Cladistics

Cladistics 31 (2015) 50–70 10.1111/cla.12068

The thorax of Mantophasmatodea, the morphology of flightlessness, and the evolution of the neopteran insects

Benjamin Wipflera,*, Rebecca Klugb, Si-Qin Gea,*, Ming Baia,Jurgen€ Gobbels€ c, Xing-Ke Yanga and Thomas Hornschemeyer€ b

aKey Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China bJohann-Friedrich-Blumenbach Institut fur€ Zoologie und Anthropologie, Universitat€ Gottingen,€ Berliner Straße 28, Gottingen,€ 37073, Germany; cBundesanstalt fur€ Materialforschung Berlin, Unter den Eichen 87, Berlin, 12205, Germany Accepted 18 December 2013

Abstract

Mantophasmatodea was described as a new insect order in 2002. Since then, this small group of wingless insects has developed into one of the best investigated insect taxa. Nevertheless, many aspects of mantophasmatodean morphology as well as their evolutionary relationships remain ambiguous. To determine the phylogenetic relationships of Mantophasmatodea based on an extended character set and to elucidate possible morphological adaptions towards flightlessness, we investigated the thoracic morphology of two species, Austrophasma caledonensis and Mantophasma sp. The morphological similarity between these two species is striking and no differences in musculature were found. The mantophasmatodean thorax strongly resembles that of ice crawlers (Grylloblattodea), especially with respect to the presence of pleural processes in the meso- and metathorax, branched furcae in all segments, and similar muscle equipment. In a cladistic analysis containing all major lineages of Neoptera, the monophyly of Polyneoptera is supported by the presence of an anal fan and several modifications of the wing joint. Within Polyneoptera, a sister-group relationship between stoneflies and the remaining Polyneoptera is supported. A clade comprising Mantophasmatodea and the Grylloblattodea gains strong support from thoracic morphology and can be considered assured. Potential thoracic apomorphies include prothoracic paracoxal invaginations, pterothoracic pleural arms that originate from the epimeron, and a unique metathoracic sterno-coxal musculature. The monophyly of Orthoptera and Dictyoptera is further sup- ported while the deeper polyneopteran nodes remain unresolved. Among the wingless taxa investigated we found few general morphological adaptations whereas, in other aspects, especially in the musculature, strong differences could be observed. How- ever, much more research on the strongly neglected topic of flightlessness is required to make reliable statements. © The Willi Hennig Society 2014.

Introduction sition of important data types (e.g. Deans et al., 2012). As a result of these combined efforts some major Interest in high-rank insect systematics has increased breakthroughs have been achieved. The highlights strongly in the last decade, largely due to advances in include resolving the basal split within the Holometa- molecular techniques: sequencing has become faster bola (e.g. Wiegmann et al., 2009; Beutel et al., 2010) and cheaper and, as a result, there has been a signifi- or solving the “Strepsiptera-problem” (Niehuis et al., cant increase in the number of published studies focus- 2012). However, several long-standing problems ing on molecular phylogenetics (for an overview see remain unresolved, most strikingly the relationships at Trautwein et al., 2012). Morphologists have also devel- the base of Neoptera (Klass, 2007) and the affiliations oped new methods, which have facilitated faster acqui- among the Eumetabola (Holometabola + Paraneoptera) and the so-called lower neopteran or polyneopteran *Corresponding authors: groups: Orthoptera (grasshoppers, crickets, and katyd- E-mail addresses: benjamin.wipfl[email protected]; [email protected]. ids), Dermaptera (earwigs), Plecoptera (stoneflies),

© The Willi Hennig Society 2014 B. Wipfler et al. / Cladistics 31 (2015) 50–70 51

Phasmatodea (stick and leaf insects), Mantophasmato- acters, comprising all major lineages of Neoptera, dea (heel walkers), Grylloblattodea (ice crawlers), Em- including all traditional polyneopteran orders and a bioptera (web spinners), Zoraptera (bark lice), and mayfly. While the reduction of wings occurs in virtu- Dictyoptera (roaches and mantids) are completely ally all insect orders (Roff, 1990), Mantophasmatodea unresolved. In fact, little further knowledge has been is one of the few completely wingless groups. To pro- gained since Kristensen (1991) summarized lower vide a deeper understanding of the morphological neopteran relationships as an unresolved phylogenetic transitions that take place during wing reduction, we comb. Despite the fact that some clades have been compared the results from the species studied with sev- repeatedly recovered in recent years (e.g. Xenonomia, eral other wingless lower neopterans and searched for Eukinolabia), the deeper nodes of Polyneoptera remain general patterns in the diverse morphological modifica- completely unresolved, and for several traditional tions accompanying flightlessness. orders, such as Plecoptera or Zoraptera, nearly all pos- sible sister-group relationships have been proposed. These severe problems are partly caused by the extre- Material and methods mely rapid radiation of the groups in the distant past (Whitfield and Kjer, 2008) and the general lack of Taxon sampling morphological data. The latter was previously addressed by comprehensive cladistic analyses (Gorb The present study is based on ethanol-fixed speci- and Beutel, 2001; Beutel and Gorb, 2006; Wipfler mens of Austrophasma caledonensis Klass et al., 2003 et al., 2011; Yoshizawa, 2011; Blanke et al., 2012, and an undescribed species, Mantophasma sp., from 2013; Friedemann et al., 2012; Wipfler, 2012). The Langeberg East, South Africa. For the sake of descrip- present study continues this work by adding the tho- tion, species are identified below by their generic name racic morphology as a fourth character system after only. the adhesive structures, the head, and the wing joint. For comparison, we propose a homologization of Our morphological investigation focuses on the tho- the musculature of the specimens in supplementary rax of Mantophasmatodea. Previous to their descrip- Appendix S1 with the works of (in alphabetical order) tion by Klass et al. (2002), it had been 88 years since Badonnel (1934), Barlet (1985a,b,c,d), Bharadwaj and the last description of a new insect order (Grylloblat- Chadwick (1974), Carbonell (1947), Czihak (1957), todea by Walker, 1914). Accordingly, there was strong Friedrich et al. (2008), Friedrich and Beutel (2008), general interest and the group is well on its way to Jeziorski (1918), Kleinow (1966), Levereault (1938), becoming one of the best-studied groups of insects. Maki (1935, 1936, 1938), Matsuda (1956, 1970), During the last 10 years 18 species in 12 genera have Mickoleit (1961), Rahle€ (1970), Snodgrass (1929), Vil- been described from Tanzania, Mali, Namibia, and helmsen (2010), Vilhelmsen et al. (2010), Voß (1905), South Africa (e.g. Klass et al., 2003; Wipfler et al., Walker (1938), and Wittig (1955). As reference termi- 2012a). Additionally, information from a broad field nology we use Friedrich and Beutel (2008). of disciplines was gathered, including: morphology (e.g. Klass et al., 2003; Baum et al., 2007; Klass and Scanning electron microscopy (SEM) Eulitz, 2007; Beutel and Gorb, 2008; Eberhard et al., 2011), sperm ultrastructure (Dallai et al., 2003), Prior to scanning, specimens were transferred to postembryonic development (Hockmann et al., 2009), 100% ethanol, critically point dried (Austrophasma: vibrational communication (Eberhard and Picker, Balzer CPD 030 critical point dryer; Mantophasma: 2008), metabolic details (Chown et al., 2006), and Emitech K850) and sputter coated with gold (Autroph- information about neuropeptides (Gade€ et al., 2005; asma: Balzers SCD 050; Mantophasma: Emitech Predel et al., 2012). Predel et al. (2012) also presented K500). Scanning was done with a LEO 438VP (Au- an intraordinal phylogeny including all described spe- strophasma) or Philips XL30 environmental scanning cies and posited a detailed biogeographical scenario electron microscope (Mantophasma) using a special for the group. However, little was known about the sample holder (Pohl, 2010). Final images were edited mantophasmatodean thorax. Klug (2008) presented using Helicon Soft Helicon Focus, Adobe Photoshop some results on the pterothorax of Austrophasma cale- CS5, and Adobe Illustrator CS5. donensis, a species we reinvestigate here. To supple- ment the information on the thoracic morphology we Micro computed-tomography (l-CT) studied the entire thorax of two species of Mantophas- matodea (Austrophasma caledonensis and Mantophas- A specimen of Austrophasma was critical point dried ma sp.), which represent the two major lineages within (Balzer CPD 030) and scanned at the Federal Institute Mantophasmatodea (Predel et al., 2012). We con- of Research and Testing (BAM), Berlin, with a spatial ducted a cladistic analysis based on 119 thoracic char- resolution of 2.5 lm. The three-dimensional model 52 B. Wipfler et al. / Cladistics 31 (2015) 50–70 was created using Visage Imaging Amira 5.2, Bitplane and of Ensifera is included. The same applies to Phas- Imaris 7.2.1, and Autodesk Maya 2013. Final images matodea, where the monophyly with respect to Timema were edited using Adobe Photoshop CS5 and Adobe was questioned, for example, by Yoshizawa and John- Illustrator CS5. son (2005). Appendix S2 provides muscle inventories for all species included in the analysis. For the wing Dissection and terminology base characters (nos. 38–70), not all studied species were available, and thus in some cases closely related One specimen of Mantophasma was manually dis- species were used instead. The characters of the wing sected in 70% alcohol under a Zeiss Stemi SV 11 with (nos. 32–37) were selected according to the analyses of an additional Euromex Illuminator EK-1 lightning sys- Beutel and Gorb (2006) and Yoshizawa (2011) to pro- tem. The terminology of skeletal elements follows vide compatibility with existing studies. The data Friedrich and Beutel (2008). In the results section, the matrix is presented in nexus format in Appendix S3. muscles are described and numbered subsequently. In Appendix S5 and the character discussion (Appendix Appendix S1 we propose a homologization of the mus- S4) provide a detailed account of the source of infor- cular terminology of Friedrich and Beutel (2008) with mation for each character. that of various other authors. In the discussion, the The cladistic parsimony analysis was conducted with terminology of Friedrich and Beutel (2008) is used to Winclada/Nona (Ratchet [Island Hopper], 1000 itera- provide comparability to other authors. Additionally, tions) (Goloboff, 1999; Nixon, 1999, 2000, 2002), TNT the general terminology of Matsuda (1970) is pre- (Wagner trees, 99 999 random seeds, 1000 replications) sented. We are aware that all supra-generic entities are (Goloboff et al., 2000). All approaches resulted in the artificial but we will use them for the sake of descrip- same minimum-length trees. Bremer support (Bremer, tion. 1994) was calculated with TNT. The character changes were retrieved from Nona (only unambiguous High-resolution photography changes).

One mid-sagittally cut specimen of Mantophasma was photographed in 70% alcohol with a Nikon D 90 Morphological results digital SLR equipped with a 40-mm and with a 63- mm Zeiss Luminar macro lens, plus an adjustable Austrophasma caledonensis extension bellow. The specimen was illuminated by two flashlights fitted with a transparent cylinder for The three thoracic segments are all moveable against even and soft light. Helicon Focus Mac Pro X64 was each other. The pro- and mesonotum are of approxi- used to combine a stack of several partially focused mately equal size while the metanotum is two-thirds images. the length of the former. The nota, pleura, coxae, and sterna of all segments are more or less densely covered Phylogenetic analysis with setae.

The data matrix for the phylogenetic analysis con- Prothorax tains the following taxa: Austrophasma caledonensis (Mantophasmatodea), Siphlonurus sp. (Ephemerop- The prothorax is connected to the head capsule via tera), Perla sp. (Plecoptera), Grylloblatta campodeifor- the cervical membrane. Two lateral cervical sclerites or mis (Grylloblattodea), Periplaneta americana laterocervicalia are present. The first (c1: Figs 1 and 2) (Blattodea), Stagmomantis carolina (Mantodea), Eubo- is a long and slender bar that articulates anteriorly rellia annulipes (Dermaptera), Gryllus domestica with the head capsule. It continues ventro-posteriorly (Orthoptera, Ensifera), Dissosteira carolina (Orthop- where it attaches to the second laterocervicale (c2: tera, Caelifera), Zorotypus hubbardi (Zoraptera), Embia Figs 1 and 2). The latter is club-shaped with its slender sp. (Embioptera), Timema sp. (Phasmatodea), Megacr- part articulating with the first sclerite. Posteriorly it is ania tsudai (Phasmatodea), Stenopsocus stigmaticus connected with two parts of the anepisternum (aes: (Psocoptera), Phloeothrips coreaceus (Thysanoptera), Fig. 2): the first near the anterior end of the paracoxal Palpares libelluloides (Neuroptera), and Macroxyela ridge (pcr: Figs 1 and 2) and the second one with the ferruginea (Hymenoptera). Representatives of Odonata antero-dorsal anepisternum, directly ventrad of the and primarily wingless insects are not considered in this pronotum. Dorsal or ventral cervical sclerites are study because of the lack of a reliable homologization absent. hypothesis for the thoracic musculature. To test the The pronotum (n1: Figs 1, 2, and 4) is a nearly proposed paraphyly of Orthoptera as suggested by squared plate densely covered with setae. Anteriorly, a Xiao et al. (2012), a representative both of Caelifera transversal pronotal ridge (prr: Figs 1 and 2) runs B. Wipfler et al. / Cladistics 31 (2015) 50–70 53

Fig. 1. Electron micrograph showing lateral view of the thorax of Austrophasma caledonensis. Abbreviations: c1, first laterocervicalia; c2, second laterocervicalia; cx1, procoxa; cx2, mesocoxa; cx3, metacoxa; hc, head capsule; ipa, invagination of pleural arm; mer, mesonotal ridge; mtr, metanotal ridge; n1, pronotum; n2, mesonotum; n3, metanotum; pcr, paracoxal ridge; pr, pleural ridge; prr, pronotal ridge; sp, spiracle; tr, troch- antin.

(a)

(b)

Fig. 2. Photographs of exoskeleton of Austrophasma caledonensis. (a) Lateral view; (b) dorsal view, notae removed. Abbreviations: aes, anepis- ternum; afa, anterior arm of furca; app, paracoxal process; bs, basisternum; c1, first laterocervicalia; c2, second laterocervicalia; cx1, procoxa; cx2, mesocoxa; cx3, metacoxa; em, epimeron; ipa, invagination of pleural arm; kes, katepisternum; n1, pronotum; n2, mesonotum; n3, metano- tum; nI, notum of abdominal segment 1; nII, notum of abdominal segment 2; pa, pleural arm; pca, pleuro-coxal articulation; pcr, paracoxal ridge; pes, preepisternum; pfa, posterior arm of furca; pr, pleural ridge; prr, pronotal ridge; pp, pleural process; s1, prospina; s2, mesospina; sp, spiracle; ten, tendon of m66; tr, trochantin. across it. The region in front of this ridge covers the The pleuron consists of a sclerotized anterior area posterior part of the head capsule (hc: Fig. 1) and the and a membranous posterior area. The anterior area occipital ridge (occ: Fig. 4). Posteriorly, the pronotum is divided into an anterior episternum and a posterior overlaps the mesonotum. The pronotum is not con- epimeron (em: Fig. 2) by the pleural ridge (pr: Figs 1 nected laterally to a membrane but rather directly to and 2) which runs slightly antero-ventrally. The base the sclerotized pleura along its entire length. of the pleural arm (pa: Fig. 2) is externally visible as 54 B. Wipfler et al. / Cladistics 31 (2015) 50–70 a small invagination (ipa: Figs 1 and 2). Ventrally, it the anterior part, the area posterad of the ridge is forms the pleural part of the pleuro-coxal articulation not covered by setae. (pca: Fig. 2). Internally, it is a projection that runs The pleural region is strongly sclerotized. The pleu- slightly posteriorly. The epimeron is a slim and undi- ral ridge (pr: Figs 1 and 2) is oblique, running from vided plate directly posterad of the pleural ridge and antero-dorsal to the postero-ventral pleuro-coxal artic- much longer than broad. The episternum is divided ulation (pca: Fig. 2). It divides the pleura in an ante- by a well-developed paracoxal ridge (pcr: Figs 1 and rior anepisternum (aes: Fig. 2) and a posterior 2) in a dorsal anepisternum (aes: Fig. 2) and a ven- epimeron (em: Fig. 2). Anteriorly, the pleural ridge tral katepisternum (kes: Fig. 2). Anteriorly, the parac- forms an internal process, the pleural process (pp: oxal ridge ends at the articulation with the second Fig. 2), before fusing with the segmental border. The laterocervicale. The anepisternum reaches far dorsally invagination of the pleural arm (ipa: Figs 1 and 2) is and is in contact with the pronotum. In the dorso- located posterad of the pleural ridge on the epimeron. anterior area it forms the second articulation with the Internally, it is a well-developed arm (pa: Fig. 2) that second laterocervicale and extends to the anterior reaches close to the anterior furcal arm. The anepister- pronotal margin. Dorso-posteriorly it extends to the num is much larger than the epimeron. Ventrally, a end of the pronotum, thus extending further posterad triangular preepisternum (pes: Fig. 2) attaches to it. than the epimeron. A membranous connection This element covers the postero-ventral body wall. between the anepisternum and the pronotum is not Posteriorly, the trochantin is attached, laterally the present. Internally, the anepisternum has an inward mesanepisternum, and mesally the basisternum. The projection, the paracoxal process (app: Fig. 2) which mesothoracic spiracle lies on a separated sclerite ante- reaches under the pronotum. Its posterior end is con- rior to the episternum. tinuous with the pleural arm (Fig. 2). It serves as The trochantin (tr: Figs 1 and 2) is sickle-shaped. It attachment side for pleuro-coxal and tergo-pleural articulates anteriorly with the anepisternum near its muscles. border with the preepisternum. Its anterior margin is The trochantin (tr: Figs 1 and 2) is a sickle- close to the posterior border of the preepisternum. shaped sclerite which articulates antero-dorsally Posteriorly, it articulates with the coxa. It is reinforced with the antero-ventral edge of the katepisternum. by an internal ridge. The trochatino-coxal articulation is located ventro- The mesosternal region is more strongly sclerotized posteriorly. The trochantin is reinforced by a longi- than the prothoracic region. The basisternum (bs: tudinal ridge. Figs 2 and 4) is an oval sclerotization which narrows The sternal region is mainly membranous. Posteri- posteriorly. Like the prothoracic basisternum its bor- orly, a small oval basisternum (bs: Figs 2 and 4) is ders cannot clearly be defined due to weak sclerotiza- present. Its borders are not clearly delimited. Laterally, tion. The massive furcal arms are located at its it holds the two furcal arms (f1: Fig. 3), the invagina- posterior edge (f2: Fig. 3). Externally their bases are tion sites of which are externally visible. Internally, well visible as two invaginations. Internally, the furcal each furcal arm is composed of a basal part, a short arms are composed of a basal part, an anterior arm posterior arm (pfa: Fig. 2), and a long anterior arm (afa: Fig. 2) that reaches further anterior than the (afa: Fig. 2) that reaches far anteriorly and very close pleural arm, and a posterior arm (pfa: Fig. 2) that to the pleural arm. The anterior arm is distally divided runs posterior-ventrally and ends close to the postero- in a ventral and a dorsal process. Posterad of the ba- lateral coxal rim. Slightly posterad the furcal arm, the sisternum, the prospina (s1: Figs 2–4) is positioned on mesal spina (s2: Figs 2–4) is located. Externally it is an a small furcasternite. Internally the spina forms a well- invagination and internally represented as a well-devel- developed apodeme. The procoxae (cx1: Figs 1, 2, and oped oval apodeme. 4) are large and elongate. Their bases are well sepa- The mesothoracic musculature is presented in rated. Table 1. The prothoracic musculature is presented in Table 1. Metathorax Mesothorax In its general shape, the metathorax resembles the The mesonotum (n2: Figs 1, 2, and 4) is as long mesothorax, being only slightly shorter. The descrip- as the pronotum which overlaps it anteriorly. Poste- tion of the mesothorax also applies to the metathorax, riorly, the mesonotum extends above the metanotum. with the exception that it is shorter and stouter than A small postero-lateral portion is delimited by a the mesothorax. A metaspina is not present and the mesonotal ridge (mer: Fig. 1). This ridge does not metathoracic basisternum is much broader than the reach the mesal area of the notum. In contrast to mesothoracic one. The pleural arm is located even B. Wipfler et al. / Cladistics 31 (2015) 50–70 55

(a)

(b)

(c)

(d)

Fig. 3. Musculature of Austrophasma caledonensis derived from l-CT, mesal view of the right thoracic half, digestive and nervous system removed. (a) Inner layer; (b) inner middle layer; (c) outer middle layer; (d) outer layer. Abbreviations: f1, profurca; f2, mesofurca; f3, metafurca; m, muscle abbreviation following those in the text; s1, prospina; s2, mesospina. 56 B. Wipfler et al. / Cladistics 31 (2015) 50–70

(a)

(b)

(c)

Fig. 4. Anatomy of Austrophasma caledonensis derived from l-CT. (a) Lateral view, thoracic exoskeleton transparent; (b) ventral musculature, notae removed, prothorax separated from meso- and meta thorax; (c) mesal view of the nervous and digestive system. Abbreviations: bs, basist- ernum; cx1, procoxa; cx2, mesocoxa; cx3, metacoxa; dg, digestive tract; g1, prothoracic ganglion; g2, mesothoracic ganglion; g3, metathoracic ganglion; m, muscle abbreviation following those in the text; n1, pronotum; n2, mesonotum; n3, metanotum; nI, notum of abdominal segment 1; occ, occipital ridge; p1, prophragma; p2, mesophragma; p3, metaphragma; s1, prospina; s2, mesospina. more posterad on the epimeron than in the mesotho- Essentially the entire description including the muscu- rax. lature for Austrophasma also applies to Mantophasma. The metathoracic musculature is presented in Differences can be found in the meso- and metanotum Table 1. where no ridge is present in Mantophasma and the setation continues towards their posterior edge. Addi- Mantophasma sp tionally, the invaginations of the pleural arms are much larger than in Austrophasma. In the sternal The overall resemblance between the thorax of region, the basisterna of Mantophasma are broader Austrophasma and Mantophasma (Fig. 5) is striking. than those of Austrophasma. B. Wipfler et al. / Cladistics 31 (2015) 50–70 57

Table 1 Musculature of Austrophasma caledonensis with origin and insertion and potential homology (Hom*) with the terminology of Friedrich & Beutel (2008)

Muscle Hom* Origin Insertion

Prothorax Prothoracic dorsal longitudinal muscles m1 Idlm1 In several bundles on the prophrgama Dorso-mesally on the occiput m2 Idlm2 + Idlm4 Mesally along the entire pronotum Dorso-mesally on the occiput, very close to m1 m3 Idlm3 + Idlm5 Along the entire anterior half of the Mesally on the prophragma pronotum including the anterior pronotal margin, directly posterad of the pronotal ridge Prothoracic dorsoventral muscles m4 Idvm2 Anterior region on second lateral cervical Dorso-lateral occipital region sclerite m5 Idvm3 Posteriorly on second lateral cervical sclerite Dorso-lateral occipital region together with m4 m6 Idvm4 With two bundles on the posterior tentorial On the anterior pronotum, bundle 1 mesally arms and bundle 2 on the ventral pronotal edge, postero-laterad of bundle 1. m7 Idvm5 Pronotal ridge Posterior second cervical sclerite, postero- mesad the origin of m5 m8 Idvm6 Pronotum, slightly posterad pronotal ridge Anterior part of first lateral cervical sclerite m9 Idvm10 Posterior arm of profurca Anterior part of mesopleura m10 Idvm13 Broad area of mesal pronotum Posterior prothoracic trochantin, close to the coxal articulation m11 Idvm14 Pronotum, dorso-laterad of m10 Posterior prothoracic trochatin, close to trochantino-coxal articulation, slightly posterad of m10 m12 Idvm15 Anterior pronotum, directly anterad of m10 Anterior procoxal rim m13 Idvm16 Meso-lateral part of pronotum Posterior procoxal rim m14 Idvm17 Laterally in the posterior third of the Posterior procoxal rim, slightly laterad pronotum, postero-laterad of m13 of m13 m15 Idvm18 Laterally posterior end of the pronotum, Posterior procoxal rim, antero-laterad postero-laterad of Idvm17 of m14 m16 Idvm19 With several bundles on the anterior Tendon of the prothoracic trochanter pronotum Prothoracic tergo-pleural muscles m17 Itpm3 Antero-lateral part of pronotum, posterad of Antero-dorsally on episternal wing m16 m18 Itpm4 Antero-lateral pronotum, ventro-laterad of One bundle on the distal tip of the pleural the anterior parts of m16 apodeme and one on the dorsal area of the anterior part of the profurca m19 Itpm6? Posterior pronotum, laterad of the insertion Antero-dorsal area of mesanepisternum of m3 Prothoracic sterno-pleural and pleuro-coxal muscles m20 Ispm1 Anterior arm of the profurca Propleural arm m21 Ispm2 Posterior part of prospina Ventral mesepisternum m22 Ipcm4 Broadly along the episternal wing Anterior procoxal rim, directly posterad of the insertion of m12 m23 Ipcm8 Pleural apodeme and dorsal pleural ridge Tendon of the trochanter Prothoracic ventral longitudinal muscles m24 Ivlm3 Ventral apodeme of the tentorium Posterior arm of profurca m25 Ivlm4 Posterior arm of profurca Prospina m26 Ivlm6 Basal part of profurca Mesospina m27 Ivlm7 Posterior arm of profurca Anterior arm of mesofurca m28 Ispm3? Prospina Spriacle Prothoracic sterno-coxal muscles m29 Iscm1 Prosternum, directly anterad of profurcal Anterior procoxa base m30 Iscm2 Base of profurcal arm Posterior procoxa m31 Iscm3 Ventral part of anterior profurcal arm Mesal procoxal rim 58 B. Wipfler et al. / Cladistics 31 (2015) 50–70

Table 1 (Continued)

Muscle Hom* Origin Insertion

m32 Iscm4 Distal tip of posterior arm of profurca Postero-lateral procoxal rim, close to m13 and m14 m33 Iscm4 Posterior arm of profurca Lateral procoxal rim m34 Iscm6 Ventral area of the anterior part of the Tendon of the trochanter profurca Mesothorax Mesothoracic dorsal longitudinal muscles m35 IIdlm1 With several bundles mesally on the With several bundles mesally on prophragma mesophragma m36 IIdlm2 Postero-lateral area of mesonotum Laterally on the mesophragma Mesothoracic dorsoventral muscles m37 IIdvm2 Mesophragma and anterior part of Trochantin, shared tendon with m38 mesonotum m38 IIdvm3 Mesonotum, posterad of m37 Trochantin, shared tendon with m37 m39 IIdvm4 With two bundles, bundle 1 on the Posterior mesocoxal base prophragma and the anterior mesonetum, bundle 2 in the posterior third of the mesonotum m40 IIdvm5 Central mesonotum, in between the bundles Posterior mesocoxal base, laterad of m39 of m39 m41 IIdvm6 Lateral mesonotum Posterior mesocoxal base, slightly laterad of m40 m42 IIdvm7 Anterior mesonotum, reaching far laterally Trochanter Mesothoracic tergo-pleural muscles m43 IItpm1 Prophragma Mesopleural wing process m44 IItpm2 Pleural ridge below pleural wing process Prophragma m45 IItpm3 Anterior mesonotum Distal tip of mesopleural wing process, distally of m43 m46 IItpm4 Anterior mesopleural ridge, posterad of m44 Anterior mesonotum, posterad of origin of m45 m47 IItpm5 Pleural ridge and basal part of pleural arm Lateral margin of the central area of the mesonotum m48 IItpm10? Postero-lateral region of mesonotum Postero-dorsal area of mesanepimeron Mesothoracic sterno-pleural and pleuro-coxal muscles m49 IIspm1 Anterior region of mesanepisternum Anterior part of mesosternum m50 IIspm2 Mesofurca Mesopleural arm m51 IIspm3 Mesospina Anterior region of metanepisternum m52 IIspm5 Mesospina Intersegmental membrane between metathorax and abdominal sternite 1 m53 IIpcm3 Dorso-posterior part of mesanepisternum Antero-lateral mesocoxal rim, close and posterior pleural ridge to m54 m54 IIpcm4 Anterior part of mesanepisternum and Antero-lateral mesocoxal rim, close anterior part of mesopleural ridge to m53 m55 IIpcm5 Antero-dorsal mesanepisternum Trochanter Mesothoracic ventral longitudinal muscles m56 IIvlm3 Mesofurca Metafurca m57 IIvlm5 In two bundles on the mesospina In two bundles on the metafurca Mesothoracic sterno-coxal muscles m58 IIscm1 Mesosternum, directly anterad of base of Anterior mesocoxal rim furcal arm m59 IIscm3 Distal tip of posterior arm of mesofurca Mesal mesocoxal rim m60 IIscm4 Mesofurca Lateral mesocoxal rim, close to pleuro-coxal articulation m61 IIscm5 Mesospina Postero-lateral mesocoxal rim m62 IIscm6 Ventral side of the anterior arm of the Trochanter mesofurca m63 IIscm7 Mesospina Antero-laterad metacoxal rim Metathorax Metathoracic dorsal longitudinal muscles m64 IIIdlm1 With several bundles mesally on the With several bundles mesally on mesophragma metaphragma m65 IIIdlm2 Postero-lateral area of metanotum Laterally on the metaphragma B. Wipfler et al. / Cladistics 31 (2015) 50–70 59

Table 1 (Continued)

Muscle Hom* Origin Insertion

Metathoracic dorsoventral muscles m66 IIIdvm2 Metaphragma and anterior part of With a very strong tendon on the metanotum trochantin m67 IIIdvm3? With two bundles on the central part of the Posterior metacoxal base metanotum m68 IIIdvm5 Central metanotum, in between the bundles Posterior metacoxal base, laterad of m67 of m67 m69 IIIdvm6 Lateral metanotum Posterior metacoxal base, slightly laterad of m68 m70 IIIdvm7 Anterior metanotum, reaching far laterally Trochanter Metathoracic tergo-pleural muscles m71 IIItpm1 Mesophragma Metapleural wing process m72 IIItpm2 Metapleural ridge below pleural wing Mesophragma process m73 IIItpm3 Anterior metanotum Distal tip of metapleural wing process, distally of m71 m74 IIItpm4 Anterior metapleural ridge, posterad of m72 Anterior metanotum, posterad of origin of m73 m75 IIItpm5 Pleural ridge and basal part of pleural arm Lateral margin of the central area of the metanotum m76 IIItpm10? Postero-lateral region of metanotum Postero-dorsal area of metanepimeron Metathoracic sterno-pleural and pleuro-coxal muscles m77 IIIspm1 Anterior region of metanepisternum Anterior part of metasternum m78 IIIspm2 Metafurca Metapleural arm m79 IIIspm5 Metafurca Intersegmental membrane between metathorax and abdominal sternite 1, dorso-laterad of insertion of IIspm5 m80 IIIpcm3 Dorso-posterior part of metanepisternum Antero-lateral metacoxal rim, close and posterior pleural ridge to m54 m81 IIIpcm4 Anterior part of metanepisternum and Antero-lateral metacoxal rim, close anterior part of pleural ridge to m53 m82 IIIpcm5 Antero-dorsal metanepisternum Trochanter Metathoracic ventral longitudinal and sterno-coxal muscles m83 IIIvlm2 Metafurca Second abdominal sternite m84 IIIscm1 Metasternum, directly anterad of base of Anterior metacoxal rim furcal arm m85 IIIscm3 Distal tip of posterior arm of metafurca Mesal metacoxal rim m86 IIIscm4 Metafurca Lateral metacoxal rim, close to pleuro-coxal articulation m87 IIIscm5 Ventral segmental boder, postero-mesal Postero-lateral metacoxal rim of base of metafurcal arm m88 IIIscm6 Ventral side of the anterior arm of the metafurca Trochanter

Hom*, proposed homologization with the terminology of Friedrich and Beutel (2008).

In the thoracic musculature no differences compared I Eumetabola [Bremer Support (BS) 2] with Austrophasma could be found. 68.1: Articulation between basalare and ventral bas- isubcostale present (reversal in Macroxyela). Results of the phylogenetic analysis 71.0: Idlm3 absent (also absent in Megacrania; reversal in Palpares;?inTimema). The cladistic analysis results in four equally parsi- 82.0: Itpm2 absent (also absent in Grylloblatta, monious cladograms with 321 steps. Figure 6 shows Austrophasma, Stagmomantis, Embia,andMegacra- their strict consensus tree with Bremer support values nia;?inTimema and Stenopsocus). in Arabic numbers. The data matrix is found in 104.0: IIpcm2 absent (also absent in Grylloblatta, Appendix S3 while Appendix S4 provides a detailed Austrophasma, Periplaneta, Euborellia, Zorotypus, character discussion. The autapomorphies for all nodes and Embia; ? for Macroxyela). (indicated by Roman numbers in Fig. 6) are listed 114.0: IIIpcm2 absent (also absent in Grylloblatta, below. Unambiguous apomorphies are marked in Austrophasma, Euborellia, Zorotypus, and Embia;? bold. for Macroxyela). 60 B. Wipfler et al. / Cladistics 31 (2015) 50–70

(a)

(b)

(c)

Fig. 5. Electron micrographs showing exoskeleton of Mantophasma sp. (a) Dorsal view; (b) ventral view; (c) lateral view. Abbreviations: bs, ba- sisternum; c1, first laterocervicalia; c2, second laterocervicalia; cx1, procoxa; cx2, mesocoxa; cx3, metacoxa; hc, head capsule; if, invaginationof furca; ipa, invagination of pleural arm; is, invagination of spina; n1, pronotum; n2, mesonotum; n3, metanotum; nI, notum of abdominal seg- ment 1; nII, notum of abdominal segment 2; pcr, paracoxal ridge; pr, pleural ridge; prr, pronotal ridge; sp, spiracle; tr, trochantin.

II Psocoptera + Holometabola (BS 2) 96.1: Iscm6 present (also present in Perla, Austroph- asma, Euborellia and Gryllus). 63.1: Basiradiale, distal to convex axillary folding 102.1: IIspm6 present (? for Macroxyela). line with membranous region (also present in Eubo- 105.1: IIpcm3 present (also present in Grylloblatta, rellia). Austrophasma, Periplaneta, Stagmomantis, Orthop- 79.0: Idvm14 absent (also absent in Grylloblatta, tera, Zorotypus, Embia, and Phasmatodea; ? for Euborellia, and Dermaptera; ? in Phasmatodea and Macroxyela). Siphlonurus). 111.1: IIscm7 present (also present in Grylloblatta, 95.1:Iscm5 present (also present in Orthoptera and Austrophasma, Periplaneta, Euborellia, Gryllus, Zo- Periplaneta;?inTimema). rotypus, and Timema;?inEmbia and Macroxyela). B. Wipfler et al. / Cladistics 31 (2015) 50–70 61

Fig. 6. Strict consensus tree from the four minimum-length cladograms with 321 steps based on 119 thoracic characters. Bremer support values above 1 are presented as Arabic numbers for each node. Potential apomorphies for each clade (Roman numbers) are presented in the text.

115.1: IIIpcm3 present (also present in Grylloblatta, 8.0: Paracoxal ridge present (reversal in Periplaneta, Austrophasma, Periplaneta, Stagmomantis, Orthop- Zorotypus,andMegacrania;?inEuborellia, Orthop- tera, Zorotypus, Embia, and Phasmatodea; ? for tera, Stenopsocus, Macroxyela, and Palpares). Macroxyela). 34.1: Hind wing vannus distinctly enlarged (reversal in Embia and Zorotypus; inapplicable to unwinged species). III Holometabola (BS 8) 44.1: Dorsal part of humeral plate membranous (inapplicable to unwinged taxa). 2.1: Cervical sclerites and pleura partly or com- 50.1: Articulation between antemedian notal wing pletely fused (? in Euborellia). process and Ax 1 present and side-by-side (reversal 18.1: Mesosternum invaginated. [state 0] in Zorotypus; new state [state 2] in Orthop- 19.1: Ventral mesosternal process (below mesofurca) tera; inapplicable to unwinged taxa). forming sterno-coxal joint present. 52.1: Proximal tail of body of Ax 1 long, articulated 20.1: Mesocoxae closely adjacent medially. with median notal wing process along long margins 28.1: Metasternum invaginated. (reversal [state 0] in Megacrania; new character state 29.1: Ventral metasternal process (below metafurca) [2] in Stagmomantis and Gryllus; inapplicable to un- forming sterno-coxal joint present. winged taxa). 31.1: Metacoxae adjacent or almost adjacent medi- 59.1: Basiradiale and head of Ax 1 articulated (rever- ally. sal in Periplaneta, Stagmomantis,andMegacrania;? 41.0: Posterior notal wing process separated to in Dissosteira; inapplicable to unwinged taxa). notum (also present in Siphlonurus, Orthoptera, and 107.1: IIscm3 present (reversal in Periplaneta, Stag- Zorotypus; inapplicable to unwinged taxa). momantis, and Euborellia;?inEmbia, Megacrania, 48.1: Basal hinge running between notum and pos- and Macroxyela). terior notal wing process (also present in Zorotypus and Embia; inapplicable to unwinged taxa). V Pliconeoptera or Paurometabola (BS 2) 80.0: Idvm18 absent (? for Timema and Phloeo- thrips). 1.2: two pairs of lateral cervical sclerites present 94.0: Iscm3 absent (also absent in Euborellia; ? for (reversal in Gryllus; also present in Stenopsocus and Timema). Palpares;?inEuborellia). 14.0: Median mesonotal suture absent (? in Peri- IV Polyneoptera (BS 2) planeta, Stagmomantis, Euborellia, Embia, Phloeo- thrips, and Palpares). 3.1: Dorsal cervical sclerites present (reversal in 22.1: Mesospina on distinct spinasternite (reversal Grylloblatta, Austrophasma, Euborellia, and Megacr- in Austrophasma and Orthoptera; ? in Euborellia; ania). inapplicable to species without a mesospina). 62 B. Wipfler et al. / Cladistics 31 (2015) 50–70

35.1: Folding of vannus pleated (reversal in Zoroty- 114.0: IIIpcm2 absent (also absent in Euborellia, pus; inapplicable to unwinged species). Stenopsocus, Phloeothrips, and Palpares;?inMacr- 36.1: Forewing with moderately sclerotized tegmina oxyela). (reversal [state 0] in Zorotypus, Embia, and partly Mantodea; new character state [state 2] in Euborel- VII Xenonomia (BS 5) lia). 75.1: Idvm5 present (reversal in Periplaneta, Embia, 3.0: Dorsal cervical sclerites absent (also absent in and Megacrania; also present in Palpares;?inTim- Siphlonurus, Megacrania, Stenopsocus, Phloeothrips, ema). Palpares, and Macroxyela). 91.1: Ivlm6 present (reversal in Stagmomantis, Diss- 9.1: Paracoxal process of the prothorax. osteira, and Megacrania; also present in Stenopso- 17.1: Invagination of mesopleural arm on epimeron cus). (not applicable to species without pleural arm). 103.1: IIpcm1 present (reversal in Zorotypus, Embia, 26.1: Invagination of metapleural arm on epimeron and Timema; also present in Stenopsocus, Palpares, (not applicable to species without pleural arm). and Macroxyela). 32.0: wings absent (also absent in Timema). 105.1: IIpcm3 present (also present in Stenopsocus 109.1: IIscm5 present (also present in Stagmomantis, and Palpares;?inZorotypus and Macroxyela). Periplaneta, Gryllus, and Dissosteira;?inPhloeo- 111.1: IIscm7 present (reversal in Stagmomantis, thrips and Macroxyela). Dissosteira, and Megacrania; also present in Steno- 116.0: IIIscm2 absent. psocus;?inEmbia and Macroxyela). 119.1: IIIscm5 present (? in Siphlonurus). 112.1: IIIdvm3 present (reversal in Gryllus;?in Siphlonurus, Austrophasma, and Stagmomantis). VIII Phasmatodea (BS2) 115.1: IIIpcm3 present (reversal in Euborellia;also present in Stenopsocus and Palpares;?inMacroxyela). 6.1: Prothoracic defence gland present. 30.1: Abdominal sternum 1 and metasternum fused VI Zoraptera + Embioptera + Xenonomia (BS 2) (not applicable to Stenopsocus, Macroxyela, and Phloeothrips). 33.1: Fewer than five costal cross veins (also present 99.1: IIdvm9 present (? in Macroxyela). in Periplaneta, Euborellia, Stenopsocus,andPhloeo- 101.1: IIspm3 present (also present in Grylloblatta, thrips; inapplicable to unwinged taxa). Austrophasma, Periplaneta, and Zorotypus;?inEm- 34.0: Hind wing vannus not enlarged (also present bia and Macroxyela). in Siphlonurus, Stenopsocus, Phloeothrips, Palpares, 108.0: IIscm4 absent (also absent in Periplaneta, and Macroxyela; inapplicable to species without Dissosteira, Zorotypus, and Palpares;?inEmbia wings). and Macroxyela). 36.0: Sclerotization of forewing absent (also absent 110.0: IIscm6 absent (also absent in Grylloblatta in Perla, Siphlonurus, Stenopsocus, Phloeothrips, and Siphlonurus;?inMacroxyela). Palpares, and Macroxyela; inapplicable to species 118.0: IIIscm4 absent (also absent in Perla, Steno- without wings). psocus,andPalpares;?inEmbia). 42.1: Tegula strongly sclerotized (inapplicable to species without wings). IX Orthoptera (BS 6) 48.1: Basal hinge running between notum and pos- terior notal wing process (also present in Macroxy- 4.1: Pronotum saddle like, cryptopleura. ela and Palpares; inapplicable to species without 13.0: Prospina on posterior part of basisternum or wings). connected with furca (also present in Embia;?in 54.1: Head of first axillary sclerite enlarged (also Perla; inapplicable to species without prospina). present in Palpares;?inSiphlonurus, Dissosteira, 16.1: Mesopleural arm arm-like structure projecting and Phloeothrips; inapplicable to species without in thoracic cavity (also present in Austrophasma, wings). Periplaneta, and Stagmomantis; inapplicable to spe- 101.1: IIspm3 present (also present in Periplaneta cies without pleural arm). and Phasmatodea; ? in Embia and Macroxyela). 22.0: Mesospina on posterior part of basisternum 103.0: IIpcm1 absent (also absent in Siphlonurus, or connected with furca (also present in Perla, Perla, Phloeothrips, and Palpares;?inMacroxyela). Austrophasma, Stenopsocus, and Macroxyela; inap- 104.0: IIpcm2 absent (also absent in Periplaneta, plicable to species without mesospina). Euborellia, Stenopsocus, Phloeothrips, and Palpares; 25.1: Metapleural arm arm-like structure projecting ?inMacroxyela). in thoracic cavity (also present in Austrophasma, B. Wipfler et al. / Cladistics 31 (2015) 50–70 63

Periplaneta, and Stagmomantis; inapplicable to spe- 69.1: Posterior corner of basalare strongly swelling cies without pleural arm). (also present in Euborellia, Embia, Stenopsocus,and 37.1: Pronounced precostal field present (also pres- Palpares;?inPhloeothrips; inapplicable to species ent in Megacrania; inapplicable to species without without wing joint). wings). 73.1: Idlm6 present (also present in Siphlonurus;?in 41.0: Posterior notal wing process separated from Perla and Phasmatodea). notum (also separated in Siphlonurus, Zorotypus, 92.1: Ivlm8 present (also present in Dissosteira and Palpares,andMacroxyela; inapplicable to unwinged Stenopsocus;?inGryllus and Timema). species). 107.0: IIscm3 absent (also absent in Siphlonurus, 45.0: Humeral plate and ventral basisubcostale Euborellia, Stenopsocus, Phloeothrips,andPalpares; widely separated (also present in Siphlonurus;?in ?inMacroxyela and Embia). Megacrania and Phloeothrips; inapplicable to unwin- 109.1: IIscm5 present (also present in Orthoptera, ged species). Grylloblatta and Mantophasma;?inPhloeothrips 62.1: Bending region of basiradiale constricted (? in and Macroxyela). Palpares and Macroxyela; inapplicable to unwinged species). 79.0: Idvm14 absent (also absent in Grylloblatta, Euborellia, Stenopsocus, Palpares, and Macroxyela; Discussion ?inSiphlonurus and Phasmatodea). 83.0: Itpm3 absent (also absent in Phloeothrips;?in The mantophasmatodean thorax Grylloblatta, Timema, and Palpares). 88.1: Ipcm2 present (also present in Grylloblatta, Kristensen (1991) considered Grylloblattodea as Stenopsocus, and Macroxyela;?inTimema). the “most homogenous insect order”. However, this 95.1: Iscm5 present (also present in Periplaneta, Steno- title might be challenged by Mantophasmatodea, psocus, Macroxyela, and Palpares;?inTimema). which shows a remarkable similarity among its spe- 109.1: IIscm5 present (also present Grylloblatta, cies both in head (Baum et al., 2007; Wipfler et al., Mantophasma, Periplaneta, and Stagmomantis;?in 2011) and in thorax morphology. The differences Phloeothrips and Macroxyela). found in the thoracic skeleton are extremely small and none was found in the muscular system of the X Dictyoptera (BS 3) two species studied, which represent the two principal lineages within Mantophasmatodea (Predel et al., 16.1: Pleural arm arm-like structure projecting in 2012). thoracic cavity (also present in Austrophasma, Gryl- Little research has been conducted in regard to the lus,andDissosteira; inapplicable to species without mantophasmatodean thorax. Aside from species pleural arm). descriptions, the only work examining features of the 25.1: Metapleural arm arm-like structure projecting thoracic morphology was presented by Klug (2008) in thoracic cavity (also present in Austrophasma, who studied the pterothorax of Austrophasma caledon- Gryllus, and Dissosteira; inapplicable to species ensis, a species we reinvestigated here. While the previ- without pleural arm). ous findings on the thoracic skeleton are confirmed, 45.2: Humeral plate and ventral basisubcostale our reinvestigation reveals that several muscles had fused (also present in Stenopsocus and Macroxyela; been overlooked (see Appendix S1). inapplicable to species without wing joint). We consider the description of the mantophasmato- 49.1: Articulation between anterior notal wing pro- dean thorax presented here a tentative reconstruction cess and first axillary sclerite along the long margin of the ground plan for the group with the following of neck of the axillary sclerite (also present in uncertainties: the meso- and metanotal ridges are pres- Stenopsocus and Macroxyela; inapplicable to species ent in Austrophasma but absent in Mantophasma. Since without wing joint). they are also absent in other lower neopterans such as 59.0: Basiradiale and head of Ax 1 not articulated Grylloblatta (Walker, 1938) we suggest that they are (also present in Siphlonurus, Megacrania, Stenopso- not part of the mantophasmatodean ground plan. For cus, Phloeothrips, Palpares, and Macroxyela;?in the other differences between Austrophasma and Man- Dissosteira; inapplicable to species without wing tophasma (see above) no reliable character polarization joint). can be presented. 61.1: First axillary sclerite and antero-proximal cor- The mantophasmatodean thorax shares the general ner of second axillary sclerite clearly separated by features of lower neopteran insects such as moveable membrane (? in Phloeothrips; inapplicable to species segments or the well-developed pro- and mesothoracic without wing joint). spinae and their associated muscles. However, it also 64 B. Wipfler et al. / Cladistics 31 (2015) 50–70 shows several apomorphic characteristics, the most Reduction of wings obvious one being the absence of wings (discussed below). A pleural process observed in the present Reduction of wings is a common feature among specimen in the meso- and metathorax was not Pterygota (Brodsky, 1994) and in most cases is associ- observed in any other wingless insect with the excep- ated with ground dwelling, a parasitic lifestyle, high tion of Grylloblatta (Walker, 1938) where the struc- altitudes or a life in low temperatures. Various grada- ture is identical and described as “pleural wing tions of this phenomenon, moving from partial to process”. However, despite the resemblance and the complete flightlessness and from sexual dimorphism to attaching tergo-pleural muscles, a clear homology periodic production of alates, can be observed in lower with the pleural wing process of winged insects can- neopteran insects and occur at any systematic level. not be assumed. There is a remarkable resemblance Well-known examples of wingless lower neopterans between Grylloblatta and Mantophasmatodea with include the dermapteran Hemimerus, the phasmatode- respect to this structure. Additionally, both groups an Timema, and various roaches and mantids. Other share the paracoxal process (“anapleural wing” of interesting cases are the termites, where winged indi- Walker, 1938) in the prothorax: an internal apodeme viduals are produced periodically, Zoraptera with alate that unites with the pleural ridge and serves as site and wingless morphs, and Embioptera and certain spe- for muscle attachment. This complex structure was cies of Phasmatodea and Mantodea where a sexual not found in any other insect studied. Extreme simi- dimorphism with winged males and wingless females larities are also observed in the furcae, which show a occurs. Many studies have addressed different aspects distinct separation into two arms in all three seg- of wing reduction (for reviews, see Roff, 1990; Wagner ments and in the epimeral origin of the meso- and and Liebherr, 1992), but little is known about the metapleural arm. The most interesting similarity comparative morphology of wingless species. Arbas might be the presence of IIIscm5, a muscle connect- (1983) addressed morphological differences between a ing the metathoracic spina with the coxa. While brachypterous and an alate species in Orthoptera, and being well developed in Grylloblatta (Walker, 1938), Friedrich and Beutel (2008) compared winged and mantophasmatodeans have no distinct metaspina. wingless morphs of Zoraptera. However, no broad, in- However, the muscle attaches far postero-mesally to terordinarial morphological comparison of unwinged the furcal arm near the border between metathorax species has been attempted so far. Thus, we compare and abdominal segment 1, exactly at the same loca- the meso- and metathoracic morphology and muscula- tion as it does in Grylloblatta (Walker, 1938). We ture of Mantophasmatodea with those of previously therefore homologized this muscle in both groups studied flightless lower neopterans: the grylloblattode- despite the absence of the distinct spina in Manto- an Grylloblatta campodeiformis (Walker, 1938), the phasmatodea. The presence of the metaspina was orthopteran Cephalocoema albrechti (Zolessi, 1968), considered one of the unique plesiomorphies of the and the dermapteran Hemimerus talpoides (Giles, 1963; group (Walker, 1938). Kristensen (1991), however, Barlet, 1985d). Klug (2008) studied the pterothoracic considered it to be a neoformation, as nothing com- morphology in a whole series of both winged and parable is found in any other hexapod. Considering wingless phasmatodean representatives. Of special the observed situation in Mantophasmatodea, it interest are the species that are dimorphic concerning appears likely that the muscle reaching from the flight: females of Embia are wingless while males are furca to the posterior coxal rim (IIIscm2) shifted its alate (Barlet, 1985a,b,c) and, in Zoraptera, winged and origin from the furca to the posterior edge of the wingless morphs have been studied for two species sternite in both groups. In Grylloblattodea, a second- (Friedrich and Beutel, 2008). Appendix S1 provides an ary spina formed to provide a higher and more sta- attempt to homologize the muscles in these groups. ble point of attachment. In favor of this hypothesis, General trends in wingless species include the fusion IIIscm2 is present in all neopterans except Gryllob- or lacking subdivision of the pterothoracic tergites, lattodea and Mantophasmatodea (see appendix 1). loss of wing base sclerites, a distinctly lower degree of The change in function associated with this shift of sclerotization, the less extensive phragmata, and the origin in Grylloblattodea and Mantophasmatodea distinctly reduced pleural arms (Friedrich and Beutel, cannot be answered yet. Unique features in the mus- 2008). Most of these adaptations can also be found in culature of two studied mantophasmatodeans are: Mantophasmatodea. However, the pleural arms of the the presence of II/IIIspm5, which is found in Blatto- pterothorax are well developed in both species studied dea, Isoptera, and Mantodea according to Matsuda (Fig. 2). A similar condition is found in the flightless (1970) but absent in Stagmomantis (Levereault, 1938) orthopteran Cephalocoema albrechti (Zolessi, 1968) and Periplaneta (Carbonell, 1947) and the absence of while in many other unwinged taxa the pleural arms II/IIIdvm8 (present in all studied species except Siph- are extremely short (Grylloblattodea: Walker, 1938; lonurus and Dissosteira). Zoraptera: Friedrich and Beutel, 2008). It is possible B. Wipfler et al. / Cladistics 31 (2015) 50–70 65 that the strong pleural arms are connected to the and in phasmids capable of flight such as Pseudophas- climbing lifestyle in shrubs observed in both Manto- ma, Sipyloidea, or male Phyllium, its neurons are only phasmatodea and Cephalocoema, rather than to flight- active during flight (R. Klug and R. Hustert, pers. lessness. This hypothesis might be supported by the commun.). Thus, we assume that this muscle has a dif- fact that short pleural arms are also found in good fly- ferent functionality in Mantophasmatodea and Gryl- ers such as Macroxyela (Vilhelmsen, 2010; Vilhelmsen loblattodea. et al., 2010) or Palpares (Czihak, 1957). However, at In the tergopleural muscles, Friedrich and Beutel the current state of knowledge we can only speculate (2008) considered the presence of II/IIItpm4, II/II- about this correlation and more research specifically Itpm7, III/IItpm9, and III/IItpm10 to be dependent on focusing on the thoracic adaptations towards flightless- flight in Zoraptera while Klug (2008) observed a corre- ness is needed to address this issue. lation between wings and the presence of IIItpm3, II- Another peculiarity of Mantophasmatodea is the Itpm4, II/IIItpm5, II/IIItpm6, and II/IIItpm9 in presence of a pleural process (the potential homology Phasmatodea. Our comparison supports the fact that with the pleural wing process of winged insects is dis- II/IIItpm9 is linked to the presence of a flight appara- cussed above) while all other sclerites of the wing joint tus. In Phasmatodea alone, apparently several inde- have been reduced or fused. This process is commonly pendent reductions occurred (Jeziorski, 1918; found in winged forms but absent in unwinged morphs Marquardt, 1939; Klug, 2008). This muscle is also of Zorotypus (Friedrich and Beutel, 2008), unwinged present in winged morphs of Zorotypus (Friedrich and earwigs (Giles, 1963), females of Embia (Barlet, 1985a, Beutel, 2008) but absent in males of Embia (Barlet, b), and wingless stick insects. In the orthopteran Bar- 1985c). II/IIItpm7, however, is also absent in all stud- ytettix, which has vestigial wings in the mesothorax, ied wingless forms but is generally found only in a few Arbas (1983) did not find any trace of this process winged insects such as Perla (Wittig, 1955) or Oligo- either. The only other wingless taxon studied here that toma (Maki, 1938); so no clear correlation with flight- has a similar process is Grylloblatta (Walker, 1938). lessness can be made. II/IIItpm6 shows a close linkage The presence of such a process, including several asso- to wing reduction in Phasmatodea (Klug, 2008) and is ciated tergo-pleural muscles, in species that are com- missing in all other studied wingless species with the pletely wingless indicates that it was repurposed for a exception of Hemimerus (Barlet, 1985d) and possibly different function. However, we currently have no idea Embia (Barlet, 1985a,b,c) where the situation is not what this might be. clear. However, it is also absent in around 50% of the The muscle groups most affected by the presence of winged species investigated (e.g. Thrips: Mickoleit, wings are the dorso-ventral (dvm), sternopleural 1961). This also applies to IItpm4 and II/IIItpm5, (spm), and tergopleural (tpm) muscles. The most obvi- which on the one hand are linked to the presence of ous adaptation towards flightlessness is therefore the wings in Phasmatodea (Klug, 2008) or Zoraptera reduction of II/IIIdvm1, which is absent in all wingless (Friedrich and Beutel, 2008), respectively, but display insects studied but present in winged zorapterans no obvious patterns in other groups. IIItpm3 and II- (Friedrich and Beutel, 2008), in males of Embia (Bar- Itpm4, on the other hand, are absent in all winged let, 1985c), and in the flying males of the phasmatode- groups with the exception of Mantophasmatodea. an Phyllium (Klug, 2008). The sternopleural basalar Both muscles are linked to flight in Phasmatodea muscle (II/IIIspm1), which is considered to act as an (Klug, 2008) as is IIItpm4 in Zoraptera (Friedrich and indirect flight muscle (Friedrich and Beutel, 2008), is Beutel, 2008). also absent in all wingless taxa studied, with the nota- In general, it can be stated that while certain mor- ble exception of Mantophasmatodea and Grylloblatto- phological adaptations toward flightlessness such as dea (Walker, 1938). The status of this adaptation in simplified terga, reduced phragmata, and the reduction females of Embia (Barlet, 1985a,b) is not clear. On the of II/IIIdvm1 are found in all groups studied, there other hand, this muscle is also reduced in the winged are several different specifications among wingless species of entire clades such as Dermaptera (Kleinow, insects. However, it is interesting that within Phas- 1966; Bharadwaj and Chadwick, 1974), Dictyoptera matodea (Klug, 2008) the reduction of wings has, (Levereault, 1938; Carbonell, 1947), and Psocoptera apparently, always followed the same pattern in which (Badonnel, 1934; Maki, 1938). However, the apparent the same muscles were affected. The two zorapterans multiple reductions of this muscle in wingless phas- studied also have the same muscular equipment in matodeans (Klug, 2008) as well as its absence in un- alate and wingless morphs (Friedrich and Beutel, winged morphs of Zoraptera (Friedrich and Beutel, 2008). We therefore tentatively conclude that the mor- 2008) underline its importance as a flight muscle and phological transformations tied to wing reduction are the trend of reduction in wingless species. Muscle II/II- more dependent on the initial morphology and func- Ispm1 acts as the physiological depressor and pronator tional constraints preceding wing loss in a group than of the fore and hind wing in locusts (Burrows, 1996), following a general rule or pattern. However, we want 66 B. Wipfler et al. / Cladistics 31 (2015) 50–70 to point out the poor sampling the current study is polyneopteran taxon sample date from the latter per- based on compared with the abundance and various iod (Wipfler et al., 2011; Yoshizawa, 2011; Blanke degrees of wing reduction in insects, and cannot over- et al., 2012, 2013; Wipfler, 2012). The present investi- emphasize the importance of addressing the morpho- gation continues this trend by recovering a monophy- logical adaptions towards flightlessness on a broader letic Polyneoptera including Zoraptera with a base and conducting more specific research on this moderate Bremer support of 2. We confirm two of the strongly neglected topic. four potential apomorphies presented by Yoshizawa (2011): a dorsally membranous humeral plate and a Phylogeny of the neopteran insects long proximal tail of the body of the first axillary sclerite that articulates with the median notal wing The present study compares the thoracic morphol- process along its long margins (with a reversal in Me- ogy of species representing all major lower neopterous gacrania). The other two derived characters proposed lineages. by Yoshizawa (2011) (a side by side articulation of the In recent years, a broad consensus has been achieved median notal wing process and the body of the first for both the internal relationships of Holometabola axillary and an at-a-point articulation between the ba- (Wiegmann et al., 2009; Beutel et al., 2010) and Para- salare and the humeral plate) are considered a neopter- neoptera (Yoshizawa and Saigusa, 2001). Additionally, an apomorphy or only support Polyneoptera under a closer relationship between these two groups (= Eu- delayed character optimization (DELTRAN). How- metabola) finds broad support (e.g. Letsch et al., ever, the present study finds an additional wing joint 2012). The present study supports the monophyly of character for Polyneoptera: a side-by-side articulation Eumetabola and Holometabola by confirming several between the antemedian notal wing process and the of the apomorphies presented by Friedrich and Beutel first axillary sclerite with a reversal in Zoraptera (char- (2010). The monophyly of Paraneoptera, however, is acter 13 of Yoshizawa, 2011). Additionally, the pres- not supported. Nevertheless, the latter is well sup- ence of a distinctly enlarged hind wing vannus (e.g. ported by molecular (e.g. Ishiwata et al., 2011) and Kristensen, 1991; Klass, 2007) is confirmed, and dorsal morphological (e.g. Yoshizawa and Saigusa, 2001) evi- cervical sclerites, a paracoxal ridge, and the presence dence, and the limited taxon sampling within the of IIscm3 are proposed as potential unambiguous apo- group might have influenced the results of the present morphies of Polyneoptera. However, all of these char- study. Reconstructing the evolution and phylogeny of acters have several reversals within the group and the remaining neopteran taxa constitutes a severe most of them might be artificial, created by the taxon problem, and the monophyly of Polyneoptera is con- sampling: a dorsal cervical sclerite is present in certain sidered to be the key question. Potential reasons for representatives of holometabolan groups such as Neu- the difficulty in understanding the evolution of the roptera or Hymenoptera (Matsuda, 1970; Vilhelmsen lower neopteran insects are the rapid radiation of this et al., 2010), a paracoxal ridge occurs in Coleoptera group (Whitfield and Kjer, 2008) and an insufficient (Matsuda, 1970), and IIscm3 is present in several holo- morphological treatment. However, the discovery of metabolan species such as Nannochorista (Friedrich Mantophasmatodea (Klass et al., 2002) greatly revived and Beutel, 2009). In addition to the characters in this interest in lower neopteran evolution. In the last study, the presence of euplantulae (Gorb and Beutel, 10 years, several molecular as well as morphological 2001; Beutel and Gorb, 2006) and a heart with both studies have been published on this issue (e.g. Kjer, ex- and incurrent ostia (Pass et al., 2006) were pro- 2004; Terry and Whiting, 2005; Kjer et al., 2006; Ma posed as derived characters for Polyneoptera. But sim- et al., 2009; Wipfler et al., 2011; Yoshizawa, 2011; ilar to most other characters in favour of Blanke et al., 2012). During this period an interesting Polyneoptera, they show a considerable amount of change took place in the observed results. While the variability in the group: euplantulae are absent in Em- papers published before 2010 agree on the paraphyly bioptera and most groups of Dermaptera. In Gryllob- of Polyneoptera with respect to Holometabola and lattodea, they are largely reduced and differ distinctly Paraneoptera (e.g. Kjer, 2004; Yoshizawa and John- from typical polyneopteran tarsal pads as found in son, 2005; Beutel and Gorb, 2006; Kjer et al., 2006; Plecoptera (Beutel and Gorb, 2006). Excurrent ostia Misof et al., 2007), the studies in the last few years are missing in Zoraptera and show a variation both in support its monophyly (Simon et al., 2010; Ishiwata number and in position among the groups where they et al., 2011; Yoshizawa, 2011; Wipfler et al., 2011 are present (Pass et al., 2006). In general, the experi- [without Zoraptera]; Blanke et al., 2012, 2013 [without ence gained from analysing morphological data sets of Zoraptera]; Simon and Letsch, 2013). From a morpho- Holometabola implies that the most reliable results are logical perspective, a possible explanation for this obtained when data from several different character change is that all those cladistic works that studied a systems are combined in one analysis (Beutel et al., specific morphological character system for a broad 2010). However, such a large comparative study is still B. Wipfler et al. / Cladistics 31 (2015) 50–70 67 lacking for Polyneoptera. The present study contrib- Yoshizawa and Johnson, 2005; Kjer et al., 2006; Misof utes an important step toward this goal by providing et al., 2007; Simon and Letsch, 2013) but final confir- the fourth studied character system following the mation is still needed. adhesive structures (Beutel and Gorb, 2006, 2008), Xenonomia (sensu Terry and Whiting, 2005 = Gryl- wing joint (Yoshizawa, 2011), and the cephalic mor- loblattodea + Mantophasmatodea) on the other hand phology (Wipfler et al., 2011; Blanke et al., 2012, can be considered as assured. The present study adds 2013; Friedemann et al., 2012; Wipfler, 2012). From a several unambiguous and complex apomorphies such molecular point of view, monophyletic polyneopterans as the prothoracic paracoxal process, the epimeral ori- are supported by nuclear coding protein genes (DNA gin of the pterothoracic pleural arms and the presence polymerase delta and two subunits of RNA polymer- of IIIscm5 (see discussion above). In general, the mor- ase II; elongation factor 1-a) and complete mitochon- phological similarity of the thorax in both groups, drial genomes (Ma et al., 2009; Simon et al., 2010; including the pleural processes in the meso- and meta- Ishiwata et al., 2011), whereas studies based on 18S, thorax and branched furcae in all segments, is striking 28S, and/or Histone3 result in its non-monophyly and fits the evidence from other character systems. In (Kjer, 2004; Terry and Whiting, 2005; Yoshizawa and fact, several recent studies support Xenonomia, and Johnson, 2005; Kjer et al., 2006; Misof et al., 2007). potential apomorphies include sperm structure (Dallai So far, no transcriptomic study has included all lower et al., 2005), several modifications in the labium and neopteran lineages but the investigations by Letsch hypopharynx (Wipfler et al., 2011), and a unique et al. (2012) and Simon and Letsch (2013) point structure of the antennal heart (Wipfler et al., 2011), towards monophyletic Polyneoptera. In summary, we added to evidence from neuropeptides (Gade€ and Sim- can state that several arguments from both a molecu- ek, 2010) and molecular data (e.g. Terry and Whiting, lar and a morphological perspective point towards the 2005; Kjer et al., 2006; Ishiwata et al., 2011; Djernaes monophyly of the group. To finally corroborate these et al., 2012). The competing hypothesis, a closer rela- results, data from a genomic study including all repre- tionship between Mantophasmatodea and Phasmato- sentatives of this group and a broad morphological dea, is based on similarities in the adhesive structures analysis are necessary. (Beutel and Gorb, 2008) as well as on mitochondrial Within Polyneoptera, the present study supports a genomic data (Ma et al., 2009; Tomita et al., 2011; sister-group relationship between Plecoptera and the Wan et al., 2012). Komoto et al. (2012) placed remaining polyneopterans (= Pliconeoptera or Pauro- Mantophasmatodea as sister group to a clade metabola). In addition to some highly homoplasious Timema + (Embioptera + Euphasmatodea). However, characters, potential synapomorphies discovered by these investigations suffer from a comparatively poor the present study include a mesospina on a distinct taxon sampling. Nevertheless, Grylloblattodea has spinasternite (with a reversal in Austrophasma and strongly reduced tarsal pads, which might be an adap- Orthoptera), a pleated folding of the vannus (with a tation towards a life between stones while Mantophas- reversal in Zoraptera), and tegmina (with a reversal in matodea and most Phasmatodea both climb in shrubs. Zorotypus, Embia, and parts of Mantodea). Tegmina, In the present study, an enforced clade Mantophas- however, are extremely difficult to define as the transi- matodea + Phasmatodea would require 13 additional tion from slightly hardened forewings, as present in steps and the only potential apomorphies are the pres- most insects which superimpose their wings at rest ence of Itpm2 and the absence of IIspm3, two states (Zwick, 2009), to strongly sclerotized forewings as that are highly homoplasious and are also found in found in, for example, Dermaptera is continuous. Grylloblattodea. However, it can be observed that certain groups of The strongly supported monophyly of Orthoptera lower neopteran insects show a high tendency towards (e.g. Fenn et al., 2008) and Dictyoptera (e.g. Wipfler sclerotized forewings. et al., 2012b) gains additional support from our study. The phylogenetic position of Plecoptera is among Recently, the monophyly of Phasmatodea has been the most disputed of all lower neopteran insects and questioned by several studies that place Timema out- might only be challenged in the number of sister-group side of the remaining phasmatodeans (e.g. Yoshizawa hypotheses by Zoraptera (for an overview see Zwick, and Johnson, 2005; Plazzi et al., 2011; Komoto et al., 2009). Next to the Pliconeoptera, proposed sister 2012). However, our study supports its monophyly groups for Plecoptera include all neopterans (e.g. Beu- (for detailed discussions see Bradler, 2009; Friedemann tel and Gorb, 2006; Zwick, 2009), Embioptera (Haas et al., 2012) but does not propose a sister group. In and Kukalova-Peck, 2001), Phasmatodea (Matsuda, recent years a closer affinity with Embioptera (= Eu- 1970), Orthoptera (Komoto et al., 2012), and Zorap- kinolabia hypothesis sensu Terry and Whiting, 2005) tera (Terry and Whiting, 2005). During the past few gained increasing support (e.g. Kjer et al., 2006; Bra- years, a clade comprising Dermaptera and Plecoptera dler, 2009; Friedemann et al., 2012; Letsch et al., gained increasing support from molecular data (e.g. 2012). 68 B. Wipfler et al. / Cladistics 31 (2015) 50–70

The deeper nodes of Polyneoptera (except Plico- Baum, E., Dressler, C., Beutel, R.G., 2007. Head structures of neoptera) are completely unresolved in the present Karoophasma sp. (Hexapoda, Mantophasmatodea) with phylogenetic implications. J. Zool. Syst. Evol. Res. 45, 104–119. study, reflecting the current state of research. While Beutel, R.G., Gorb, S., 2006. A revised interpretation of the there is a certain consensus about clades such as Dict- evolution of attachment structures in Hexapoda with special yoptera, Xenonomia, or Eukinolabia in the literature emphasis on Mantophasmatodea. Arthropod Syst. Phylogeny 61, 3–35. (see above), any profound statement about deeper Beutel, R.G., Gorb, S., 2008. Evolutionary scenarios for unusual nodes within Polyneoptera is currently impossible and attachment devices of Phasmatodea and Mantophasmatodea might not be resolvable. The present study provides (Insecta). Syst. Entomol. 33, 501–510. valuable information about the monophyly of Poly- Beutel, R.G., Friedrich, F., Hornschemeyer,€ T., Pohl, H., Hunefeld,€ F., Beckmann, F., Meier, R., Misof, B., Whiting, M.F., neoptera and, finally, confirms Xenonomia. However, Vilhelmsen, L., 2010. Morphological and molecular evidence we emphasize the preliminary nature of the findings as converge upon a robust phylogeny of the megadiverse the taxon sampling remains insufficient and a combi- Holometabola. Cladistics 27, 341–355. nation with other character systems is still missing. Bharadwaj, R.K., Chadwick, L.E., 1974. Postembryonic development of the cervicothoracic skeleton of Euborellia Nevertheless, these difficulties suggest that resolving annulipes (Lucas) (Dermaptera: Labiduridae). J. Morphol. 144, the polyneopteran affinities is among the largest and 255–268. most puzzling problems of systematic entomology. Blanke, A., Wipfler, B., Letsch, H., Koch, M., Beckmann, F., Beutel, R., Misof, B., 2012. Revival of Palaeoptera: head characters support a monophyletic origin of Odonata and Ephemeroptera (Insecta). Cladistics 28, 560–581. Acknowledgments Blanke, A., Greve, C., Wipfler, B., Beutel, R.G., Holland, B.R., Misof, B., 2013. The identification of concerted convergence in insect heads corroborates Palaeoptera. Syst. Biol. 62, 250– Klaus Klass (Dresden) and Reinhard Predel 263. (Cologne) provided the material for the present study. Bradler, S., 2009. Die Phylogenie der Stab- und Gespenstschrecken We thank Paul Frandson (Rutgers) for revising the (Insecta: Phasmatodea). SPE 2, 3–139. Bremer, K., 1994. Branch support and tree stability. Cladistics 10, English text and Hans Pohl (Jena) for the photos of 295–304. Austrophasma. Frank Friedrich (Hamburg) helped by Brodsky, A.K. 1994. The Evolution of Insect Flight. Oxford providing several cited articles. R.K. and T.H. are University Press, Oxford. grateful for general support by Rainer Willmann. We Burrows, M. 1996. The Neurobiology of an Insect Brain. Oxford University Press, Oxford. thank two anonymous reviewers for their suggested Carbonell, C.S., 1947. The thoracic muscles of the cockroach improvements to the manuscript. The present study Periplaneta americana (L.). Smithson. Misc. Collect. 107, 1–32. was supported by the National Basic Research Pro- Chown, S.L., Marais, E., Picker, M.D., Terblanche, J.S., 2006. Gas gram of China (973 Program) (No. 2011CB302102), exchange characteristics, metabolic rate and water loss of the heelwalker, Karoophasma biedouwensis (Mantophasmatodea: the National Natural Science Foundation of China Austrophasmatidae). J. Insect Physiol. 52, 442–449. (Nos. 31350110218, 31010103913, 31172143, 51305057), Czihak, G., 1957. Beitrage zur Anatomie des Thorax von Ascalaphus € the National Science Fund for Fostering Talents in macaronicus Scop. Osterr. Zool. Z. 4, 401–432. Dallai, R., Frati, F., Lupetti, P., Adis, J., 2003. Sperm ultrastructure Basic Research (Special Subjects in Animal Taxonomy, of Mantophasma zephyra (Insecta, Mantophasmatodea). NSFC-J0630964/J0109, J1210002), by a Humboldt Fel- Zoomorphology 122, 67–76. lowship (M.B.) from Alexander von Humboldt Foun- Dallai, R., Machida, R., Uchifune, T., Lupetti, P., Frati, F., 2005. dation and the DFG (grant numbers BE1789/6-1, The sperm structure of Galloisiana yuasai (Insecta, Grylloblattodea) and implications for the phylogenetic position HO2306/6-1 and HO2306/10-1). B.W. is supported by of Grylloblattodea. Zoomorphology 124, 205–212. the Chinese Academy of Sciences Fellowship for Young Deans, A.R., Miko, I., Wipfler, B., Friedrich, F., 2012. Evolutionary International Scientists (grant number 2011Y2SB05). phenomics and the emerging enlightenment of arthropod systematics. Invertebr. Syst. 26, 323–330. Djernaes, M., Klass, K.D., Picker, M.D., Damgaard, J., 2012. References Phylogeny of cockroaches (Insecta, Dictyoptera, Blattodea), with placement of aberrant taxa and exploration of out-group sampling. Syst. Entomol. 37, 65–83. Arbas, E.A., 1983. Thoracic morphology of a flightless Mexican Eberhard, M., Picker, M., 2008. Vibrational communication in two grasshopper, Barytettix psolus: comparison with the locust, sympatric species of Mantophasmatodea (heelwalkers). J. Insect Schistocerca gregaria. J. Morphol. 176, 141–153. Behav. 21, 240–257. Badonnel, A., 1934. Recherches sur l’anatomie des Psoque. Bull. Eberhard, M., Picker, M., Klass, K.D., 2011. Sympatry in Biol. France Belg. 18, 1–241. Mantophasmatodea, with the description of a new species and Barlet, J., 1985a. Le squelette pterothoracique d′une femelle d’Embia phylogenetic considerations. Org. Divers. Evol. 11, 43–59. (Insectes, Embiopteres). Bull. Soc. Roy. Sci. Liege 54, 133–139. Fenn, J.D., Song, H., Cameron, S.L., Whiting, M.F., 2008. A Barlet, J., 1985b. La musclature pterothoracique d’une Embia femelle preliminary mitochondrial genome phylogeny of Orthoptera (Insectes, Embiopteres). Bull. Soc. Roy. Sci. Liege 54, 140–148. (Insecta) and approaches to maximizing phylogenetic signal Barlet, J., 1985c. Le pterothorax du male d’Embia sucrofi Navas found within mitochondrial genome data. Mol. Phylogenet. Evol. (Insectes, Embiopteres). Bull. Soc. Roy. Sci. Liege 54, 349–362. 49, 59–68. Barlet, J., 1985d. La musculature thorarique d’Hemimerus bouvieri Friedemann, K., Wipfler, B., Bradler, S., Beutel, R.G., 2012. On the CHPD. (Dermapteres). Bull. Annls Soc. R. Belge Ent. 121, 169– head morphology of Phyllium and the phylogenetic relationships 195. of Phasmatodea (Insecta). Acta Zool. 93, 184–199. B. Wipfler et al. / Cladistics 31 (2015) 50–70 69

Friedrich, F., Beutel, R.G., 2008. The thorax of Zorotypus Kristensen, N.P. 1991. Phylogeny of extant hexapods. In: Naumann, (Hexapoda, Zoraptera) and a new nomenclature for the J.D., Carne, P.B., Lawrence, J.F. (Eds.), Insects of Australia. musculature of Neoptera. Arthropod Struct. Dev. 37, 29–54. Melbourne University Press, Melbourne, Vol. 2, pp. 125–140. Friedrich, F., Beutel, R.G., 2009. The thoracic morphology of Letsch, H.O., Meusemann, K., Wipfler, B., Schutte,€ K., Beutel, R., Nannochorista (Nannochoristidae) and its implications for the Misof, B., 2012. Insect phylogenomics: results, problems and the phylogeny of Mecoptera and Antliophora. J. Zool. Syst. Evol. impact of matrix composition. Proc. R. Soc. B 279, 3282–3290. Res. 48, 50–74. Levereault, P., 1938. The morphology of the Carolina mantis. Friedrich, F., Beutel, R.G., 2010. Goodbye Halteria? The thoracic section II: the musculature. Univ. Kans. Sci. Bull. 25, 578–633. morphology of Endopterygota (Insecta) and its phylogenetic Ma, C., Liu, C., Yang, P., Kang, L., 2009. The complete implications. Cladistics 26, 579–612. mitochondrial genomes of two band-winged grasshoppers, Friedrich, F., Farrell, B.D., Beutel, R.G., 2008. The thoracic Gastrimargus marmoratus and Oedaleus asiaticus. BMC morphology of Archostemata and the relationships of the extant Genomics 10, 156. suborders of Coleoptera (Hexapoda). Cladistics 24, 1–37. Maki, T., 1935. A study of the musculature of the phasmid Gade,€ G., Simek, P., 2010. A novel member of the adipokinetic Megacrania tsudai Shiraki. Mem. Fac. Sci. Agric. Taihoku Imp. peptide family in a “living fossil”, the ice crawler Galloisiana Univ. 12, 181–279. yuasai, is the first identified neuropeptide from the order Maki, T., 1936. Studies of the skeletal structure musculature and Grylloblattodea. Peptides 31, 372–376. nervous system of the alder fly Chauliodes formosanus Petersen. Gade,€ G., Marco, H.G., Simek, P., Marais, E., 2005. The newly Mem. Fac. Sci. Agric. Taihoku Imp. Univ. 16, 117–243. discovered insect order Mantophasmatodea contains a novel Maki, T., 1938. Studies on the thoracic musculature of insects. member of the adipokinetic hormone family of peptides. Mem. Fac. Sci. Agric. Taihoku Imp. Univ. 24, 1–343. Biochem. Biophys. Res. Commun. 330, 598–603. Marquardt, F., 1939. Beitrage€ zur Anatomie der Muskulatur und Giles, E.T., 1963. The comparative external morphology and der peripheren Nerven von Carausius (Dixippus) morosus. BR. affinities of the Dermaptera. Trans. R. Entomol. Soc. Lond. 115, Zool. Jahrb. Anat. Onto. 66, 63–128. 95–164. Matsuda, R., 1956. The comparative morphology of the thorax of Goloboff, P. 1999. NONA (no name), version 2. Published by the two species of insects. Microentomology 21, 1–65. author, Tucuman, Argentina. Matsuda, R., 1970. Morphology and evolution of the insect thorax. Goloboff, P., Farris, S., Nixon, K. 2000. TNT (Tree analysis using Mem. Entomol. Soc. Can. 102 (S76), 5–431. New Technology). Published by the author, Tucuman, Mickoleit, G., 1961. Zur Thoraxmorphologie der Thysanoptera. Argentina. Zool. Jahrb. Anat. Onto. 79, 1–92. Gorb, S., Beutel, R., 2001. Evolution of locomotory attachment Misof, B., Niehuis, O., Bischoff, I., Rickert, A., Erpenbeck, D., pads of hexapods. Naturwissenschaften 88, 530–534. Staniczek, A., 2007. Towards an 18S phylogeny of hexapods: Haas, F., Kukalova-Peck, J., 2001. Dermaptera hindwing structure accounting for group-specific character covariance in optimized and folding: new evidence for familial, ordinal and superordinal mixed nucleotide/doublet models. Zoology 110, 409–429. relationships within Neoptera (Insecta). Eur. J. Entomol. 98, Niehuis, O., Hartig, G., Grath, S., Pohl, H., Lehmann, J., Tafer, H., 445–509. Donath, A., Krauss, V., Eisenhardt, C., Hertel, J., Petersen, M., Hockmann, D., Picker, M.D., Klass, K.D., Pretorius, L., 2009. Mayer, C., Meusemann, K., Peters, R., Stadler, P.F., Beutel, Postembryonic development of the unique antenna of R.G., Bornberg-Bauer, E., McKenna, D., Misof, B., 2012. Mantophasmatodea (Insecta). Arthropod Struct. Dev. 38, 125–133. Genomic and morphological evidence converge to resolve the Ishiwata, K., Sasaki, G., Ogawa, J., Miyata, T., Su, Z.H., 2011. enigma of Strepsiptera. Curr. Biol. 22, 1309–1313. Phylogenetic relationships among insect orders based on three Nixon, K.C., 1999. The parsimony ratchet, a new method for rapid nuclear protein-coding gene sequences. Mol. Phylogenet. Evol. parsimony analysis. Cladistics 15, 407–414. 58, 169–180. Nixon, K.C. 2000. WinClada, Ver. 0.99. Cornell University, Ithaca, Jeziorski, L., 1918. Der Thorax von Dixippus morosus (Carausius). NY. Z. Wiss. Zool. 117, 727–815. Nixon, K.C. 2002. WinClada, version 1.00. 08. Published by the Kjer, K.M., 2004. Aligned 18S and insect phylogeny. Syst. Biol. 53, author, Ithaca, NY. 506–514. Pass, G., Gereben-Krenn, B.A., Merl, M., Plant, J., Szucsisch, N., Kjer, K.M., Carle, F.L., Litman, J., Ware, J.L., 2006. A molecular Togel,€ M., 2006. Phylogenetic relationships of the orders of phylogeny of Insecta. Arthropod Syst. Phylogeny 64, 35–44. Hexapoda: contributions from the circulatory organs for a Klass, K.D., 2007. Die Stammesgeschichte der Hexapoden: eine morphological data matrix. Arthropod Syst. Phylogeny 64, 165–203. kritische Diskussion neuerer Daten und Hypothesen. Denisia 20, Plazzi, F., Ricci, A., Passamonto, M., 2011. The mitochondrial 413–450. genome of Bacillus stick insects (Phasmatodea) and the Klass, K.D., Eulitz, U., 2007. The tentorium and anterior head sulci phylogeny of orthopteroid insects. Mol. Phylogenet. Evol. 58, in Dictyoptera and Mantophasmatodea (Insecta). Zool. Anz. 304–316. 246, 205–234. Pohl, H., 2010. A scanning electron microscopy specimen holder for Klass, K.D., Zompro, O., Kristensen, N.P., Adis, J., 2002. viewing different angles of a single specimen. Micros. Res. Tech. Mantophasmatodea: a new insect order with extant members in 73, 1073–1076. the Afrotropics. Science 296, 1456–1459. Predel, R., Neupert, S., Huetteroth, W., Kahnt, J., Waidelich, D., Klass, K.D., Picker, M.D., Damgaard, J., Noort, S.V., Tojo, K., Roth, S., 2012. Peptidomics-based phylogeny and biogeography 2003. The taxonomy, genitalic morphology, and phylogenetic of Mantophasmatodea (Hexapoda). Syst. Biol. 61, 609–629. relationships of Southern African Mantophasmatodea (Insecta). Rahle,€ W., 1970. Untersuchungen an Kopf und Prothorax von Entomol. Abh. 61, 3–67. Embia ramburi Rimsky-Korsakow 1906 (Embioptera, Embiidae). Kleinow, W., 1966. Untersuchungen zum Flugelmechanismus€ der Zool. Jahrb. Anat. Onto. 87, 248–330. Dermapteren. Zoomorphology 56, 363–416. Roff, D.A., 1990. The evolution of flightlessness in insects. Ecol. Klug, R., 2008. Anatomie des Pterothorax der Phasmatodea, Monogr. 60, 389–421. Mantophasmatodea und Embioptera und seine Bedeutung fur€ die Simon, S., Letsch, H., 2013. Insect phylogenomics: new insights on Phylogenie der Polyneoptera (Insecta). Species,. Phyl. Evol. 1, the relationships of lower neopteran orders (Polyneoptera). Syst. 171–217. Entomol. 38, 783–793. Komoto, N., Yukuhiro, K., Tomita, S., Bonen, L., 2012. Novel gene Simon, S., Schierwater, B., Hadrys, H., 2010. On the value of rearrangements in the mitochondrial genome of a webspinner, Elongation factor-1a for reconstructing pterygote insect Aposthonia japonica (Insecta: Embioptera). Genome 55, 222–233. phylogeny. Mol. Phylogenet. Evol. 54, 651–656. 70 B. Wipfler et al. / Cladistics 31 (2015) 50–70

Snodgrass, R.E., 1929. The thoracic mechanism of a grasshopper Xiao, B., Chen, A.H., Zhang, Y.Y., Jiang, G.F., Hu, C.C., Zhu, and its antecedents. Smithson. Misc. Collect. 82, 1–111. C.D., 2012. Complete mitochondrial genomes of two Terry, M.D., Whiting, M.F., 2005. Mantophasmatodea and phylogeny cockroaches, Blattella germanica and Periplaneta americana, and of the lower neopterous insects. Cladistics 21, 240–257. the phylogenetic position of termites. Curr. Genet. 58, 65–77. Tomita, S., Yukuhiro, K., Komoto, N., 2011. The mitochondrial Yoshizawa, K., 2011. Monophyletic Polyneoptera recovered by wing genome of a stick insect Extatosoma tiaratum (Phasmatodea) and base structure. Syst. Entomol. 36, 377–394. the phylogeny of polyneopteran insects. J. Insect Biotechnol. Yoshizawa, K., Johnson, K.P., 2005. Aligned 18S for Zoraptera Sericology 80, 79–88. (Insecta): phylogenetic position and molecular evolution. Mol. Trautwein, M.D., Wiegmann, B.M., Beutel, R., Kjer, K.M., Yeates, Phylogenet. Evol. 37, 572–580. D.K., 2012. Advances in insect phylogeny at the dawn of the Yoshizawa, K., Saigusa, T., 2001. Phylogenetic analysis of postgenomic era. Annu. Rev. Entomol. 57, 449–468. paraneopteran orders (Insecta: Neoptera) based on forewing base Vilhelmsen, L., 2010. Before the wasp-waist: comparative anatomy structure, with comments on monophyly of Auchenorrhyncha and phylogenetic implications of the skeleto-musculature of the (Hemiptera). Syst. Entomol. 26, 1–13. thoraco-abdominal boundary region in basal Hymenoptera Zolessi, L.C., 1968. Morphologie, endosquelette et musculature d’un (Insecta). Zoomorphology 119, 185–221. acridien aptere (Orthoptera: Proscopiidae). Trans. R. Entomol. Vilhelmsen, L., Miko, I., Krogmann, L., 2010. Beyond the wasp- Soc. Lond. 120, 55–113. waist: structural diversity and phylogenetic significance of the Zwick, P., 2009. The Plecoptera – who are they? The problematic mesosoma in apocritan wasps (Insecta: Hymenoptera). Zool. J. placement of stoneflies in the phylogenetic system of insects. Linn. Soc. Lond. 159, 22–194. Aquat. Insects 31, 181–194. Voß, F., 1905. Uber€ den Thorax von Gryllus domesticus mit besonderer € € Berucksichtigung des Flugelgelenks und dessen Bewegung. Zweiter Supporting Information Teil: Die Muskulatur. Z. Wiss. Zool. 78, 355–521. Wagner, D.L., Liebherr, J.K., 1992. Flightlessness in insects. Trends Ecol. Evol. 7, 216–220. Additional Supporting Information may be found in Walker, E.M., 1914. A new species of Orthoptera, forming a new the online version of this article: genus and family. Can. Entomol. 46, 93–99. Walker, E.M., 1938. On the anatomy of Grylloblatta campodeiformis Appendix S1. Proposed homology of the thoracic Walker: 3. exoskeleton and musculature of the neck and thorax. musculature with the works of (in alphabetical order): Ann. Entomol. Soc. Am. 31, 588–640. Badonnel (1934), Barlet (1985a,b,c,d), Bharadwaj and Wan, X., Kim, M.I., Kim, M.J., Kim, I., 2012. Complete Chatwick (1974), Carbonell (1947), Czihak (1957), mitochondrial genome of the free-living earwig, Challia fletcheri (Dermaptera: Pygidicranidae) and phylogeny of Polyneoptera. Friedrich et al. (2008), Friedrich and Beutel (2008), PLoS ONE 7, e42056. Jeziorski (1918), Kleinow (1966), Leverault (1938), Whitfield, J.B., Kjer, K.M., 2008. Ancient rapid radiations of Maki (1935, 1936, 1938), Matsuda (1956, 1970), insects: challenges for phylogenetic analysis. Annu. Rev. € Entomol. 53, 449–472. Mickoleit (1961), Rahle (1970), Snodgrass (1929), Vil- Wiegmann, B.M., Trautwein, M.D., Kim, J.W., Cassel, B.K., helmsen (2010), Vilhelmsen et al. (2010), Voß (1905), Bertone, M.A., Winterton, S.L., Yeates, D.K., 2009. Single-copy Walker (1938), and Wittig (1955). nuclear genes resolve the phylogeny of the holometabolous Appendix S2. Muscle presence according to the pro- insects. BMC Biol. 7, 34. Wipfler, B. 2012. Polyneopteran head morphology and its posed homology in Appendix S1 in Siphlonurus colum- phylogenetic implications. PhD thesis, Friedrich-Schiller- bianus, Perla marginata, Grylloblatta bifractellata, Universitat€ Jena. Austrophasma caledonensis, Periplaneta americana, € Wipfler, B., Machida, R., Muller, B., Beutel, R.G., 2011. On the Stagmomantis carolina, Euborellia annulipes, Gryllus head morphology of Grylloblattodea (Insecta) and the systematic position of the order, with a new nomenclature for the head domesticus, Dissosteira carolina, Zorotypus hubbardi, muscles of Dicondylia. Syst. Entomol. 1, 10–20. Embia sp., Timema nevadense, Megacrania tsudai, Wipfler, B., Pohl, H., Predel, R., 2012a. Two new genera and two Stenopsocus stigmaticus, Phloeothrips coreaceus, Pal- new species of Mantophasmatodea (Insecta, Polyneoptera) from + Namibia. ZooKeys, 75, 75–98. pares libelluloides, and Macroxyela ferruginea. , mus- Wipfler, B., Wieland, F., DeCarlo, F., Hornschemeyer,€ T., 2012b. cle present; À, muscle absent; ?, no information or Cephalic morphology of Hymenopus coronatus (Insecta: unclear homology. Mantodea) and its phylogenetic implications. Arthropod Struct. Appendix S3. Data matrix in nexus format. Dev. 41, 87–100. Wittig, G., 1955. Untersuchungen am Thorax von Perla abdominalis Appendix S4. Character discussion. Burm. (Larve und Imago) unter besonderer Berucksichtigung€ des Appendix S5. Source of information for each charac- peripheren Nervensystems und der Sinnesorgane. Zool. Jahrb. ter. Anat. Onto. 74, 491–570.