evaluated the level of substitution saturation apply- model and generates a nexus-formatted data file, which ing a saturation test using the F84 model implemented in composes a model applicable for MrBayes, and a phylip DAMBE v5.2.40 (Xia and Lemey, 2009). We constructed file for RAxML, we used those outputs from Kakusan4 the data sets for each codon position (nt1, nt2, nt3) and for subsequent Bayesian analyses with MrBayes and ML combined the sequences (nt12 and nt123). Prior to the analyses with RAxML. According to the software instruc- saturation test, we accounted for invariant sites, which tions, we assumed that the branch lengths are proportional provide a more reasonable estimation of potential satura- among genes for Bayesian analysis. tion within the data sets. The critical value for substitution Aminosan (Tanabe, 2011) was used to choose amino saturation index (Iss.c) was based on simulation results, acid substitution models and partitioning strategies. For which was limited to less than 32 operational taxonomic both Bayesian and ML analyses, partitioning the data was units (OTUs) using the method of Xia and Xie (2001). supported for each gene, and proper amino acid substitu- Because our data set included 45 OTUs, DAMBE ran- tion models were chosen according to the BIC and AIC domly sampled 4, 8, 16, and 32 OTUs multiple times and for Bayesian and ML analyses, respectively. performed the test for each subset.

Reconstruction of phylogenetic trees RESULTS Tree searches on nucleotide and amino acid sequences were performed using Bayesian inference and ML analy- The mitochondrial genome of Extatosoma tiara- ses using the data sets described above. Bayesian infer- tum ences were conducted using MrBayes 3.1.2 (Ronquist and We determined the entire mtDNA sequence of E. tiara- Huelsenbeck, 2003). For nucleotide data sets, we ran four tum, which is 16,537 bp in length. The mitogenome con- concurrent Bayesian analyses of 2 × 107 generations with sists of 13 PCGs, two ribosomal RNA genes, 23 tRNA eight chains each (seven heated and one cold) using dif- genes, and an A+T-rich region (Fig. 1). The gene order ferent random starting trees (nruns = 4), and sampled ev- and the strands, in which each PCG is encoded, were ery 100 generations. We examined the Markov chain identical to that proposed as a symplesiomorphic arrange- Monte Carlo (MCMC) samples in Tracer 1.5 (Rambaut ment of Pancrustacea (Boore et al., 1998). The overall and Drummond, 2009) to determine stationarity, conver- A+T content was 75.8%, and the AT skew and GC skew gence, and adequate mixing of the Markov chains. To ver- were 0.213 and −0.263, respectively. These three values ify convergence, the average deviation of split frequencies are within the range of other Verophasmatodea mitoge- from the four runs was confirmed to be below 0.01. From nomes that were 74.3 ~ 79.2, 0.148 ~ 0.261 and −0.125 ~ each data set, we discarded the first 100,000 samplings as −0.246, respectively (Kômoto et al., 2011). The A+T-rich 82 Tomita et al.

region consisted of tandem sequence repeats containing three 189-bp units flanked by an incomplete 77-bp unit.

Phylogenetic analyses Results from the phylogenetic inferences performed are displayed in Fig. 2. The tree topologies from each analysis are not strictly congruent but they share many important features. First, the monophyly of Phasmatodea (Timema+ Verophasmatodea sensu Zompro 2004) was supported in all analyses and Timema was distantly placed among Phasmatodea. Xenonomia (Sclerophasma+Grylloblatta) was recovered either as a monophyletic or a paraphyletic group depending on the data set and analytical methods used. Nonetheless, the monophyly of (Phasmatodea+ Fig. 1. Mitochondrial genome map of Extatosoma tiaratum. Sclerophasma+Grylloblatta) was supported in these analy- Genes on the outer circumference are transcribed clockwise and genes on the inner circumference are transcribed coun- ses. Monophyletic Dictyoptera was supported with high terclockwise. bootstrap values (BS) in the ML analyses and applying

Fig. 2. Polyneopteran phylogenetic trees based on mitochondrial genome sequences. (A) Phylogenetic tree inferred by ML analysis of the amino acid data set. The same tree topology was obtained by Bayesian analysis. ML bootstrap values (1,000 replicates) are shown in each node. Bayesian posterior probability was shown only when it was smaller than 1.00. (B) ML results based on the nt12 data set. (C) Bayesian results based on the nt12 data set. (D) ML results based on the nt123 data set. (E) Bayesian results based on the nt123 data set. (B-E) Applicable BS or PP are shown in ML or Bayesian analyses, respectively. Polyneopteran phylogeny based on mitogenome 83 84 Tomita et al. posterior probabilities (PP) in Bayesian inferences (100 monophyletic Ensifera was recovered in the analyses with and 1.00, respectively), and it appeared to be a sister nt12 data set (Fig. 2 BC). to the (Phasmatodea+Sclerophasma+Grylloblatta) clade. Five previously reported clades within Phasmatodea In most analyses, the monophyly of Orthoptera and (Kômoto et al., 2011) were stably recovered in these anal- Caelifera were supported, but not for Ensifera, which was yses. Clade I included three Phraortes species (Diapher- appeared to be a paraphyletic group. Pteronarcys (Plecop- omeridae: Lonchodinae) and Phyllium giganteum (Areolatae: tera) was placed as sister to Orthoptera, which is a sister to Phylliidae: Phylliinae). Clade II contained Phobaeticus ((Phasmatodea+Sclerophasma+Grylloblatta)+Dictyoptera). serratipes (: Clitumninae), Megacrania alpheus Since ML and Bayesian inference are not exhaustive (Phasmatidae: Platycraninae), and E. tiaratum (Phasmati- but heuristic methods to identify tree topologies, we have dae: Extatosomatinae). Clade III included Sipyloidea sipy- statistically tested the paraphyly vs. monophyly of Xenon- lus, Micadina phluctainoides (both : omia and Ensifera (Fig. 3). We used the amino acid data ), and Neohirasea japonica (Diapheromeridae: set and the phylogenetics inferred from this data set, in Lonchodinae). The two species (Areola- which both Xenonomia and Ensifera appeared as paraphy- tae), Heteropteryx dilatata (Heteropteryginae) and Orestes letic. The initial ML tree was compared to alternative mouhotii (Dataminae), were clustered in Clade IV. Entoria trees, whereby either one or both of Xenonomia and En- okinawaensis and the two Ramulus species, all belonging sifera are monophyletic. We found that tree2, in which to Clitumnini (Phasmatidae: Clitumninae), formed Clade V. Xenonomia is monophyletic and Ensifera is paraphyletic, In addition, two Bacillus species (Bacillidae: Bacillinae) was supported considerably (P values were 0.028, 0.025, also formed a clade. These clades were strongly supported and 0.124 in the AU, KH, and SH tests, respectively). in each analysis (> 70 BS and 1.00 PP). The interrelation- These results provide weak but significant support for the ships of these clades were, however, not stable. Limiting monophyly of Xenonomia. However, trees 3 and 4, in the taxa to Verophasmatodea, which should have sup- which Ensifera appeared as a monophyletic group, re- pressed the compositional bias among OTUs (Kômoto et ceived minimal statistical support. It should be noted that al., 2011), did not improve the stability (data not shown).

Fig. 3. Statistical testing of tree topology. The congruent topology derived from the ML and BI analyses of the amino acid data set is presented as tree 1, in which both Xenonomia and Ensifera are shown as paraphyletic (shaded in gray). Tree 2 and 4 assume Xenonomia is monophyletic and trees 3 and 4 assume Ensifera is monophyletic. The table below the trees shows the log likelihood calculated in Tree-Puzzle and the scores of au (approximately unbiased), kh (Kishino-Hasewaga), and sh (Shimodaira-Hasegawa) tests by CONSEL. Polyneopteran phylogeny based on mitogenome 85

Table 2. Results from the substitution saturation test of ran- DISCUSSION domly sampled 32 OTUs. Iss.ca Use of mitogenomics for polyneopteran phyloge- Issb netics Sym Asym The mitochondrial genome has been used for a wide nt1 0.388 0.808 0.550 nt2 0.231 0.808 0.550 range of phylogenetic studies (Lavrov et al., 2004, 2005; nt3 0.716 0.808 0.550 Singh et al., 2009). One negative aspect for using the mi- nt12 0.287 0.814 0.570 togenome to investigate phylogenies is the lineage-specific nt123 0.516 0.817 0.571 compositional heterogeneity of mitochondrial genome se- aIss.c: Critical index of substitution saturation, computed for two quences. Heterogeneity in A+T content, and AT and GC extreme topologies: a symmetrical (Sym) and an extremely asym- skews, can lead to erroneous results in phylogenetic infer- metrical tree (Asym). b ence (Gibson et al., 2005; Jones et al., 2007; Masta et al., Iss: Index of substitution saturation, when this value is significantly smaller than Iss.c, substitution saturation is not taken as serious in 2009). Another disadvantage is that its accelerated substi- the dataset. When an observed Iss value falls above the Iss.c, level tution rates may also play a role in masking and eroding of saturation is taken as significant. the phylogenetic signals through unrecognized homoplasy. These problems have been thought to limit the phyloge- acid data set produced a stable result. When using amino netic resolving power of the mitogenome (Cameron et al., acid data sets, results from ML and Bayesian analyses are 2004; Castro and Dowton, 2007). Recently, sophisticated congruent in tree topology, while the use of nt123 or nt12 evolutionary models such as heterogeneous CAT (Lartillot data sets results in different topologies between ML and and Philippe, 2004), and the derived CAT-BP model Bayesian analysis, even when similar evolutionary models (Blanquart and Lartillot, 2008), were shown to reduce the are applied. effects of compositional biases (Rota-Stabelli et al., 2010). Increasing the number of OTUs is also crucial to break Phylogenetic position of E. tiaratum and phasmid long branches since it allows better elucidation of homo- phylogeny plasy. In all phylogenetic analyses performed in this study, E. The phylogenetic relationships among polyneopteran tiaratum was grouped with P. serratipes and M. alpheus. species inferred in this study varied depending on the data The support for this clade was quite high and stable (> 90 sets used. Previous reports suggested that using whole nu- BS and 1.00 PP). Although these three species belong to cleotide data sets, including all the three codon positions the same family, each belongs to distinct subfamilies. (nt123), is beneficial when analyzing intraordinal relation- Note that P. serratipes is classified as Clitumninae, and ships of orthopteran and phasmatodean species (Fenn et other Clitumninae species included in this study formed a al., 2008; Kômoto et al., 2011). When analyzing deeper distinct cluster (Clade V). Within Clade V, R. hainanense relationships such as interordinal phylogenetics among was placed as a sister to R. irregulariterdentatus + E. oki- polyneoptera, base substitutions of the codon third posi- nawaensis with firm support (100 BS and 1.00 PP). This tions in PCGs from mitogenomes may be saturated, re- may suggest the necessity of revising the genera Ramulus sulting in retrieval of little or no phylogenetic signals. We and Entoria. The comparison of the three Phraortes sam- performed a substitution saturation test on each codon po- ples suggests the possibility that they speciated during the sition (nt1, nt2, and nt3) and the combined data sets (nt12 northward expansion of their distribution. On the other and nt123). The test results showed a significant level of hand, E. okinawaensis distributes widely among the Nansei saturation among the third codon positions (nt3) (Table 2), Islands without allopatric speciation. To further investigate suggesting the inclusion of the third codon position may the speciation process of Phraortes species in association have disturbed the analysis, and the recovered topology with island formation, it is necessary to examine other was negatively affected by the noise, rather than the sig- Phraortes populations in the Nansei Islands. nal, from the nt3 data. In contrast, because the nt1, nt2, Analyses performed in this study confirmed that Time- and nt12 data sets showed little saturation, they should be matodea and Verophasmatodea are sister groups, regard- suitable for phylogenetic analyses. Accordingly, Plazzi et less of the data sets used. In our previous study, we failed al. (2011) demonstrated significant levels of saturation to recover the monophyletic Phasmatodea (Verophasmatodea among the codon third position in mtDNA of Polyneop- +Timematodea) using the nt123 data set (Kômoto et al., tera, and suggested that a fully partitioned amino acid 2011). We believe this was caused by an artifact resulting data set is best for estimating phylogenetic relationships. from an improper outgroup choice, which was circum- Since their data set was essentially a subset of ours, the vented in this study by increasing OTUs and selecting amino acid data set likely would also perform best in our proper outgroups (Rota-Stabelli and Telford, 2008). Still, analyses. Indeed, unlike nucleic acid data sets, this amino a deep branch node exists between T. californicum and 86 Tomita et al. the other phasmids, suggesting that Timematodea is dis- placed as sister to Orthoptera. Plecoptera has been hy- tantly related to Verophasmatodea, and evoking the need pothesized to be a sister to Dermaptera or (Dermaptera+ for sampling species from Agathemerodea to fully resolve Zoraptera) (Kristensen, 1981; Terry and Whiting, 2005) the phasmid phylogeny. and our data matrix, lacking species from these two or- ders, unable to test these hypotheses. In addition, since Phylogenetic relationships among Polyneoptera only one plecopteran mitogenome was included in this Among Polyneoptera various supraordinal taxa have study, sampling more species is needed to resolve the been proposed but Dictyoptera (Mantodea+Blattodea+ phylogenetic position of Plecoptera. 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