The Function and Phylogenetic Implications of the Tentorium in Adult Neuroptera (Insecta)

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Arthropod Structure & Development 40 (2011) 571e582

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Arthropod Structure & Development

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The function and phylogenetic implications of the tentorium in adult Neuroptera (Insecta)

Dominique Zimmermann a,*, Susanne Randolf a, Brian D. Metscher b, Ulrike Aspöck a a Natural History Museum, 2nd Zoological Department, Burgring 7, 1010 Vienna, Austria b Department of Theoretical Biology, University of Vienna, Althanstrasse 14, 1090 Wien, Austria article info abstract

Article history: Despite several recent analyses on the phylogeny of Neuroptera some questions still remain to be Received 11 April 2011 answered. In the present analysis we address these questions by exploring a hitherto unexplored Accepted 12 June 2011 character complex: the tentorium, the internal cuticular support structure of the insect head. We described in detail the tentoria of representatives of all extant neuropteran families and the muscles Keywords: originating on the tentorium using 3D microCT images and analyzed differences in combination with Neuroptera a large published matrix based on larval characters. We ﬁnd that the tentorium and associated Tentorium musculature are a source of phylogenetically informative characters. The addition of the tentorial Musculature Phylogeny characters to the larval matrix causes a basad shift of the Sisyridae and clearly supports a clade of all Function Neuroptera except Sisyridae and Nevrorthidae. A sister group relationship of Coniopterygidae and the Laminatentorium dilarid clade is further corroborated. A general trend toward a reduction of the dorsal tentorial arms and the development of laminatentoria is observed. In addition to the phylogenetic analysis, a correlation among the feeding habits, the development of the maxillary muscles, and the laminatentoria is demonstrated. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction In light of previous work we concentrate on the following three points: Neuroptera are a small but biologically and morphologically highly heterogeneous insect order. Together with Megaloptera and 1. The monophyly of Hemerobiiformia. The Hemerobiiformia, Raphidioptera, they constitute the superorder Neuropterida, which comprising 12 families, were recognized as “hemerobioid larval is one of the oldest holometabolan lineages (Aspöck et al., 2001; type” by MacLeod (1964) on the basis of features of the larval Grimaldi and Engel, 2005). In recent years many analyses have head anatomy. In a holomorphological cladistic analysis per- been conducted to illuminate the phylogeny of the Neuroptera, formed by Aspöck et al. (2001) these features were interpreted including molecular (Haring and Aspöck, 2004) morphological, as synapomorphies of this suborder. In a molecular analysis by (Aspöck et al., 2001; Aspöck and Aspöck, 2008; Beutel et al., 2010b, Haring and Aspöck (2004) the clade Hemerobiiformia was 2010a), and combined analyses (Winterton et al., 2010; review and fractured as the Sisyridae and the Osmylidae emerged as two documentation: Aspöck and Aspöck, 2010). In short, two contro- separate branches, a view also held in an analysis based on versial approaches have been conveyed: the triple suborder concept genital sclerites by Aspöck and Aspöck (2008). Keeping things with Neuroptera comprising three monophyletic suborders, Nev- complicated, Beutel et al. (2010b) re-established the mono- rorthiformia, Myrmeleontiformia, and Hemerobiiformia; and Hem- phylum Hemerobiiformia. erobiiformia as paraphyletic with respect to Myrmeleontiformia. 2. The position of the Sisyridae. Traditionally, the Sisyridae are Within Hemerobiiformia the Sisyridae and the Coniopterygidae part of the Hemerobiiformia. The position as sister group of the compete for the most conﬂicting status. Coniopterygidae in Aspöck et al. (2001) is strongly affected by the incorrect interpretation of the enlarged labial palps which are, in fact, an independent acquisition (Zimmermann et al., 2009). In the molecular analysis of Haring and Aspöck (2004) * þ þ Corresponding author. Tel.: 1 43 1 521 77 316; fax: 1 43 1 521 77 302. the Sisyridae emerge as sister group of all others except E-mail addresses: [email protected] (D. Zimmermann), [email protected] (S. Randolf), [email protected] Nevrorthidae, and the same relationships are hypothesized by (B.D. Metscher), [email protected] (U. Aspöck). Aspöck and Aspöck (2008). The molecular-morphological

combined analysis of Winterton et al. (2010) results in combination with a previously published matrix based on larval Coniopterygidae þ [(Osmylidae þ (Sisyridae þ Nevrorthidae)) þ characters (Beutel et al., 2010b). In addition, we discuss evolu- (all other families)]. tionary changes of the tentorium in the light of their adaptive value 3. The position of the Coniopterygidae. Due to reductions corre- and their correlation with feeding habits. lated with their small body size the Coniopterygidae are prob- ably the most difficult neuropteran family to classify. They have 2. Material and methods taken various positions: as sister group of all other Neuroptera (Withycombe, 1925; Winterton et al., 2010), as sister group of 2.1. Specimens examined the Sisyridae (Aspöck et al., 2001), as sister group of the Dilar- idae (possibly a result of a long-branch attraction; Haring and At least one species from each of the 17 neuropteran families Aspöck, 2004), and finally as sister group of the whole dilarid was examined. A detailed list of the material is given in Table 1.As clade (Aspöck and Aspöck, 2008; Beutel et al., 2010b). far as possible the same species as in the larval analysis by Beutel et al. (2010b) were used. Otherwise the availability of fresh mate- In the present study we address these questions by exploring rial determined our species choice. a hitherto unexplored character complex in Neuroptera: the ten- For morphological investigations the specimens were killed in torium, the inner skeleton of the insect head (Fig. 1). The tentorium 90% alcohol, fixed in alcoholic Bouin’s fluid for 3 h, and stained in 1% serves as a stabilizing element and muscle attachment structure for iodine-ethanol solution overnight. The specimens were microCT muscles of the antennae, pharynx, maxillae, labial and hypopha- scanned in 95% alcohol. The specimens are vouchered in the ryngeal muscles which are also considered in the course of this Natural History Museum Vienna. study. The tentorium is formed by the intersegmental invagination of the head capsule at two anterior and two posterior points which 2.2. Imaging and 3D-Reconstruction are externally visible as anterior and posterior tentorial pits. The anterior and posterior tentorial arms converge to the tentorial The specimens were imaged with an Xradia MicroXCT x-ray bridge. Dorsal tentorial arms are evaginations of the anterior ten- microtomography system (University of Vienna, Department of torial arms (Snodgrass, 1928, 1935). They are connected to the head Theoretical Biology) with a tungsten or rhodium source at 40- capsule by fibrillae in the antennal area (Staniczek, 2001). 80kVp and 4-8W and images were reconstructed using the soft- The tentorium has been studied in adults and larvae of various ware provided with the microCT system. The microCT data were insect taxa and has provided informative characters for recon- reconstructed with 2 2 pixel binning to reduce noise and file size, structing phylogenies for certain taxa (e.g. Klass and Eulitz, 2007; and reconstructed volume images were exported as TIFF image Koch, 2000; Symmons, 1950). In Neuroptera the tentoria and its stacks. The software Amira 5.1 was used for 3D-visualization and muscles have thus far only been described for a few species in the analysis of the data. MicroCT image stacks and histological sections scope of morphological investigations of the adult head (Ferris, are deposited in the Natural History Museum Vienna. 1940; Korn, 1943; Morse, 1931; Beutel et al., 2010a), but were The classification of musculature follows von Kéler (1963). never analyzed further. The aim of our study is to elucidate the tentorial complex in an 2.3. Cladistic analysis evolutionary and phylogenetic context and to help clarify the classification of the Neuroptera and the Neuropterida. To this end For the phylogenetic analysis we only scored differences that we have used 3D microtomographic (microCT) images to obtain could be defined as qualitative characters, in order to minimize detailed morphological data for adult tentoria and associated subjectivity. As these characters alone are insufficient to infer musculature in all 17 Neuropteran families. In order to test the a phylogeny, we added them to a large published matrix based phylogenetic value of the tentorial characters we analyzed them in on larval characters (Beutel et al., 2010b). We chose the study of

Fig. 1. Scheme of neuropteran head with tentorium. Abbreviations: af - antennal foramen, ata - anterior tentorial arm, atp - anterior tentorial pit, dta - dorsal tentorial arm, lt - laminatentorium, pta - posterior tentorial arm, tb - tentorial bridge. D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582 573

Table 1 List of taxa examined.

Family Species Sex Method Ascalaphidae Libelloides macaronius (Scopoli, 1763) ? microCT Ascalaphidae Libelloides coccajus (Denis & Schiffermüller, 1775) ? dissection Berothidae Podallea vasseana (Navás, 1910) male microCT, histological sections Chrysopidae Chrysoperla carnea (Stephens, 1836) female microCT, histological sections Chrysopidae Chrysopa dorsalis Burmeister, 1839 female microCT, histological sections Coniopterygidae Coniopteryx sp. female microCT, histological sections Dilaridae Nallachius sp. male microCT Hemerobiidae Micromus variegatus (Fabricius, 1793) female microCT, histological sections Hemerobiide Hemerobius humulinus Linnaeus, 1758 male microCT Ithonidae Ithone sp. ? microCT, histological sections Mantispidae Mantispa styriaca (Poda, 1761) male microCT, histological sections Myrmeleontidae Myrmeleon hyalinus distinguendus Rambur, 1842 male microCT, histological sections Nemopteridae Nemoptera sinuata Olivier, 1811 male microCT, histological sections Nevrorthidae Nevrorthus apatelios Aspöck et al., 1977 female microCT, histological sections Nymphidae Nymphes sp. female microCT, histological sections Osmylidae Osmylus fulvicephalus (Scopoli, 1763) ? microCT Polystoechotidae Polystoechotes punctata (Fabricius, 1793) male microCT, histological sections Psychopsidae Psychopsis sp. female microCT, histological sections Rhachiberothidae Mucroberotha vesicaria Tjeder, 1968 male microCT, histological sections Sisyridae Sisyra nigra (Retzius, 1783) ? microCT, histological sections Sisyridae Sisyra terminalis Curtis, 1854 female histological sections

Beutel et al. (2010b) for two main reasons: 1) it is the largest 3.1.2. Sisyra nigra (Sisyridae) (Fig. 2A) morphological data set of Neuroptera analyzed to date and 2) it is The anterior tentorial arms converge continuously toward the the data set with the largest congruence of taxa at the genus level tentorial bridge and are slightly flattened dorsoventrally at the with respect to the present study. We avoided chimeric data and anterior ends. All parts of the tentorium are well sclerotized and analyzed only the genera that were present in the original larval hollow tubes, but the lumen is very small in the anterior flat- matrix, thus excluding the tentorial characters of the family Ber- tened part of the anterior arms. Laminatentoria are absent. Dorsal othidae (Podallea in our analysis, Lomamyia in Beutel et al., 2010b). tentorial arms are well developed. The tentorial bridge is rather Furthermore we did not add data for the following taxa considered broad and distinctly long. It is located right behind the dorsal in Beutel et al. (2010b): Helicoconis (Coniopterygidae), Pterocroce tentorial arms. (Nemopteridae), Croce (Nemopteridae), as well as for the outgroups Neohermes (Corydalidae), Ochtebius (Coleoptera) and Catops 3.1.3. Nevrorthus apatelios (Nevrorthidae) (Coleoptera). The tentorium of Nevrorthus resembles very much the tento- The cladistic analyses were performed with TNT (Goloboff et al., rium of Sisyra with the difference that in Nevrorthus the tentorial 2008). Multistate characters were treated as non-additive. The bridge is distinctly shorter and the anterior arms are round parsimony analysis with equal weights, the analyses with implied throughout and not flattened in the anterior part as in Sisyra. weights (Goloboff, 1993) and the bootstrap value calculations were conducted by exhaustive search. Bremer support values were 3.1.4. Ithone sp. (Ithonidae) calculated with heuristic search (100,000 replications, 1000 TBR The anterior tentorial arms are distinctly widened and curved branch swapping replications). For character optimization and for outwards near the anterior tentorial pits and otherwise rather slim the calculation of the retention indices Winclada (Nixon, 2002) and evenly converging toward the tentorial bridge. Laminatentoria was used. are present. Dorsal tentorial arms are weakly developed. The tentorial bridge is very broad and long. 3. Results 3.1.5. Polystoechotes sp. (Polystoechotidae) 3.1. Description of the tentoria and the attaching muscles The anterior tentorial arms converge on the anterior half and are nearly straight toward the tentorial bridge. Dorsal tentorial arms 3.1.1. General structure of the Neuropteran tentorium (Fig. 1) are weakly developed. Laminatentoria are present and located The tentorium takes diverse shapes within Neuroptera. It always rather posteriorly. The tentorial bridge is moderately broad and consists of long anterior tentorial arms (ata), strong and short moderately long. posterior tentorial arms (pta), and a tentorial bridge (tb) that varies in width and length. Dorsal tentorial arms (dta) can be present, and 3.1.6. Osmylus fulvicephalus (Osmylidae) if so, antennal muscles (M1, M4) and the pharyngeal muscle M. The anterior tentorial arms converge uniformly toward the tentoriobuccalis lateralis (M49) can originate on them. Another tentorial bridge. They are distinctly flattened and broadened structure present in various lineages is the laminatentorium (lt) at dorsoventrally in the anterior half, forming indistinct shallow the median region of the anterior tentorial arms. It serves as laminatentoria at about midlength. The dorsal tentorial arms are attachment area of the maxillary muscles M17 and M18 and may located above the laminatentoria at about midlength of the also serve as attachment area of antennal muscles. The anterior, anterior tentorial arms. The tentorial bridge is moderately broad posterior, and dorsal arms, as well as the tentorial bridge are and long. O. fulvicephalus is described in detail in Beutel et al. tubular, hollow throughout. In posterior region of the anterior arms (2010a). there is often an area where the dorsal part of the tube is extremely thin. 3.1.7. Chrysopa dorsalis (Chrysopidae) An overview of the musculature originating on the tentorium is The anterior tentorial arms converge toward the laminatentoria given in Table 2. and are bowed outward between the laminatentoria and the Table 2 List of origin, attachment and function of muscles originating on the tentorium in Neuroptera.

# (Kéler, Muscle Sisyra Nevrorthus Ithone Polystoechotes Osmylus Chrysopa Chrysoperla Hemerobius Micromus Psychopsis Nemoptera Nymphes Libelloides Myrmeleon Coniopteryx Mantispa Nallachius Mucroberotha Podallea Insertion Function 1963) M1 M. tentorioscapalis ata þ ata þ dta ata þ ata þ dta ata þ ata ata ata þ dta ata ata þ dta ata ata ata ata ata ata ata ata ata basal margin of depressor and anterior dta dta dta scapus flexor of antenna M2 M. tentorioscapalis ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata posterior basal levator of posterior margin of scapus antenna M3 M. tentorioscapalis absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent depressor and lateralis flexor of antenna M4 M. tentorioscapalis dta dta dta dta dta dta dta dta ata dta ata dta ata dta ata ata ata ata ata mesal basal depressor and medialis margin of scapus extensor of antenna M14a M. zygomaticus ata ata ata ata ata ata ata ata ata absent ata ? ata ata ata absent ata ata ata dorsal inner adductor and mandibulae surface of the proprioreceptor mandibular base of mandible M14b M. zygomaticus ata ata ata ata ata ata absent ata ata ata ata ? ata ata ata ata ata absent absent ventral inner adductor and mandibulae surface of the proprioreceptor mandibular base of mandible M14c M. zygomaticus absent absent ata ata ata ata ata ata ata ata ata ? ata ata absent ata absent ata ata primary adductor and mandibulae mandibular joint proprioreceptor of mandible M17 M. tentoriocardinalis tb tb ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata cardo, near flexor of maxilla cardostipital ata þ lt ata þ lt ata ata ata ata ata mesalsuture margin adductor, levator M18 M. tentoriostipitalis ata þ ata ata þ ata þ lt ata þ ata þ lt ata þ lt ata ata þ lt ata þ lt ata ata þ lt of stipes and retractor pta lt tb of stipes M29 M. tentoriopraementalis occiput pta pta pta pta pta pta pta pta pta absent occiput occiput pta occiput occiput absent? occiput occiput posterior outer adductor of inferior angles praementum of prementum M30 M. tentoriopraementalis absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent absent adducto rof superior prementum M41a M. frontohypopharyngalis frons frons frons frons frons frons frons frons frons frons frons frons frons frons frons frons frons frons frons apex of lateral dilator suspensorial of anatomical sclerites mouth M41b M. frontohypopharyngalis ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata ata apex of lateral dilator suspensorial of anatomical sclerites mouth M42 M. tentoriohypopharyngalis occiput pta pta pta pta pta pta occiput occiput tb-pta absent occiput occiput pta occiput occiput absent occiput occiput lateral wall of adductor of salivarium hypopharynx M48 M. tentoriobuccalis tb tb tb tb tb tb tb tb tb tb tb tb tb tb tb tb tb tb tb ventral buccal ventral dilator of anterior wall anterior pharynx M49 M. tentoriobuccalis dta dta absent absent dta absent absent absent absent absent absent absent absent absent absent absent absent absent absent lateral buccal lateral dilator of lateralis wall anterior pharynx M50 M. tentoriobuccalis tb-pta tb-pta tb tb ata tb tb tb tb tb absent? tb ata tb ata-tb tb tb ata tb ventral ventral dilator of posterior pharyngeal wall anterior pharynx M52 M. tentoriopharyngalis pta pta-occ pta pta pta occiput occiput tb tb-pta tb pta tb-pta absent? occiput absent? ata-tb ? pta ata ventral dilator of postcerebral postcerebral pharyngeal wall pharynx D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582 575 tentorial bridge. Thin dorsal tentorial arms are located at the laminatentoria are located rather anteriorly. Dorsal tentorial arms posterior end of the laminatentoria which are around midlength of are absent. the anterior tentorial arms. The tentorial bridge is moderately broad and very short. It has a small median process on its anterior 3.1.17. Mantispa styriaca (Mantispidae) (Fig. 2E, 3B) margin. The anterior tentorial arms converge uniformly between the anterior pits and the laminatentoria and then continue straight and 3.1.8. Chrysoperla carnea (Chrysopidae) (Fig. 2B) slim to the tentorial bridge. Prominent quadrangular lami- Tentorium and muscles as in C. dorsalis with the only difference natentoria are located around midlength. Dorsal tentorial arms are that the M14 is tripartite in C. carnea. absent. The tentorial bridge is moderately long and broad.

3.1.9. Hemerobius humulinus (Hemerobiidae) (Fig. 3A) 3.1.18. Nallachius sp. (Dilaridae) (Fig. 2F) The anterior tentorial arms converge towards the lami- The anterior tentorial arms converge uniformly between the natentoria and are slightly bowed outward between them and the anterior tentorial pits and the tentorial bridge. Laminatentoria tentorial bridge. The anterior tentorial arms are constricted at the and dorsal tentorial arms are absent. The tentorial bridge is pits. The laminatentoria are distinctly pointed and located rather extraordinarily narrow and long. anteriorly. Short dorsal tentorial arms are located midway between the laminatentoria and the tentorial bridge. The tentorial bridge is 3.1.19. Mucroberotha sp. (Rhachiberothidae) moderately broad and long. It has a small median process on its The anterior tentorial arms converge uniformly from the ante- anterior margin. rior tentorial pits to the tentorial bridge. Dorsal tentorial arms are absent. Prominent trapezoid laminatentoria are located around 3.1.10. Micromus variegatus (Hemerobiidae) midlength. The tentorial bridge is narrow and moderately long. The anterior tentorial arms converge towards the laminatentoria and are bowed outward between the laminatentoria and 3.1.20. Podallea vasseana (Berothidae) the tentorial bridge. The laminatentoria are triangular and located The anterior tentorial arms converge uniformly between the rather anteriorly. Dorsal tentorial arms are absent. The tentorial anterior tentorial pits and the tentorial bridge. Dorsal tentorial bridge is moderately broad and rather short. It has a distinct arms are absent. The laminatentoria are small and roundish and are median process on its anterior margin. located rather anteriorly. The tentorial bridge is narrow and moderately long. 3.1.11. Psychopsis sp. (Psychopsidae) The anterior tentorial arms converge strongly between the anterior tentorial pits and the laminatentoria and then continue 3.2. Characters analyzed straight to the tentorial bridge. Prominent laminatentoria are located around midlength. Dorsal tentorial arms are weakly 3.2.1. Dorsal tentorial arms: present (0) (Fig. 2AeC), absent (1) developed. The tentorial bridge is broad and moderately short. (Fig. 2DeF) [character 65] Dorsal tentorial arms are a groundplan feature of the Ptery- 3.1.12. Nemoptera sinuata (Nemopteridae) gota and are present in most orders (Beutel and Vilhelmsen, The tentorium is distinctly U-shaped with straight stick-like 2007: Hymenoptera; Hörnschemeyer et al., 2002: Coleoptera; anterior tentorial arms and an extremely broad and short tento- Beutel et al., 2009: Mecoptera), but also often reduced within the rial bridge. Prominent quadrangular laminatentoria are located orders (Beutel et al., 2009). Within the Neuropterida dorsal rather anteriorly. Dorsal tentorial arms are absent, only a very thin tentorial arms are present in Raphidioptera (Achtelig, 1967: lamina protrudes from the anterior tentorial arms where the dorsal Raphidia flavipes), in Megaloptera (Röber, 1942: Sialis flavilatera; tentorial arms origin in the other families. Maki, 1936: Chauliodes), and in all neuropteran families except members of the dilarid clade (Mantispa, Podallea, Dilar, Nalla- 3.1.13. Nymphes sp. (Nymphidae) chius, Mucroberotha), Coniopteryx, Libelloides and Micromus.Itis The anterior tentorial arms converge between the anterior notable that within Hemerobiidae both character states occur: in tentorial pits and the laminatentoria and then continue straight to contrast to H. humulinus the dorsal tentorial arms are absent in the tentorial bridge. Laminatentoria are located around midlength. M. variegatus. This conforms to the more specialized male genital Dorsal tentorial arms are weakly developed. The tentorial bridge is sclerites of Micromus (pers. obs. Aspöck). Dezaly (1960) describes broad and short. short dorsal tentorial arms for Libelloides coccajus. We could not find any dorsal tentorial arms in L. coccajus and interpret the 3.1.14. Libelloides macaronius (Ascalaphidae) structure described by Dezaly as a laminatentorium (compare The anterior tentorial arms are plate-like and converge strongly Dezaly, 1960, Fig. 7). on the anterior two thirds, whereas they are roundish and converge moderately on the posterior third. The anterior tentorial pits are 3.2.2. Dorsal tentorial arms: well developed (0) (Fig. 2A), weakly closed. Dorsal tentorial arms are absent. Laminatentoria are small. developed (1) (Fig. 2B, C) [character 66] The tentorial bridge is broad and short. If present, dorsal tentorial arms can be differently developed: They are long and thick in Nevrorthus and Sisyra, short and thick in 3.1.15. Myrmeleon hyalinus (Myrmeleontidae) (Fig. 2C, Fig. 4B) Hemerobius and weakly developed in Osmylus, Chrysopa, Chrys- The tentorium resembles very much the tentorium of Libelloides operla, Ithone, Polystoechotes, Nymphes, Psychopsis and Myrmeleon macaronius with the difference that in M. hyalinus thin dorsal (Myrmeleon: in contrast to Sundermeier (1940) who described no tentorial arms are present. dorsal tentorial arms for Myrmeleon europaeus ). In Nemoptera the thin epidermis that protrudes from the anterior tentorial arm is 3.1.16. Coniopteryx sp. (Coniopterygidae) (Fig. 2D) understood as relic of a dorsal tentorial arm. The dorsal tentorial The tentorium is distinctly U-shaped with straight stick-like arms are connected with fibrillae to the anterolateral head capsule anterior arms and an extremely broad and short bridge. Small near the ocular diaphragm. 576 D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582

Fig. 2. 3-dimensional reconstructions of tentoria in sublateral view. A: Sisyra nigra,B:Chrysoperla carnea,C:Myrmeleon hyalinus,D:Coniopteryx sp., E: Mantispa styriaca, F: Nallachius sp.

3.2.3. Laminatentorium: absent (0) (Fig. 2A, 2F), present (1) Chauliodes (Maki, 1936) Raphidia (Achtelig, 1967) and Sialis (Röber, (Fig. 2BeE) [character 67] 1942), but present in Trachypachus (Dressler and Beutel, 2010). Median extensions of the tentorium occur in various hexapod When present, the maxillary muscles (M17 þ M18), and in Nemo- orders (Comstock and Kochi, 1902: Blattodea; Beutel et al., 2003: ptera and Coniopteryx also antennal muscles attach on it. Hydraenidae, Coleoptera; Hoke, 1923: Plecoptera; Klass and Eulitz, 2007: Dictyoptera and Mantophasmatodaea; Yuasa, 1920: Ortho- 3.2.4. Anteriorly projecting median process on the tentorial bridge: ptera, Blattodea, Mantodea; Hudson, 1945: Blattidae, Mantidae). A absent (0), present (1) [character 68] laminatentorium is present in all studied specimens except Nev- In both investigated species of Hemerobiidae and Chrysopidae rorthus, Sisyra, Nallachius and Dilar. It is absent in the outgroups there is an anteriorly projecting median process on the tentorial D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582 577

Fig. 3. 3-dimensional reconstruction of the tentorium, the antennal muscles and the maxillary muscles in Hemerobius humulinus (A) and Mantispa styriaca (B). Abbreviations: ata e anterior tentorial arm, atp e anterior tentorial pit, dta e dorsal tentorial arm, pta e posterior tentorial arm, tb e tentorial bridge, 1 - M. tentorioscapalis anterior, 2 - M. tentorioscapalis posterior, 4 - M. tentorioscapalis medialis, 17 - M. tentoriocardinalis, 18 - M. tentoriostipitalis. bridge. Oswald (1993) already described this process as “sagittal arm whereas in Sialis (Röber, 1942) and all Neuroptera it originates tentorial process” for Notiobiella multifurcata (Hemerobiidae). only on the anterior tentorial arm.

3.2.5. Anterior tentorial pits: open (0) (Fig. 2A), slit-like constricted 3.2.9. Origin of M. tentorioscapalis medialis (M4): dta (0) (Fig. 3A), (1), trumpet-like (2), closed (3) (Fig. 2C) [character 69] ata (1) (Fig. 3B) [character 73] The anterior tentorial pits are constricted in both hemerobiid When dorsal arms are present, the antennal muscle M. tento- species and widened like a trumpet in Ithone and Polystoechotes.In rioscapalis medialis (M4) originates on them with the exception of Libelloides and Myrmeleon the anterior tentorial pits are closed Nemoptera where a relic of the dorsal tentorial arm is recognizable while the rest of the anterior tentorial arm is a hollow tube (as in all in histological section but no muscle originates on it. The M4 also other Neuroptera). This is a remarkable and apparently unique originates on the dorsal tentorial arms in the outgroups Chauliodes feature of these two families e we found no indications of such (Maki, 1936) and Raphidia (Achtelig, 1967). [We do not agree with tentorial pits in other insects. the homologization of M4 (Maki, 1936) with M2 (von Kéler, 1963) and M5 (Maki, 1936) with M4 (von Kéler, 1963)byAchtelig (1967), 3.2.6. Anterior tentorial arms: cylindrical tubes (0) (Fig. 2A, B, DeF), but we assume M4 (Maki, 1936) to be homologous with M4 (von ﬂat hollow plates (1) (Fig. 2C) [character 70] Kéler, 1963) and M5 (Maki, 1936) with M2 (von Kéler, 1963)]. The anterior tentorial arms are cylindrical tubes in most studied Neuroptera, they are ﬂat hollow plates in Myrmeleon and Libelloides. 3.2.10. M. zygomaticus mandibulae, component a: present (0), absent (1) [character 74] 3.2.7. Origin of M. tentorioscapalis anterior (M1): ata (0) (Fig. 3B), This and the following characters are three bundles of one ata þ dta (1) (Fig. 3A) [character 71] muscle, the M. zygomaticus mandibulae. As these bundles have The shift of the M1 dorsal tentorial arm-component to the three separate attachment points, it is generally questionable to anterior tentorial arm in Neuroptera seems to be correlated with the summarize them as one muscle. The M. zygomaticus mandibulae is reduction of the dorsal tentorial arm: In the two families with a well not mentioned in the descriptions of the outgroups with exception developed dorsal tentorial arm (Nevrorthus, Sisyra) and in a part of of Trachypachus, where it is absent (Dressler and Beutel, 2010). The the families with a weakly developed dorsal tentorial arm (Osmylus, component “a” originates at the dorsal inner surface of the Hemerobius, Ithone, Polystoechotes, Psychopsis) the M1 originates on mandibular base. It is absent in Mantispa and Psychopsis. In litera- the anterior and the dorsal tentorial arm. And in the other part with ture the same muscle is also described as as M. hypopharyngo- a weakly developed dorsal tentorial arm and in the families with no mandibularis (M13) (e.g. Beutel and Friedrich, 2008: Neohermes; dorsal tentorial arm it originates again solely on the anterior tento- Beutel and Baum, 2008: Nannochorista). rial arm. However, in the outgroups Raphidia (Achtelig, 1967), Chauliodes (Maki, 1936) and Sialis (Röber, 1942), the M1 originates 3.2.11. M. zygomaticus mandibulae, component b: present (0), solely on the anterior tentorial arm, although a dorsal tentorial arm is absent (1) [character 75] present. A shift of the antennal muscles from the dorsal tentorial arm The component “b” originates at the ventral inner surface of the tothe anterior tentorial armwithout reduction of the dorsal tentorial mandibular base. It is absent in Chrysopa, Podallea and Mucroberotha. arm occurs also in Phasmatodea: While in Carausius morosus (Lochodinae) the muscles M1 and M2 originate on the dorsal ten- 3.2.12. M. zygomaticus mandibulae, component c: present (0), torial arms (Scholl,1969), in Phryganistria virgea (Clitumninae) these absent (1) [character 76] muscles origin on the anterior tentorial arm while the dorsal ten- The component “c” originates at the primary mandibular joint. It torial arm serve exclusively as stabilizing element (Strenger, 1942). is absent in Nevrorthus, Sisyra, Nallachius and Coniopteryx. This is contradictory to our results as we have no specimen with a dorsal tentorial arm and no muscle originating there. 3.2.13. Origin M. tentoriocardinalis (M17): pta (0), tb (1), ata (2) (Fig. 3A,B) [character 77] 3.2.8. Origin of M. tentorioscapalis lateralis (M2): ata þ dta (0), ata The M17 originates on the posterior tentorial arms in Raphidia (1) (Fig. 3A, B) [character 72] (Achtelig, 1967), on the tentorial bridge in Chauliodes (Maki, 1936), The M2 originates in Raphidia (Achtelig, 1967) and Chauliodes Sialis (Röber, 1942), Nevrorthus and Sisyra and on the anterior ten- (Maki, 1936) on the anterior tentorial arm and the dorsal tentorial torial arm in all other Neuroptera. 578 D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582

3.2.14. Anterior component of M. tentoriostipitalis (M18): present of relationships on higher phylogenetic level, parallel evolution (0) (Fig. 3A), absent (1) [character 78] does deﬁnitely occur. In particular, reductions are likely to evolve In the outgroup Trachypachus (Dressler and Beutel, 2010) and in independently (e.g. Beutel, 1995; Dressler and Beutel, 2010), and Neuroptera an anterior component of the M18 is present. The structures such as the laminatentorium are unlikely to be actual anterior component is absent in Sialis (Röber, 1942) and in Raphidia homologs throughout the insect orders. (Achtelig, 1967) which might be correlated with the prognathous In the present analysis the higher retention index of the struc- head of Raphidioptera and Megaloptera. tural characters in comparison to the muscular characters (Table S2) suggests that the tentorium contains more information 3.2.15. Posterior component of M. tentoriostipitalis (M18): present for a phylogeny on family level than the muscles. A sound analysis (0) (Fig. 3A), absent (1) (Fig. 3B) [character 79] of the consistency of different character groups on higher phylo- The posterior component of M18 is absent in Nevrorthus, Ithone, genetic level, as has been done for echinoids (Coppard et al., 2010), Nemoptera, Coniopteryx, and the members of the dilarid clade, and is highly desirable but would necessitate broader taxon sampling. present in the remaining neuropteran families. However, in our analysis both the tentorium and its muscles do contribute informative characters to the resolution of neuropteran 3.2.16. Origin of the posterior component of M. tentoriostipitalis phylogeny on family level. Returning to the three major open (M18A): pta (0), tb (1), ata (2) [character 80] questions in the phylogeny of Neuroptera (see 1.1), the tentorial The M18A originates on the postgular ridge in Trachypachus characters are inconclusive in the case of Hemerobiiformia but do (Dressler and Beutel, 2010), on the posterior tentorial arm in the help to place the two critical taxa Sisyridae and Coniopterygidae. outgroup Raphidia (Achtelig, 1967), and in Sisyra, Hemerobius, Micromus, Chrysopa, Chrysoperla, Psychopsis, and Nymphes, and on 4.1.1. Hemerobiiformia the transition of the tentorial bridge and the anterior tentorial arm In our analysis we retrieve paraphyletic Hemerobiiformia with in the outgroup Sialis (Röber, 1942), as well as in the neuropteran respect to Myrmeleontiformia, because the Myrmeleontiformia families Polystoechotes, Osmylus, Myrmeleon, Ascalaphus. emerge as a sister group of the dilarid clade þ Coniopterygidae. However, the support for this sister group relationship is rather 3.2.17. M. frontohypopharyngalis (M41): non-partitioned (0), weak and lacks plausibility: the presence of 5 larval corneae (Fig. 4, bipartite (1) [character 81] 16.2) is retrieved as synapomorphy, although 5 corneae are only In the outgroups (Maki, 1936: Chauliodes; Röber, 1942: Sialis; present in Coniopterygidae (Coniopteryx, Helicoconis) and Psych- Achtelig, 1967: Raphidia) the M41 is non-partitioned, whereas in all opsis. In Nemopteridae (Nemoptera, Croce, Pterocroce), Myrmeleon, Neuroptera it is bipartite. It is notable that in neuropterid larvae the Libelloides and Nymphes (partim) seven corneae appear as the M41 is bipartite in Raphidia (Beutel and Ge, 2008) and even derived state. This is inconsistent with the current view that seven tripartite in Nevrorthus (Beutel et al., 2010b). corneae are the plesiomorphic state within Neuroptera (Paulus, 1986; Aspöck, 1995). Additionally, within the dilarid clade three 3.2.18. M. tentoriobuccalis lateralis (M49): present (0), absent (1) more states occur (2, 1, or no corneae). Altogether, the number of [character 82] corneae provides weak support for Myrmeleontiformia þ (dilarid The M49 is present in Chauliodes (Maki, 1936), Sisyra, Nevrorthus clade þ Coniopterygidae). and Osmylus. It is not mentioned for Sialis (Röber, 1942), and absent The other synapomorphy concerns the antennal muscle M1 of the in Raphidia (Achtelig, 1967) and in all remaining neuropteran adults (Fig. 4 , 71.0) which is originating only on the anterior tentorial families. The loss of M49 is likely to be correlated with the reduc- arms in Myrmeleontiformia þ (dilarid clade þ Coniopterygidae) and tion of the dorsal tentorial arm. not on the anterior and dorsal tentorial arms. However, this is also the The character state matrix is given in Table S1, and the analyzed case in Chrysopa and in the outgroups Raphidia, Sialis, and Trachy- matrix including larval characters is provided in Nexus format in pachus. Without these synapomorphies the relationship between File S1. Myrmeleontiformia and Hemerobiiformia is unresolved, which admits once more the hypothesis of monophyletic Hemerobiiformia 3.3. Cladistic analysis (with exclusion of Sisyridae; for discussion see Aspöck, 1995; Beutel et al., 2010b). The combined cladistic analysis with equal weights of our tentorial characters and the larval characters from Beutel et al. 4.1.2. Position of the Sisyridae (2010b) results in two most parsimonious trees, each with While the Sisyridae appear deeply nested within monophyletic a length of 174 steps and a Consistency Index (CI) of 0.59. These two Hemerobiiformia in Beutel et al. (2010b), they are pushed to a very trees differ in the internal relationships of Nemopteridae basal position by adding the tentorial characters to the analysis. A (Nemoptera þ Pterocroce versus Croce þ Pterocroce). One of these rather basal position of the Sisyridae has been suggested already in two trees (Nemoptera þ Pterocroce) is also retrieved with implied previous works (Aspöck and Aspöck, 2008; Haring and Aspöck, weighting (K2-K6) and is thus our preferred tree (Fig. 4). The strict 2004), but in both of them Sisyridae emerge as sister group to all consensus tree with Bootstrap and Bremer support values is shown Neuroptera except Nevrorthidae. in Fig. 5. The retention indices (ri) for the 18 tentorial characters are The position of Sisyridae as sister group of all other Neuroptera given in Table S2. including Nevrorthidae is new in our tree. It is supported by two unique, although debatable synapomorphies of all neuropteran 4. Discussion families except Sisyridae: the larvae of all other families have a poison gland (Fig. 4, 53.1) and a poison channel in the maxillary 4.1. Phylogenetic importance stylet (Fig. 4, 37.1). It is most likely that these two characters were reduced secondarily in the aquatic larvae of Sisyridae, which use A number of studies have been performed on the tentorium in their piercing mouthparts to feed on freshwater sponges. The insects, and have made its phylogenetic relevance clear (e.g. Yuasa, possession of a poison gland and channel certainly was directly 1920; Symmons, 1950; Hudson, 1951; Koch, 2000; Klass and Eulitz, related to the evolution of the sucking tubes and thus belongs to the 2007). However, despite its undisputed value for the reconstruction ground pattern. The support for a very basal position of the D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582 579

Fig. 4. Combined analysis of tentorial characters and the larval characters of Beutel et al. (2010b): One of two most parsimonious trees produced by exhaustive search under equal weights with unambiguous character optimization (except for the basalmost branches where apomorphies are retrieved by comparison of fast and slow optimization). Branches without support were collapsed after analysis. An identical topology is retrieved with implied weights K2-K6. Black boxes indicate unique synapomorphies, white boxes homoplastic ones. Tentorial characters highlighted gray.

Sisyridae might thus be a result of secondary losses e basal Nev- complexes, have not been studied exhaustively and it cannot be rorthidae are retrieved at a cost of only one additional step. In excluded that they can also facilitate other functions than the addition, it should be considered that this very basal position of the resorption of water as an adaptation to dry habitats. Sisyridae would imply either the unlikely scenario of an independently evolved open head capsule in Sisyridae or a reversal of this 4.1.3. Position of the Coniopterygidae character in Nevrorthidae (Aspöck, 1992: “maxillary head”; Beutel As in the larval character analysis of Beutel et al. (2010b),our et al., 2010b). In any case, the support for monophyletic Neuroptera combined analysis retrieves a sister group relationship of Conio- without Sisyridae and Nevrorthidae is clearly enhanced by the pterygidae and the dilarid clade. The single larval synapomorphy addition of tentorial characters, speciﬁcally by the development of (Fig. 4, 28.2: larval sucking stylets with straight distal inner margin a laminatentorium (Fig. 4, 67.1), the weakening of the dorsal ten- and external margin concave) is endorsed by three e though torial arms (Fig. 4, 66.1), and the anterad shift of the M17 (Fig. 4, homoplastic e characters of the tentorium: the absence of dorsal 77.2) in the clade Neuroptera excluding Sisyridae and Nevrorthidae. tentorial arms (Fig. 4, 65.1, also in Libelloides and Micromus), the As a result of a basad shift of the Sisyridae, the evidence of their origin of the M4 on the anterior tentorial arm (Fig. 4, 73.1, also in larvae as being secondarily aquatic (Gaumont, 1976) becomes Nemoptera and Libelloides), and the absence of a posterior component further compromised: it is now at least equally parsimonious to of M18 (Fig. 4, 79.1, also in Nemoptera, Ithone and Nevrorthus). This assume that their aquatic larvae represent, together with the clade receives comparatively high bootstrap and bremer support aquatic larvae of Nevrorthidae, the groundplan of Neuroptera, as values (68/4). A sister group relationship of Coniopterygidae and the already hypothesized in Aspöck et al. (2001) and Aspöck (2002).We dilarid clade was suggested already by Aspöck and Aspöck (2008). interpret the presence of a cryptonephridic malpighian tubule as an Our result further weakens the hypothesis suggested in the independent adaptation to their feeding on freshwater sponges and recent combined analysis of Winterton et al. (2010) of a very basal Bryozoans (which was studied in detail by Weibmair, 2005). The position of the Coniopterygidae. A basal position is retrieved at excretory systems of insects, and particularly cryptonephridic a cost of 11 additional steps. 580 D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582

Fig. 5. Combined analysis of tentorial characters and the larval characters of Beutel et al. (2010b): Strict consensus of two most parsimonious trees produced by exhaustive search under equal weights with bootstrap values over 50% (above) and bremer support values (below, bold).

Finally we can confirm the presence of a tentorial component of maxillae, the prominence of the muscles M17 and M18 and the size the M. frontohypopharyngalis being a synapomorphy of Neuro- of the laminatentorium. In contrast to this we are confronted with ptera, as already hypothesized by Beutel et al. (2010a). predatory feeding behavior in Libelloides and Myrmeleon. In these families food uptake is carried out mainly with the mandibles, 4.2. Functional correlations which show unambiguous adaptations for the capture of fast-flying or well sclerotized insects (Stelzl, 1991). The maxillary muscles M17 The tentorium fulfills two main functional requirements in the and M18 are much less developed (measured as volume) and the insect head: it stabilizes the head capsule and it serves as a muscle laminatentorium is comparatively small. attachment structure. Both functions might influence the shape of However, less pronounced differences in the feeding habits are the tentorium. For example, Nemoptera are well studied highly primarily reflected in the mouthparts and not necessarily in the specialized pollen feeders (Popov, 2002; Krenn et al., 2008) with tentorium. Within the family Chrysopidae, the species C. carnea is a snout-like head and the most deviant tentorium: it is strictly U- palyno-glycophagous (pollen, honeydew, nectar) and C. dorsalis is shaped with very thin arms, a short bridge, and extremely large carnivorous, feeding mainly on aphids and other small arthropods laminatentoria. Krenn et al. (2008) demonstrated that the pollen (Canard, 2001). The differences in the feeding habits correspond to uptake is carried out mainly by fast pro- and retraction of the differences in the mandibles, which are larger and asymmetrical maxillae. The stipes is strongly elongated and the brush-shaped with a strong incisor on the left side used to capture and to hold lacinia functions as pollen collecting organ and forms a functional prey in C. dorsalis, and symmetrical without any incisor and unit with the galea and the maxillary palps. The other mouthparts, a spoon-like lacinia for the consumption of honeydew and pollen in the labium and the mandibles, play no major role in food uptake. C. carnea (Canard, 2001). In spite of these conspicuous changes in The laminatentoria e if present e serve as origin of the maxillary the mandibles, the tentoria are very similar. Specializations in the muscles M17 and M18 which are most prominent in Nemoptera. feeding habits thus seem to be reflected primarily in the mouth- Thus there seems to be a clear correlation between the use of the parts, and secondarily in the musculature and in the tentorium. D. Zimmermann et al. / Arthropod Structure & Development 40 (2011) 571e582 581

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