Cladistics

Cladistics 27 (2011) 341–355 10.1111/j.1096-0031.2010.00338.x

Morphological and molecular evidence converge upon a robust phylogeny of the megadiverse Holometabola

Rolf G. Beutela,*, Frank Friedrichb, Thomas Ho¨ rnschemeyerc, Hans Pohla, Frank Hu¨ nefelda, Felix Beckmannd, Rudolf Meiere, Bernhard Misof f, Michael F. Whitingg and Lars Vilhelmsenh aEntomology Group, Institut fu¨r Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, FSU Jena, Erbertstrasse 1, 07743 Jena, Germany; bBiozentrum Grindel und Zoologisches Museum, Martin-Luther-King-Platz 3, Universita¨t Hamburg, 20146 Hamburg, Germany; cInstitut fu¨r Zoologie und Anthropologie der Universita¨t, Berlinerstr. 28, 37073 Go¨ttingen, Germany; dInstitute for Materials Research GKSS-Research Center, c ⁄ o GKSS at DESY, Notkestr. 85, 22607 Hamburg, Germany; eDepartment of Biological Sciences, National University of Singapore, 14 Science Dr 4, Block S2 #02-01, Singapore 117543; fZoologisches Forschungsmuseum Alexander Ko¨nig, Abteilung Molekulare Biodiversita¨tsforschung, Adenauerallee 160, 53113 Bonn, Germany; gDepartment of Biology, 693 Widtsoe Building, Brigham Young University, Provo, UT 84602, USA; hNatural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, DK-2100, Denmark Accepted 26 June 2010

Abstract

We present the largest morphological character set ever compiled for Holometabola. This was made possible through an optimized acquisition of data. Based on our analyses and recently published hypotheses based on molecular data, we discuss higher- level phylogeny and evolutionary changes. We comment on the information content of different character systems and discuss the role of morphology in the age of phylogenomics. Microcomputer tomography in combination with other techniques proved highly efficient for acquiring and documenting morphological data. Detailed anatomical information (356 characters) is now available for 30 representatives of all holometabolan orders. A combination of traditional and novel techniques complemented each other and rapidly provided reliable data. In addition, our approach facilitates documenting the anatomy of model organisms. Our results show little congruence with studies based on rRNA, but confirm most clades retrieved in a recent study based on nuclear genes: Holometabola excluding , Coleopterida (= Strepsiptera + Coleoptera), Neuropterida excl. Neuroptera, and . Mecopterida (= Antliophora + ) was retrieved only in Bayesian analyses. All orders except Megaloptera are monophyletic. Problems in the analyses are caused by taxa with numerous autapomorphies and ⁄or inapplicable character states due to the loss of major structures (such as wings). Different factors have contributed to the evolutionary success of various holometabolan lineages. It is likely that good flying performance, the ability to occupy different habitats as larvae and adults, parasitism, liquid feeding, and co-evolution with flowering plants have played important roles. We argue that even in the ‘‘age of phylogenomics’’, comparative morphology will still play a vital role. In addition, morphology is essential for reconstructing major evolutionary transformations at the phenotypic level, for testing evolutionary scenarios, and for placing fossil taxa. Ó The Willi Hennig Society 2010.

Holometabola or are ‘‘the most suc- what is known today is doubtlessly only the tip of the cessful lineage of living organisms’’ according to Kris- iceberg. Erwin (1997) estimated 7.5 million extant tensen (1999a). They comprise ca. 780 000 described species for Coleoptera, based on investigations carried species (Grimaldi and Engel, 2005), which is equivalent out in the tropical forests of Central and South America, to more than 50% of the kingdom. Moreover, and a recent survey of dipteran diversity also provides evidence for a large number of undescribed species in most biogeographical regions (Pape et al., 2009). *Corresponding author: Holometabola also includes groups of almost unparal- E-mail address: [email protected] leled medical and economic importance (e.g. biting flies,

Ó The Willi Hennig Society 2010 342 R.G. Beutel et al. / Cladistics 27 (2011) 341–355 bees, leaf beetles, weevils). The most conspicuous Giribet et al., 2005; Meier and Lim, 2009; see also characteristic of the group as a whole, and possibly Wheeler, 2008) in order to make further progress in one reason for its unrivalled evolutionary success, is the inter-ordinal-level phylogenetics. occurrence of complete metamorphosis, with a pupal A comprehensive morphological data matrix focused stage preceding the adult (Cameron et al., 2009). specifically on the phylogeny of Holometabola has not The monophyly of Holometabola has never been previously been compiled. Our primary aim in this study seriously questioned. However, as pointed out by is to provide an extensive and well documented mor- Kristensen (1999a,b), convincing morphological aut- phological character set for a carefully chosen sample apomorphies are scarce. The interrelationships of the across all holometabolan orders and several outgroup orders are also far from being settled. Widely accepted taxa. Further aims are to describe how the acquisition of phylogenetic hypotheses, originating with Hennig (1969) anatomical data can be optimized, to compare mor- and thoroughly re-evaluated by Kristensen (e.g. 1981, phology-based phylogenies with those based on molec- 1999a; see also Beutel and Pohl, 2006), were challenged ular data, to evaluate the phylogenetic information by the results of molecular studies using different content of different subsets of data, and to discuss analytical approaches and data sets (Wheeler et al., possible factors that may have contributed to the 2001; Whiting, 2002a,b; Kjer, 2004; Savard et al., 2006; extreme diversification in holometabolan lineages. Cameron et al., 2009; Wiegmann et al., 2009). Argu- The work was carried out in the framework of a ably, morphology-based reconstructions of holometa- project funded by the Deutsche Forschungsgemeinschaft bolan interrelationships have recently fallen behind (DFG; German Research Foundation). It was an molecular studies, especially as only limited new com- international cooperation with research groups in Ger- parative anatomical data had been acquired, and some many, Denmark and the USA. Notably, the cooperation morphological analyses were either not numerical with the Deutsches Elektronen-Synchrotron (DESY) was (Hennig, 1969; Kristensen, 1999a; Beutel and Pohl, crucial for assembling the morphological data within a 2006) or employed only moderately sized data sets time span of 30 months. The data presented here are coded across the entire Hexapoda (Beutel and Gorb, based mainly on the results of 20 original studies on 2001, 2006). In contrast, the past decade has seen a larval and adult morphology (see Appendix S3 in considerable number of studies presenting new molec- Supporting Information), two PhD theses, and a series ular data sets analysed with state-of-the-art methodol- of unpublished Diploma theses. A positive side-effect of ogy (see references above). the project was intensive training of a new generation of Beutel and Friedrich (2008b) pointed out that morphologists who employ innovative anatomical tech- detailed insect morphology flourished in the first two- niques. A combined effort and a strong synergy effect thirds of the 20th century, but has suffered a decline between the research groups finally resulted in the since then. This development was arguably linked with largest and arguably best documented morphological the rise of molecular systematics (e.g. Scotland et al., data set ever used in insect systematics. 2003). The novelty of molecular techniques, the opportunities of throwing new light on phylogenetic problems that had proved difficult to solve with Materials and methods morphological techniques (such as the ‘‘Strepsiptera problem’’; see Kristensen, 1999a), and the ability to Taxa examined generate large data sets across extensive taxon samples in a comparatively short time all contributed to the For a list of taxa examined, see Appendix S1. We appeal of molecular systematics. Some analyses yielded selected mainly so-called ‘‘basal’’ representatives of taxa peculiar results (such as non-monophyly of Coleoptera; as exemplars, that is, those that have a large number of Whiting et al., 1997), which may have contributed putative plesiomorphies splitting off close to the basal indirectly to the realization that morphological data node of each order (e.g. Nevrorthidae for Neuroptera, are still needed. However, the controversial hypotheses Xyelidae for Hymenoptera, Micropterigidae and Agathi- also provided a healthy shake-up and forced morphol- phagidae for , Mengenillidae for Strepsi- ogists to re-evaluate time-honoured hypotheses and to ptera). We included the largest number of terminals for generate new morphological data. Furthermore, in Mecoptera, as the monophyly and intra-ordinal phy- recent years new technologies such as computer-based logeny of this group are controversial (see e.g. Whiting, 3D reconstruction, confocal laser scanning microscopy 2002b). The outgroup comprises representatives of (CLSM), and microcomputer tomography (l-CT) have Plecoptera (Pteronarcyidae, Pteronarcys californica become available to reinvigorate morphological sys- Newport, 1848); Orthoptera (Tettigoniidae, Tettigonia tematics. It is conceivable that this will lead to sp.); Zoraptera (Zorotypidae, Zorotypus hubbardi increased recognition of the need to produce compre- Caudell, 1918); and Psocoptera (Caeciliusidae) (see hensive combined data sets (Wheeler et al., 2001; Appendix S1). R.G. Beutel et al. / Cladistics 27 (2011) 341–355 343

Morphological techniques Parsimony analyses were carried out with TNT (Goloboff et al., 2008)—100 random addition tradi- The morphological techniques applied in the tional searches; NONA (Goloboff, 1995)—ratchet, 1000 project—histology, scanning electron microscopy replicates; and PAUP* 4.0b10 (Swofford, 2001)—heu- (SEM), CLSM, l-CT, and 3D computer reconstruc- ristic search, stepwise addition random, 100 replicates. tion—are outlined in detail by Friedrich and Beutel Bremer support (BS) values were calculated with Auto- (2008, 2010a,b), Friedrich et al. (2008, 2009, 2010), and Decay 5.0 (Eriksson, 2003). All characters were initially Beutel and Friedrich (2008a,b). equally weighted and unordered. In order to test whether the tree topology was sensitive to weighting Phylogenetic analysis gains and losses of structures differentially, we also used Dollo parsimony and step matrices to assign a weight of The majority of the entries in the data matrix 2 to gains and a weight of 1 to losses for two sets of (Appendix S3) were based on original observations in characters. The first set consisted of absence ⁄presence one species (see list in Appendix S1). Additional infor- muscle characters (20, 21, 36, 37, 40–42, 74, 90, 99, 100, mation was taken from detailed taxon-specific studies 105, 180, 183, 184, 189–198, 202, 206, 208–210, 212, 213, (e.g. Hannemann, 1956; Mickoleit, 1969; Kristensen, 215, 216, 218, 220–226, 229, 230, 232, 234, 335), the 1984; Vilhelmsen, 1996). In only a few cases (e.g. other set of characters pertaining to complex structures Malpighian tubules, ovarioles, sperm ultrastructure), (8, 15, 18, 35, 44, 46, 47, 49, 53, 59, 70, 85, 98, 122, 142, entries were based on information available only from 147, 149, 156, 165). For those characters with several other members of more inclusive groups (e.g. four free ‘‘presence’’ states, the weight for the transitions between Malpighian tubules in Trachypachidae, as in other these states was set to 1. These analyses were carried out Adephaga; Lawrence, 1982). The main data sources in PAUP* 4.0b10 (Swofford, 2001) (heuristic search, are shown in Appendix S2. A list of all 356 characters stepwise addition random, 100 replicates). Node support with detailed comments is presented in Appendix S4. was assessed in PAUP* 4.0b10 (same search settings as As adults and larvae of the same species (or genus, e.g. above) using BS as implemented in TreeRot (Sorenson Archostemata) were not available for study in some cases, and Franzosa, 2007), jackknifing (250 replicates), and we used a groundplan approach (Bininda-Emonds et al., bootstrapping (250 replicates). Partitioned Bremer sup- 1998; Beutel and Gorb, 2001, 2006; Beutel and Leschen, port (PBS) analyses using TreeRot and PAUP* were 2005) with families as terminal taxa (with the exception of also used to determine the relative support contributions the highly heterogeneous boreid genera Caurinus and of larval and adult characters as well as the character Boreus; Beutel et al., 2008b). Conditions probably repre- partitions pertaining to head, thorax, wing, abdomen, senting the groundplan of a polymorphic taxon were and remaining characters mostly pertaining to the coded as one character state (e.g. simplified compound digestive tract (Appendix S6). eyes present in larvae of Macroxyela considered as ancestral for Xyelidae, absent in Xyela and other endo- phytic xyelid larvae). If the groundplan condition could Results not be estimated with high probability, the character was scored as polymorphic. Almost all entries are based on External and internal characters were scored for four direct observations in one or several representatives of the outgroup and 30 ingroup taxa, the latter representing all taxon in question, or on taxon-specific information traditionally recognized orders of Holometabola. The presented in studies carried out in the framework of the aim was to examine larvae and adults of all selected project (see Appendix S4, list of characters). species within a time span of 30 months. The main focus Bayesian analysis was carried out with MrBayes 3.1.2 was on characters of the larval head (Beutel and (Huelsenbeck and Ronquist, 2001; Ronquist and Huel- Friedrich, 2008a; Beutel et al., 2009a, 2010); the head senbeck, 2003). The standard model for morphological of adults (Beutel and Baum, 2008; Beutel et al., characters as implemented in MrBayes 3.1.2 and 2008a,b); the thorax of adults (Friedrich and Beutel, proposed by Lewis (2001) and Nylander et al. (2004) 2008, 2010a,b; Friedrich et al., 2009); the female pos- was employed in its simplest version, with all state tabdomen (Hu¨ nefeld and Beutel, in press); the wing frequencies (change rates) set equal, all topologies with base; and attachment devices. Character systems repre- equal probabilities, and with unconstrained branch sented by few characters are the male genital apparatus length. Two parallel runs with four chains each were and wing venation. In the case of the former character calculated with a ratio of one cold to three hot chains. set, this was due to the lack of resources to carry out Two separate analyses with 6 and 8 million generations detailed anatomical investigations. Wing venation was were run. Temperature was set to 0.30, and 500 000 widely used in high-level phylogeny and with a focus on generations were discarded as burn-in prior to genera- holometabolan systematics by Kukalova´ -Peck and tion of the consensus tree. Lawrence (2004). Wing characters were scored only on 344 R.G. Beutel et al. / Cladistics 27 (2011) 341–355 an ordinal level by these authors. A critical re-evaluation Data analyses for a wider range of subordinal taxa will be required prior to a formal analysis. The parsimony analyses with TNT and PAUP yielded The use of a combination of SEM, histological only one tree with a length of 1144 steps (Fig. 1). sections, l-CT, and (in a few cases) CLSM turned out Holometabola is monophyletic, as are all orders except to be very efficient in providing detailed documentation Megaloptera. Hymenoptera is placed as sister-group to of the character systems used in the analysis. Notably, the remaining Holometabola, and Neuropterida are l-CT is extremely useful for examining external and monophyletic and sister-group to a taxon composed of internal features, including different types of soft parts. Amphiesmenoptera, Antliophora, Coleoptera, and Artefacts are minimal and the procedure is nondestruc- Strepsiptera. The latter are sister-groups and surpris- tive. The specimens dried at the critical point can be ingly nested within a paraphyletic Mecopterida as used for SEM or histological sectioning after l-CT sister-group of Antliophora. Reweighting the sets of scanning. The use of absorption contrast and stable characters as specified in the Materials and beams with relatively low energy yielded a very distinct methods yields a paraphyletic Holometabola with differentiation of various tissue types (see e.g. Beckmann Hymenoptera being sister-group to the outgroups. et al., 2006; Friedrich et al., 2008). 3D reconstructions Coleoptera + Strepsiptera are now nested within the and animations based on the high-quality l-CT Antliophora as sister-group to Diptera + Siphona- image stacks obtained were created using a combination ptera. Reweighting only the muscle character set yields of different software programs (see Materials and a similar tree, with the exception that Holometabola methods). monophyly is restored. PBS analyses reveal that, due Raw data, as l-CT image stacks, photographs of to its large size (196 characters) and higher per microtome sections, and SEM images, were stored in a character support (1.17), the adult character partition data bank. All original data compiled will be available contributes 85% of the total node support (229.7), for follow-up studies and will also be made available to while the larval characters have lower per character other research groups on request. (0.67) and total support (40.3). Within the adult

Mecoptera Diptera Coleoptera Lepido. Streps. Raph. Tricho. Si. Neuro. “Mega.” Bittacidae Tipulidae Bibionidae Boreus Culicidae Caurinus Trachypachidae Cupedidae Hymeno. Helophoridae Ommatidae 1/69 6/95 Mengenillidae Xenidae Eriocraniidae Agathiphagidae Raphidiidae Inocelliidae 11/99 2/51 11/100 5/77 11/100 Limnephilidae Micropterigidae Nevrorthidae Osmylidae Corydalidae Rhyacophilidae 19/100 6/90 4/82 3/72 20/100 19/100 Chrysopidae Sialidae 1/54 4/72 12/100 17/100 1/60 6/75 Tenthredinidae Diprionidae 14/100 4/86 18/100 7/67 Xyelidae 8/95 9/100 6/66

22/100 6/68 Caeciliusidae 16/100 Zorotypidae Tettigoniidae Pteronarcyidae

Fig. 1. Single minumum-length cladogram with 1144 steps. Bremer support and bootstrap values mapped on branches. R.G. Beutel et al. / Cladistics 27 (2011) 341–355 345 character partition, the highest total support is from Discussion the thorax (80.2), followed by the abdomen (79.8), wing (52.2), digestive system (12.8), and head (2.5). Phylogeny PBS per character provides a different picture because the partitions are of unequal size (abdomen: 1.60, The monophyly of Holometabola (or Endopterygota) digestive system: 1.60, wing: 0.92, thorax: 0.62, head: has never been seriously challenged. However, Kristen- 0.05). sen (1999a) pointed out that the arguments ‘‘are not The topology of trees resulting from the Bayesian numerous, and perhaps not particularly weighty either’’ analyses with 6 and 8 million generations were (see above). The analyses of our complete data set identical. At the end of the 8 million-generation confirmed several autapomorphies suggested earlier analysis, the average standard deviation of split (Hennig, 1969; Kristensen, 1999a; Beutel and Pohl, frequencies was 0.001 597 and the likelihood values 2006): the absence of ocelli in the larvae [character state for the two independent runs were )4576.65 and (char.) 7.0], the appearance of fully developed com- )4577.81. Two consensus trees were generated: one pound eyes in the pupal stage (char. 354.1) (reversal in containing only nodes with posterior probabilities of Strepsiptera), the absence of external wing buds (char. 50% or more, the other presenting all compatible 355.1) (reversal in Strepsiptera), the invagination nodes (Fig. 2). of both pterothoracic sternites (chars 138.1, 158.1), Full lists of apomorphies for major clades obtained in and the presence of ventral meso- and metasternal the parsimony analyses are given in Appendix S5. processes (chars 140.1, 162.1) (Hennig, 1969: sternales

Pteronarcyidae Pteronarcyidae Chrysopidae Chrysopidae

1.00 Nevrorthidae 1.00 Nevrorthidae 1.00 Osmylidae 1.00 Osmylidae 1.00 Sialidae 1.00 Sialidae Corydalidae Corydalidae 1.00 1.00 0.99 Raphidiidae 0.99 Raphidiidae 1.00 Inocelliidae 1.00 Inocelliidae 1.00 Ommatidae 1.00 Ommatidae 0.99 Cupedidae 0.99 Cupedidae 0.30 Helophoridae Helophoridae 0.81 Trachypachidae 0.81 Trachypachidae 0.99 0.99 Mengenillidae Mengenillidae 1.00 1.00 Xenidae 1.00 Xenidae Xyelidae Rhyacophilidae 1.00 1.00 Tenthredinidae Limnephilidae 0.42 1.00 Diprionidae 1.00 Micropterigidae

Rhyacophilidae 1.00 Agathiphagidae 1.00 Limnephilidae 0.98 Eriocraniidae Micropterigidae Boreus 1.00 1.00 0.55 1.00 1.00 Agathiphagidae Caurinus 0.98 Eriocraniidae 0.87 Nannochoristidae

Boreus 0.90 Panorpidae 0.55 1.00 0.95 Caurinus 0.98 Bittacidae 0.87 Nannochoristidae Pulicidae

0.90 Panorpidae 1.00 Culicidae 0.95 0.98 Bittacidae 1.00 Tipulidae Pulicidae 1.00 Bibionidae

1.00 Culicidae Xyelidae 1.00 Tipulidae 1.00 Tenthredinidae 1.00 Bibionidae 1.00 Diprionidae Tettigoniidae Tettigoniidae

0.99 Zorotypidae 0.99 Zorotypidae 0.99 Caeciliusidae 0.99 Caeciliusidae halfcomp allcomp

Fig. 2. Cladograms from the Bayesian analysis (two runs and four chains with 8 million generations and a burn-in of 500 000 generations), numbers on branches give the posterior probability for the respective node. A, 50% majority rule cladogram; B, cladogram with all compatible nodes resolved, even if the posterior probability is below 50%. 346 R.G. Beutel et al. / Cladistics 27 (2011) 341–355

Coxalgelenk). The true is probably another fusions in the ovipositor apparatus as apomorphies of autapomorphy but was not coded as a character here. the non-hymenopteran Holometabola. Another con- The as a usually inactive and nonfeeding last spicuous and unique plesiomorphy is the presence of a immature stage was also not included in the analysis. It relatively high number of Malpighian tubules in Hyme- was evaluated as a potential autapomorphy of Holo- noptera (char. 350.0). However, the reduced number metabola by Kristensen (1999a,b). Additional derived (eight or fewer) was not confirmed as an apomorphy of larval features revealed by our analyses are the loss of Holometabola excl. Hymenoptera in our analysis. This muscles of the glossa and paraglossa (chars 41.0, 42.0), is arguably an artefact caused by the inclusion as and the absence of intrinsic antennal muscles (char. outgroups of Zoraptera and Caeciliusidae, which pos- 26.0). The absence of the endite lobes of the labium as sess six or four Malpighian tubules, respectively. discrete structures is also a likely autapomorphic Neuropterida is clearly corroborated (BS: 14) in feature, but was not included as a separate character. agreement with the conventional view (e.g. Aspo¨ ck New potential autapomorphies of adults are the meso- et al., 2001) and Wiegmann et al. (2009), but in contrast and metacoxae being closely adjacent medially (proba- to parsimony analyses of the mitochondridal genome bly correlated with the invaginated sternites) (chars (Cameron et al., 2009; retrieved as a clade in their 144.1, 167.1), several thoracic muscular features (e.g. Bayesian analyses). Groundplan autapomorphies of absence of the pronoto-trochanteral muscle, char. 180.0; Neuropterida are the increased number of retinula cells presence of the metascutello-postnotal muscle, char. in the stemmata (char. 6.1), a medially divided meta- 222.1), and modifications in the wing base (e.g. chars postnotum, the presence of a muscle between the 257.0, 267.0, 292.1) (see also Friedrich and Beutel, metafurca and the abdominal spiracle I, and the 2010b). Interestingly, a derived feature previously sug- presence of a trichobothria field on tergum X (Aspo¨ ck gested as a synapomorphy of Hymenoptera and Mec- et al., 2001). Whether the nearly equal size of the fore opterida, the sitophore plate (Kristensen, 1999a), was and hind wings is indeed an autapomorphy of Neuro- optimized as an autapomorphic groundplan feature of pterida is questionable. The optimization of this char- Holometabola in the accelerated transformation mode acter depends on the choice of outgroups. Considering (char. 102.0), with secondary loss in Neuropterida, the functional background, it appears very unlikely that Coleoptera, and Strepsiptera. It should be noted in this the condition in Neuropterida is due to a secondary context that a sitophore plate is also present in Para- modification, as with equal-sized wings (with the . exception of Odonata) generally display moderate or All traditional orders, with the exception of Mega- poor flying ability. Posteromotorism has evolved in loptera (see below), were confirmed as monophyletic. In Coleoptera + Strepsiptera, and anteromotorism and addition, our analyses retrieved several long-recognized functional or anatomical diptery in Hymenoptera and superordinal groups (e.g. Hennig, 1969): Neuropterida, within Mecopterida. Amphiesmenoptera, and Antliophora (excl. Strepsi- The position of Neuropterida as the second branch ptera). In contrast to the traditional view (Hennig, within Holometabola (Fig. 1) remains questionable. The 1969; see also Kristensen, 1999a; Wheeler et al., 2001), potential clade comprising Coleoptera, Strepsiptera, but in agreement with Rasnitsyn and Quicke (2002), Amphiesmenoptera, and Antliophora is not supported Kukalova´ -Peck and Lawrence (2004) and recent molec- by convincing apomorphies (see below). A close rela- ular studies (Savard et al., 2006; Wiegmann et al., 2009), tionship between Neuropterida and Coleoptera (or our analyses unambiguously support a clade comprising Coleopterida) was not confirmed in our analyses, not all holometabolan lineages except for Hymenoptera. even when Strepsiptera are excluded from the analysis The non-hymenopteran Holometabola are supported by (maximum parsimony). Within Neuropterida, a clade the primarily prognathous larval head (char. 1.1); the comprising Megaloptera and Raphidioptera is corrob- presence of stemmata in the larval stage (char.5.2) (with orated, in contrast to Aspo¨ ck et al. (2001), Aspo¨ ck secondarily developed simplified compound eyes in (2002), and Cameron et al. (2009), but in agreement Mecoptera; e.g. Melzer et al., 1994); and a considerable with Achtelig and Kristensen (1973) and also Wiegmann number of autapomorphies of adults: the reduction of et al. (2009). The proposed neuropteran–megalopteran the paraglossa and its muscle (char. 98.0, 99.0), the sister-group relationship in Cameron et al. (2009) is not presence of a distinct meron (char. 146.1), modifications convincing, as only one representative of each of the of the wing base (e.g. chars 248.0, 275.0), ventromedially three neuropterid orders was included, among them a connected gonocoxae (char. 303.1), and short ventral mantispid, which is a highly derived member of sclerites of segments VIII and IX in females (equivalent Neuroptera (Aspo¨ ck and Aspo¨ ck, 2007). The mono- to gonapophsyes VIII and IX) (char. 305.0). Non- phyly of the raphidiopteran–megalopteran lineage aculeatan hymenopterans (‘‘Symphyta’’) are the only appears very likely considering the consistent support holometabolan insects with a fully functional lepisma- from independent analyses based on extensive morpho- toid ovipositor. We identify specific reductions and logical and molecular data sets. Synapomorphies are the R.G. Beutel et al. / Cladistics 27 (2011) 341–355 347 distinctly flattened larval head (char. 2.1), the progna- autapomorphies of Coleoptera and of Coleoptera thous head of adults (char. 62.1), the presence of a excluding Archostemata it appears highly unlikely that large, exposed prothoracic basisternum (char. 118.1), Strepsiptera are a subordinate group of polyphagan and specially developed hairy soles on the tarsomeres beetles, as suggested by Crowson (1981). (char. 240.1). The corydalid–raphidiopteran clade The monophyly of Mecopterida, a clade comprising (implying the paraphyly of Megaloptera) in our anal- Amphiesmenoptera and Antliophora (Hinton, 1958; yses may be an artefact caused by the advanced panorpoid orders; Hennig, 1969; Kristensen, 1975, predacious habits in the larvae of the two groups and 1999a) remains questionable. It is weakly supported in resulting modifications of head structures (Beutel and our Bayesian analyses (Fig. 2), whereas the support was Friedrich, 2008a). However, it is evident that the strong (100%) in Wiegmann et al. (2009). Potential monophyly of the Megaloptera is far from being well groundplan autapomorphies are the partial reduction of supported. the dorsal tentorial arms (well developed in Caurinus), An important result of the present study is a sister- the presence of the larval M. craniodististipitalis (see group relationship between Coleoptera and Strepsiptera Beutel et al., 2009a), the elongation of the long ventral (Coleopterida). This lineage was supported in parsi- metasternal process, the presence of a muscularis of the mony and Bayesian analyses (BS: 5, posterior probabil- spermathecal duct (different orientation of fibes in ity: 0.99). It is in agreement with earlier analyses of Antliophora and Amphiesmenoptera; char. 324.1 ⁄2), morphological data sets (Beutel and Gorb, 2001, 2006) the presence of a specific muscle attached to the genital and also with the results of Wiegmann et al. (2009), but chamber, and telescoping or at least retractable poster- in contrast to the Halteria concept (Strepsiptera + Di- ior abdominal segments in females (Hu¨ nefeld and ptera), which was supported in analyses of 18S rDNA Beutel, in press). The typical telescoping postabdomen conducted with POY (Whiting et al., 1997; Wheeler activated by hemolymph pressure (char. 317.1) is found et al., 2001; see also Whiting, 2002a). Putative synapo- in Nannochoristidae (Hu¨ nefeld and Beutel, in press), morphies of Coleoptera and Strepsiptera are the absence and a similar condition is also present in other mecopt- of the larval frontolabral muscle (char. 9.0), the com- erans (e.g. Caurinus, Panorpa) and in several dipteran plete loss of the salivary duct (char. 44.0), the vestigial or lineages. The eversion is achieved by muscle force in absent lateral cervical sclerites (char. 112.0), the lateral Amphiesmenoptera and segments IX + X are fused connection of the pronotum and propleura (char. (char. 317.2). 116.1), the missing connection of the profurcal arm Mecopterida were not supported in the parsimony and the propleura (char. 120.0), and features linked with analyses, but Coleopterida were nested within this posteromotorism (chars. 126.2, 127.2, 146.0). The lineage as sister to Antliophora. None of the putative monophyly of Coleopterida implies that posteromoto- apomorphies supporting either the entire assemblage, or rism has evolved only once in Holometabola, in contrast only Coleopterida + Antliophora, are convincing (see to anteromotorism, which probably evolved indepen- Appendix S4). All of them are losses (e.g. absence of a dently in Hymenoptera, Bittacidae, Diptera, and other typical M. frontolabralis: muscle with clypeal origin groups. present in dipterans), and almost all imply more or less Using posterior probability as a measure of branch unlikely reversals, such as the secondary gain of extrinsic support, the signal for a clade Coleopterida was strong antennal muscles (char. 25.1) or the median submental in Wiegmann et al. (2009; PP: 99%). It was found in all retractor (char. 40.1) in coleopteran larvae, or the regain analyses and recovered in nearly all single-gene analyses. of M. mesonoto-coxalis posterior in adult beetles (char. Both groups do not share a single unique morphological 196.1). character gain. Nevertheless, considering the convincing Numerous losses that probably occurred indepen- molecular evidence and the unambiguous support by dently in Strepsiptera in correlation with miniaturiza- our extensive morphological data set, the monophyly of tion (primarily in the larvae) and structural Coleopterida (Coleoptera + Strepsiptera) appears very simplification (adult males) have had a strong impact likely. This implies that several seemingly plesiomorphic on the results of the analyses. Coleoptera do not group features of Strepsiptera (Kristensen, 1999a; Beutel and within Mecopterida when Strepsiptera are removed Pohl, 2006) are in fact reversals. This applies to the from the data set. However, the mecopterid–coleopterid appearance of compound eyes (char. 353.0) and external clade turned out as stable, even when the analyses were wing buds (char. 354.0) before the pupal stage, and the rerun with increased penalty on the secondary gain of presence of a well developed abdominal segment XI once lost muscles or other structures, and even when (char. 49.1) and cerci with extrinsic muscles (char. 53.1) DolloÕs parsimony was applied not permitting such in primary larvae. reversals. Apparently, the monophyly of Mecopterida The monophyly of Coleoptera is strongly supported should be considered an open question presently, in the present analysis (BS: 20) and other studies (e.g. because analyses excluding Strepsiptera and enforcing Beutel and Haas, 2000). Considering the numerous a clade Coleoptera + Neuropterida yielded a clade 348 R.G. Beutel et al. / Cladistics 27 (2011) 341–355

Mecopterida on a tree that requires three additional pleural arm (char. 210.1). Another apomorphy almost steps. generally occurring in Antliophora is the extensive Amphiesmenoptera are exceptionally well supported reduction or loss of the dorsal tentorial arm (in larvae (Hennig, 1969; Kristensen and Skalski, 1999). New and adults). However, it is well developed in adults of autapomorphies are the spermathecal duct with a Caurinus (Beutel et al., 2008b). The muscle system of the muscularis composed mainly of circular fibres (char. antliophoran head is generally simplified in larvae and 324.2), the presence of a spermathecal gland (char. adults (Beutel and Baum, 2008; Beutel et al., 2009a). An 325.1), and a well developed bursa copulatrix extreme degree of reduction is found in some lineages of (char. 326.1). Another set of potential apomorphies is Diptera with short-lived adults, which are also charac- the occurrence of apodemes at the female abdominal terized by highly reduced mouthparts. Only eight segments VIII and IX. The telescoping postabdomen, muscles are preserved in the adult head of Deuterophle- which may possibly represent a groundplan autapomor- bia (K.S., pers. comm.), in contrast to more than 50 in phy of Mecopterida, is preserved. However, the eversion representatives of Neuropterida. is achieved not only by hemolymph pressure, as is the In contrast to Whiting (2002b), Wiegmann et al. case in Nannochorista (Hu¨ nefeld and Beutel, in press) (2009), and other studies (e.g. Kristensen, 1999a; Beutel and other antliophoran groups, but by the activity of and Pohl, 2006), we did not recover a mecopteran– abdominal muscles with modified sites of origin and siphonapteran clade, but a sister-group relationship insertion. between Diptera and Siphonaptera. Shared derived Modifications of the ovipositior and egg deposition features supporting this are the complete loss of larval apparently played an important role in Holometabola in thoracic legs, the presence of a labral food channel in general, and also in Amphiesmenoptera. The primary adults, and other features related to specialized liquid egg-laying mode of the group cannot be assessed feeding (see below and Appendix S4). unambiguously, but at least in the case of Lepidoptera, Mecoptera are a species-poor group, but arguably a primary egg deposition in narrow crevices appears key taxon within Holometabola (Hinton, 1958; Hennig, plausible. Agathiphagidae, the only group with three 1969), justifying the comparatively large taxon sample instead of two pairs of apophyses, deposit their eggs included in the present study. Mecopteran phylogeny below the cone scales of kauri pines (Kristensen, 1999b). has been much debated in the past couple of decades The apodemes are completely reduced in micropterigid (e.g. Willmann, 1987; Whiting, 2002b). Hypotheses moths, which are partly associated with moss and lay implying their nonmonophyly were suggested by Wheel- their eggs on the surface of their host plants. This er et al. (2001), Whiting (2002b), and Beutel and Baum reduction is an autapomorphy of Micropterigidae, (2008). In the former studies, which were mainly or which are confirmed here as sister-group of all the exclusively based on 18S rDNA sequences, a clade remaining Lepidoptera (Kristensen, 1999a,b). A new comprising Nannochoristidae (= Nannomecoptera) + synapomorphy of Agathiphagidae and the remaining (Boreidae + Siphonaptera) was suggested. The lineage lepidopteran groups (represented here by Eriocraniidae) comprising fleas and boreids is supported not only by is a spermathecal duct with a characteristic two- the DNA sequences analysed, but also by potential compartment-section that has a small and a strongly morphological synapomorphies, such as the partial loss extended part of the lumen and a content of mesocuticle of wings, jumping capability, secondarily panoistic in its intima. ovarioles, a silken cocoon, and a specific mode of resilin Even though the sperm pump, the character on which production (Bilin´ ski et al., 1998; Kristensen, 1999a). It the name Antliophora (= pump bearers) is based may be noted that the first two features are poorly (Hennig, 1969), was discarded as an autapomorphy defined and also occur in other lineages outside (Hu¨ nefeld and Beutel, 2005), the monophyly of the Holometabola, and the latter is very insufficiently lineage appears well founded, mainly, but not only, by documented throughout insects. Similarly to Wiegmann reductional features (e.g. Beutel and Baum, 2008; Beutel et al. (2009), our analyses support a monophyletic et al., 2009a; Friedrich and Beutel, 2010a,b). The Mecoptera, which is mainly corroborated by characters autapomorphies retrieved here include the immobilized of the postabdominal segments—specific modifications labrum (char. 70.0), the loss of the craniocardinal of the male and female genital apparatus. muscle (char. 90.0) (arguably correlated with the The group that differs most strikingly from the formation of a maxillolabial complex, char. 81.1), the remaining mecopteran taxa is Nannochoristidae. The reduced number of labial palpomeres (char. 95.2), larvae are not orthognathous, grub-like, and soil-dwelling, the massive complex around the pharynx formed by or associated with moss, as is the case in Pistillifera and the brain and subesophageal ganglion (char. 109.1), the Boreidae, but are prognathous, extremely slender, and loss of M. mesofurca-phragmalis (char. 198.0) and M. aquatic. These features were retrieved here as autapo- metafurca-phragmalis (char. 225.0), and the origin of a morphies of the family. The results of our analyses, and bundle of M. mesonoto-pleuralis posterior on the that of Wiegmann et al. (2009), also imply that striking R.G. Beutel et al. / Cladistics 27 (2011) 341–355 349 structural affinities of the adult head of Nannochorist- among more species-poor lineages (Kristensen, 1999a). idae, Siphonaptera, and Diptera have evolved indepen- This pattern is actually repeated ad infinitum as one dently. These include the presence of a labral focuses on progressively less inclusive groups, making food channel, a very strongly developed postcerebral the exercise of identifying causes for diversification pumping apparatus, a dorsally concave prelabium somewhat elusive. Nevertheless, we will attempt to forming a trough or sheath for the paired mouthparts, identify some ‘‘nodal points’’ (Hinton, 1977; see also an enlarged prementopalpal muscle, salivary channels Kristensen, 1999a), and key features that, in combina- on the laciniae (Nannochoristidae and fleas), lamelli- tion, allowed the Holometabola to become one of the form or stylet-like mandibles, and a deeply excavated dominant groups of organisms on the planet. sensory organ on the third maxillary palpomere (Nan- It is likely that the complete metamorphosis (in nochoristidae and Diptera). This is an entire series of combination with the absence of external wing buds), character gains, and intuitively it appears rather unlikely arguably a ‘‘key character’’ of Holometabola (Kristen- that these complex transformations related to liquid sen, 1999a), had a positive long-term effect on the feeding are due to parallel evolution in the groups in diversification of the group. Putative advantages are the question. However, such a scenario is suggested by the ability to exploit different resources in the immature and strong support for a monophyletic Mecoptera in Wieg- adult stages, and the greatly improved ability of the mann et al. (2009). endopterygote larvae to penetrate into crevices and to Another taxon strikingly different from the ‘‘typical move forward and backward in dense substrates (Hin- pistilliferan pattern’’ (e.g. Panorpidae) is Caurinus. The ton, 1977; Kristensen, 1999a). Additionally, the pupal head morphology appears highly plesiomorphic com- stage can serve as a possibility to efficiently endure pared with that of other Boreidae, and also pistilliferan adverse periods. It is obvious, however, that the groups such as Bittacidae or Panorpidae (Beutel et al., immediate impact on the diversification rate was very 2008b). The characteristic rostrum is entirely lacking, a limited. Megaloptera, Raphidioptera, Strepsiptera, and distinctly developed dorsal tentorial arm is present, the Mecoptera comprise only a few hundred species each, mandible is equipped with a prominent molar part, and and Neuroptera and Siphonaptera are also moderately the tentoriomandibular muscle is exceptionally well diverse taxa. Neuropterida and Mecoptera were prob- developed. Despite conspicuous structural discrepan- ably more diverse in the past (Grimaldi and Engel, 2005; cies, the inclusion of Caurinus not only in Mecoptera, Aspo¨ ck and Aspo¨ ck, 2007), but there is no fossil but also in the family Boreidae, is well founded (Figs 2 evidence indicating that any of the small holometabolan and 3; see also Beutel et al., 2008b). It was pointed out lineages came even close to the species richness of the by Beutel et al. (2008) that Caurinus is crucial for the four megadiverse orders (Rasnitsyn and Quicke, 2002; reconstruction of the groundplan of Mecoptera and Grimaldi and Engel, 2005). The branching pattern Antliophora. The omission of this extremely rare species retrieved here and by Wiegmann et al. (2009) strongly would have resulted in considerable misinterpretation of suggests that extreme diversification took place four character evolution. times independently within each of the megadiverse The clade Pistillifera is well supported by larval orders, all of which contain species-poor taxa at the features (Beutel et al., 2009a) and the specialized sperm base: the Hymenoptera (e.g. Xyelidae, Blasticotomidae), pump of adult males (Willmann, 1981, 1987, 1989; Coleoptera (Archostemata, Myxophaga), Lepidoptera Mickoleit, 2009: Spermienauspressvorrichtung). A possi- (Micropterigidae, Agathiphagidae, Heterobathmiidae), ble clade comprising Pistillifera and Boreidae is sug- and Diptera (e.g. Nymphomyiidae, Deuterophlebiidae). gested by the presence of a unique, compact intrinsic The current diversity of large subgroups within these muscle of the salivary duct. However, this grouping was orders (see Kristensen, 1999a; staphyliniform and not supported in our parsimony analysis with all cucujiform beetles, cyclorrhaphan flies, ditrysian characters equally weighted, which favoured a sister- Lepidoptera, apocritan wasps) was caused mainly by group relationship between Nannochoristidae and radiation in the , and was probably linked, to Pistillifera (see above). varying degrees, with the angiosperm diversification taking place in that period. It was pointed out by Evolution Grimaldi and Engel (2005) that most of the recent insect families appeared in the Cretaceous, and many When discussing possible causes for the present-day of them are associated with flowering plants in one diversity of any group of organisms, it is important to way or another. It is noteworthy, however, that the realize that a ‘‘successful’’ group such as the Holometa- radiations of some important groups, such as ditrysian bola usually can be broken down into a number of Lepidoptera or schizophoran flies, took place in the natural lineages, some of which are actually not partic- Cenozoic. ularly diverse (e.g. Kristensen, 1999a). Rather, the In the case of Hymenoptera, it is unlikely that a close diverse groups (e.g. within Holometabola) are scattered association with flowering plants is the primary cause 350 R.G. Beutel et al. / Cladistics 27 (2011) 341–355 for their present day diversity, at least not directly. The A key innovation in antliophoran subgroups is the main groups of herbivorous Hymenoptera (sawflies, fig acquisition of highly modified sucking mouthparts in wasps, gall wasps, bees) may be associated primarily adults. It is uncertain whether this is linked primarily to with angiosperms, but they comprise only a minority of imbibing nectar from flowering plants or to ectoparasitic the total diversity of the order. Instead, evolutionary habits and the consumption of blood. The former is success in terms of species richness was predominantly apparently the case in Nannochoristidae (Beutel and linked with parasitism. Various groups of parasitoid Baum, 2008). Females of Siphonaptera and biting flies wasps (e.g. Chalcidoidea, Ichneumonoidea) probably suck blood, but males of dipteran groups with preserved represent the largest reservoir of undescribed insect stylet-like mandibles and maxillae (e.g. Culicidae, species (Grimaldi and Engel, 2005). The wasp waist, Tabanidae) consume only nectar. Another trend in which increases the movability of the abdomen, may Antliophora is the simplification or reduction of the also have contributed to the very successful diversifica- larval legs. They are very short and delicate in Nan- tion of the group (Grimaldi and Engel, 2005). The nochoristidae, the number of segments is reduced in greater movability facilitates both precise egg deposition Boreidae and Pistillifera, and they are absent in Sipho- in concealed hosts in parasitic groups, and wielding of naptera and Diptera. In dipterans, this is apparently the sting in Aculeata. However, many of the parasitoid related to larval development in moist substrate, prob- or predacious Hymenoptera feed primarily on insects ably primarily linked with saprophagous feeding habits associated with angiosperms, so their diversity may be (Neugart et al., 2009). indirectly linked with that of flowering plants. Our analyses show that very good flying ability, The role of ‘‘angiosperm revolution’’ in the evolution linked with simplified wing venation, anteromotorism, of beetles (Farrell, 1998) is also not straightforward. It and functional (Hymenoptera) or anatomical (Diptera) was apparently largely or completely irrelevant in the diptery, has evolved twice independently. This suggests diversification of the predominantly predacious Ade- that this was a factor contributing to the success of both phaga (ca. 30 000 species), and also of Staphylinoidea Diptera and Hymenoptera. An unusual structural (ca. 35 000 species), which are either saprophagous, diversity of larvae and the ability to occupy a very mycophagous, carnivorous, or carrion-feeders (Hansen, broad variety of habitats in the immature stages (fast- 1997). It was suggested by Hunt et al. (2007) that flowing streams, mud, soil, wood, leaves, fruit, etc.) is extensive diversification in beetles may not have been characteristic for the nonrelated and extremely success- linked primarily with the Cretaceous diversification of ful orders Coleoptera and Diptera. Again, the indepen- angiosperm plants, but was rather due to ‘‘high survival dent occurrence in two megadiverse lineages makes it of lineages and sustained diversification in a variety of likely that this capacity was one factor enhancing niches’’. However, it is obvious that the role of diversification. Another feature occurring within nonre- angiosperms as a food source was a crucial factor in lated, species-rich taxa is liquid feeding with strongly the evolution of one of the most diverse polyphagan modified sucking mouthparts. Very distinctly different subgroups, the Phytophaga (Chrysomeloidea and Cur- types have evolved within Hymenoptera (Jervis, 1998; culionoidea, ca. 120 000 species) and also in the Jervis and Vilhelmsen, 2000), in the extremely species- Scarabaeoidea (ca. 35 000 species). rich Glossata, and within Antliophora (Nannochoristi- The Lepidoptera provides the most clear-cut case of dae, Siphonaptera, Diptera). Liquid feeding apparently diversification associated with angiosperms, although occurred long before the rise of angiosperms (e.g. members of the most basal lineages, Micropterigidae Archaeodictyoptera in the Palaeozoic; Grimaldi and and Agathiphagidae, feed on non-angiosperm plants. A Engel, 2005), but in the case of the groups discussed feature that apparently played an outstanding role in the here, a relationship with flowering plants and their evolution of the order is the formation of an elongate radiation in the Cretaceous and Cenozoic is obvious. proboscis with intrinsic musculature in the adult stage. As mentioned above, Hymenoptera is the only holo- This is an autapomorphy of Myoglossata, which com- metabolan order with a fully functional lepismatoid prise more than 90% of the known lepidopteran species ovipositor. It is reduced in Coleoptera and Neuropte- (Kristensen, 1999b). In this case, the link with angio- rida, and completely lost in the Mecopterida, where it is sperms as host plants is evident. Another prominent replaced by an oviscapt, a telescopic egg-laying tube feature is the characteristic plant-feeding larval stage, formed by the postabdominal segments (Scudder, 1971; the caterpillar, a larva with a globular orthognathous Hu¨ nefeld, 2009). These different types of egg-laying head, orthopteroid mouth parts, a cylindrical body, a device probably confer benefits and incur penalties on series of abdominal prolegs, and a more or less exposed different substrates. Females of many hymenopteran life style. In its overall body structure and evolutionary groups, including the basal herbivorous lineages, have to strategy, the lepidopteran caterpillar is similar to a larval cut or drill tough plant material to oviposit. The type which may be assigned to the hymenopteran ovipositor, which is often armed with teeth and is capa- groundplan. ble of executing alternating thrusts during oviposition, is R.G. Beutel et al. / Cladistics 27 (2011) 341–355 351 apparently well suited for hard substrates. The draw- the comprehensive, detailed, and well documented back is that drilling such a substrate and conveying eggs information provided in these studies can be extremely down the narrow inner lumen of the ovipositor is time- useful in systematic and evolutionary research, and also consuming and leaves the insect exposed to in other disciplines. The modern attitude to restrict for extended periods. It may also restrict egg size. In information to those data immediately relevant in a contrast, the oviscapt probably allows the insect to pass narrow context saves space, but might prevent a bigger eggs, and pass them more quickly, thus minimiz- publication from retaining its usefulness over time. ing exposure of the ovipositing female. However, insects Despite the demonstrated value of our data, it must with oviscapts are probably dependent on comparatively be noted that morphological characters alone appar- softer substrates or pre-existing holes for egg deposition. ently have their limitations in insect systematics, at least They have to evolve novel features if they want to on the level of interordinal relationships. The addition oviposit in harder substrates. of more characters to extensive data matrices will probably lead to a ‘‘phylogenetic saturation curve’’ in most cases. In the present project, the addition of more Future perspectives for morphology in insect systematics character systems did not have a strong impact on the outcome of the analyses beyond a certain point of data The present study shows that a comprehensive and assessment, and did not necessarily improve the results. well documented morphological data set can be The analyses of larval data and features of the adult acquired within reasonable time limits using a combi- head and thorax (without wing base) yielded a branch- nation of traditional and innovative techniques. The ing pattern (one tree, CI 0.43) with Neuropterida basal approach applied here may be useful not only for instead of Hymenoptera, and with a clade Nannocho- compiling morphological matrices for combined analy- ristidae + Siphonaptera + Diptera (see above), but ses, but also for documenting in detail anatomical otherwise identical with the cladogram obtained with features of life stages of model organisms, such as all characters included (Fig. 1). Adding more character Drosophila or Tribolium. The substantial congruence systems, such as the tracheal system or the innervation with the results of the molecular study of Wiegmann pattern of muscles, would possibly slightly improve the et al. (2009) and the high resolution of the single phylogenetic results, but the cost-effectiveness of this is obtained minimum-length cladogram demonstrate the disputable. In contrast, the addition of more taxa in usefulness of the data set presented here (see Appen- order to break down long branches would probably dix S2). Analyses of different subsets of characters improve results. Graybeal (1998) demonstrated with (Fig. 3) show that the most inclusive data set yields simulations that adding more taxa increases the accu- the best resolution. Larval characters, and characters of racy in phylogenetic reconstruction dramatically (see the adult head and thorax, supported most of the also Dunn et al., 2008), whereas it improves much more branches we obtained in the analyses of the complete slowly when more characters are added. In the case of data set. The resolution was lowest with the characters our project, the inclusion of more mecopteran terminals, of the wing base (more than 100 000 minimum-length notably of the family Meropidae, may have led to trees), which display a high degree of homoplasy, but different results, especially with respect to Nannocho- nevertheless may contribute to a better resolution in ristidae (see Friedrich and Beutel, 2010b); and a denser some cases. taxon sampling in Diptera (e.g. Deuterophlebiidae, Even though the acquisition of original data was a Axymyiidae, Blephariceridae) may also have affected major objective in our project, older studies based on a the outcome of the analyses. traditional approach proved invaluable. Detailed mono- A general problem occurring in phylogenetic recon- graphs such as Maki (1936) on Chauliodes or Badonnel structions based on morphological characters is similar (1934) on Psocoptera, also extensive and superbly to the phenomenon of ‘‘long-branch attraction’’ occur- illustrated anatomical works published by PhD students ring in analyses of molecular data. The inclusion of and scientists of the Weber school at the Institute of terminals with accelerated evolution rates, resulting in Zoology of the University of Tu¨ bingen (Beutel et al., an exceptionally high number of autapomorphies and 2009b), provided very detailed and useful taxon-specific erosion of signal, can lead to artificial results in the information, for example, on larval head morphology of analyses. In our project, this might apply to the Osmylus (Wundt, 1961) and Panorpa (Bierbrodt, 1942). secondarily flightless fleas and Boreidae (‘‘snow fleas’’) Interestingly, Hermann Weber urged his students to and to the highly autapomorphic endoparasitic Strep- refrain from any phylogenetic interpretations in siptera. Even though the ‘‘Strepsiptera problem’’ ap- their theses (Beutel et al., 2009b). A PhD thesis with a pears to have come full circle, it has to be noted that similar scope and approach would probably not further Coleopterida is not supported by convincing autapo- the scientific career of a young researcher today. morphies (character gains) apart from posteromotorism, Nevertheless, even several decades after publication, which has also evolved in other insect lineages. The 352 R.G. Beutel et al. / Cladistics 27 (2011) 341–355 Number of characters Number of equally trees parsimoneus Hymenoptera Neuroptera Megaloptera Raphidioptera Lepidoptera Trichoptera Mecoptera Diptera Coleoptera Strepsiptera Neuropterida Neuropterida + Coleoptera Coleopterida Amphiesmenoptera Antliophora Mecoptera excl. Nannochoristidae Mecoptera + Siphonaptera Mecoptera + Diptera Diptera + Siphonaptera + Nannochoristidae Diptera Mecopterida Holometabola excl. Hymenoptera Holometabola excl. Neuropterida + Coleoptera Holometabola Nannochoristidae + Nannochoristidae Siphonaptera

all characters 356 1

larval characters 58 63 90 50 84 88 90 22

larval head 45 48

adult characters 292 11 54 8136 1818 54 81

adult characters 235 6 excl. wing base adult head 49 5 40 80 ×

adult thorax 132 2

wing base 57 >150T 89 84 92 96 <1 79 65 74

adult abdomen 54 672 16 10 98 73 73 3

internal features 141 4 50 50 50 50

external features 211 8 25 25 25

× Neuropterida paraphyletic

Fig. 3. Clades supported by subsets of characters.

coleopterid–mecopterid and the coleopterid–antliopho- phylogenies without using morphological information. ran groupings, which we consider artificial, were appar- However, mere branching patterns, robust as they may ently caused mainly by reductional features, which have be, give only limited insights into the evolution of a very probably evolved independently in strepsipterans group. What is essential for the reconstruction of and groups belonging to Mecopterida. There is no complex evolutionary scenarios is knowledge of the simple solution to this problem, and only an extended morphological transformations. It is the phenotype, approach adding taxa (including fossils) and substantial with its morphological features, that primarily interacts molecular data will increase the possibility of deriving with the environment (Beutel et al., 2009b). strongly supported hypotheses. An antagonism between systematists using morpho- Recent reviews of systematic studies (Bybee et al., logical and molecular data, respectively, appears out of 2009) suggest that the use of morphology in insect place in any case. It is not the type of data that counts in phylogenetics loses ground when compared with molec- the first place, but rather the quality of the data, the ular systematics, whereby this trend appears to be density of the sample, and the quality of the analyses. largely due to a faster growth in the number of studies One of the most important roles morphology will play in based on DNA sequences as opposed to fewer studies the future is that of providing an independent and based on morphological characters (Meier and Lim, separately analysed character set (as with other defin- 2009). However, Wheeler (2008) points out that, even able character partitions, such as different sets of genes). when morphological contributions to combined matri- Analyses of morphological characters provide a stan- ces are seemingly dwarfed by the sheer number of dard of comparison for molecular phylogenies (and vice informative sites in extensive molecular data sets (e.g. versa), following the principle of reciprocal illumination 39.9 MB in Dunn et al., 2008), they still can have a very (wechselseitige Erhellung) as advocated by Hennig substantial impact on the reconstruction of the deeper (1966). It is almost self understood that both types of nodes of the trees, which are often not well resolved with data have their shortcomings, but conflicts allow for molecular data alone (Balke et al., 2005; von Reumont identifying sources of errors and provide compelling et al., 2009). Extensive molecular data sets including reasons for further study through additional sampling complete genomes will be available for a considerable (see Su et al., 2008). taxon sample in the foreseeable future, and these data A field in which morphology will obviously play an sets will probably enable systematists to produce robust exclusive role is paleontology. Fossils will provide R.G. Beutel et al. / Cladistics 27 (2011) 341–355 353 useful DNA sequences only in extremely rare cases. Aspo¨ ck, U., Plant, J.D., Nemeschkal, H.L., 2001. Cladistic analysis of Beutel et al. (2008a) demonstrated that the neglect of Neuroptera and their systematic position within Neuropterida (Insecta: Holometabola: Neuropterida: Neuroptera). Syst. Ento- fossils can lead to serious misinterpretations in phylo- mol. 26, 73–86. genetic reconstruction, and it is obvious that the Badonnel, A., 1934. Recherche sur lÕanatomie des Psoques. Bull. biol. knowledge and interpretation of fossils is essential for Fr. Belg. (Suppl.) 18, 1–241. understanding the evolutionary history of any group of Balke, M., Ribera, I., Beutel, R.G., 2005. The systematic position of organisms. Fossils were not included in the present Aspidytidae, the diversification of Dytiscoidea (Coleoptera, Adephaga) and the phylogenetic signal of third codon positions. analyses. However, our data matrix may serve as the J. Zool. Syst. Evol. Res. 43, 223–242. basis for future investigations of extinct holometabolan Beckmann, F., Donath, T., Fischer, J., Dose, T., Lippmann, T., lineages. Lottermoser, L., Martins, R.V., Schreyer, A., 2006. New develop- Insects, with their highly complex exoskeleton and ments for synchrotron-radiation-based microtomography at internal structures, will continue to offer rich rewards DESY. Proc. SPIE. 6318, 1–10. Beutel, R.G., Baum, E., 2008. A longstanding entomological problem for dedicated and careful morphological exploration. finally solved? Head morphology of Nannochorista (Mecoptera, Well documented data sets will have a profound impact Insecta) and possible phylogenetic implications. J. Zool. Syst. Evol. on phylogenetic reconstruction. We are confident that Res. 46, 346–367. morphology will not succumb in the near future, but will Beutel, R.G., Friedrich, F., 2008a. Comparative study of larval head continue to play a vital role in the disciplines of biology structures of Megaloptera (Hexapoda). Eur. J. Entomol. 105, 917– 938. related to systematics and evolutionary research. Beutel, R.G., Friedrich, F., 2008b. A renaissance of insect morphology – l-Ct and other innovative techniques. DGaaE Nachr. 22, 5–8. Beutel, R.G., Gorb, S., 2001. Ultrastructure of attachment specializa- Acknowledgements tions of hexapods (Arthropoda): evolutionary patterns inferred from a revised ordinal phylogeny. J. Zool. Syst. Evol. Res. 39, 177– 207. Our greatest thanks go to Prof. Dr N.P. Kristensen Beutel, R.G., Gorb, S., 2006. A revised interpretation of the evolution (Zoologisk Museum, Copenhagen). His immense posi- of attachment structures in Hexapoda (Arthropoda), with special tive input in different stages of this project cannot be emphasis on Mantophasmatodea. Syst. Phylogeny 64, overestimated. We are also very grateful to Dr E. 3–25. Beutel, R.G., Haas, F., 2000. Phylogenetic relationships of the Altenhofer (Groß Gerungs), Prof. Dr U. Aspo¨ ck suborders of Coleoptera (Insecta). Cladistics 16, 103–141. (Naturhistorisches Museum Wien), R. Bellstedt (Mu- Beutel, R.G., Leschen, R.A.B., 2005. Phylogenetic analysis of Staph- seum der Natur, Gotha), Dr T. Galloway (University of yliniformia (Coleoptera) based on characters of larvae and adults. Manitoba), Prof. Dr P. Johnson (South Dakota State Syst. Entomol. 30, 510–548. University), Dr B. Krasnov (Ben Gurion University of Beutel, R.G., Pohl, H., 2006. Endopterygote systematics – where do we stand and what is the goal (Hexapoda, Arthropoda)? Syst. Negev), Prof. Dr M. Moog (Universita¨ t fu¨ r Bodenkun- Entomol. 31, 202–219. de, Wien), Dr. M. Ohl (Museum fu¨ r Naturkunde, Berlin) Beutel, R.G., Ge, S., Ho¨ rnschemeyer, T., 2008a. On the head and Dr S. Winterton (The University of Queensland) for morphology of Tetraphalerus, the phylogeny of Archostemata providing valuable specimens, and to Dr A. Staniczek and the basal branching events in Coleoptera. Cladistics 24, 270– (Museum am Rosenstein) for arranging the loan of 298. Beutel, R.G., Friedrich, F., Whiting, M.F., 2008b. Head morphology megalopteran larvae. The very competent support with of Caurinus (Boreidae, Mecoptera) and its phylogenetic implica- the l-tomography at DESY by Dr J. Herzen is also tions. Arthropod Struct. Dev. 37, 418–433. greatly appreciated. Synthesis stipends to Dr F. Hu¨ ne- Beutel, R.G., Kristensen, N.P., Pohl, H., 2009a. Resolving insect feld and Dr F. Friedrich are gratefully acknowledged, phylogeny: the significance of cephalic structures of the Nannome- coptera in understanding endopterygote relationships. Arthropod and also the main financial support granted by the Struct. Dev. 38, 427–460. German Science Foundation (BE1789 ⁄4-1, HO2306 ⁄ Beutel, R.G., Leschen, R.A.B., Friedrich, F., 2009b. Darwin, beetles 4-1). Finally, our great thanks are due to two anony- and phylogenetics. Naturwiss. 96, 1293–1312. mous reviewers. Their comments have greatly helped to Beutel, R.G., Friedrich, F., Aspo¨ ck, U., 2010. The larval head improve this study. of Nevrorthidae and the phylogeny of Neuroptera (Insecta). Zool. J. Linn. Soc. doi: 10.1111/j.1096-3642.2009.00560.x Bierbrodt, E., 1942. Der Larvenkopf von Panorpa communis L. und seine Verwandlung, mit besonderer Beru¨ cksichtigung des Gehirns References und der Augen. Zool. Jb. Anat. 68, 49–136. Bilin´ ski, S.M., Bu¨ ning, J., Simiczyjew, B., 1998. The ovaries of Achtelig, M., Kristensen, N.P., 1973. A re-examination of the Mecoptera: basic similarities and one exception to the rule. Folio relationships of the Raphidioptera (Insecta). Z. zool. Syst. Evol.- Histochem. Cytobiol. 36, 189–195. forsch. 11, 268–274. Bininda-Emonds, O.R.P., Bryant, H.N., Russell, A.P., 1998. Supra- Aspo¨ ck, U., 2002. Phylogeny of the Neuropterida (Insecta: Holometa- specific taxa as terminals in cladistic analysis: implicit assumptions bola). Zool. Scr. 31, 51–55. of monophyly and a comparison of methods. Biol. J. Linn. Soc. 64, Aspo¨ ck, U., Aspo¨ ck, H., 2007. Verbliebene Vielfalt vergangener Blu¨ te. 101–133. Zur Evolution, Phylogenie und Biodiversita¨ t der Neuropterida Bybee, S.M., Zaspel, J.M., Beucke, K.A., Scott, C.H., Smith, (Insecta: Endopterygota). Denisia 20, 451–516. B.W., Branham, M.A., 2009. Are molecular data supplanting 354 R.G. Beutel et al. / Cladistics 27 (2011) 341–355

morphological data in modern phylogenetic studies? Syst. Ento- Hu¨ nefeld, F., Beutel, R.G., in press. The female postabdomen of the mol. 35, 2–5. enigmatic Nannochoristidae (Insecta: Mecopterida) and its phylo- Cameron, S.l., Sullivan, J., Song, H., Miller, K.B., Whiting, F.W., genetic significance. Acta Zool. 2009. A mitochondrial genome phylogeny of the Neuropterida Hunt, T., Bergsten, J., Levkanicova, Z., Papadopoulou, A., St. John, (lace-wings, alderflies and snakeflies) and their relationship to the O., Wild, R., Hammond, P.M., Ahrens, D., Balke, M., Caterino, other holometabolous insect orders. Zool. Scr. 38, 575–590. M.S., Go´ mez-Zurita, J., Ribera, I., Barraclough, T.G., Bocakova, Crowson, R.A., 1981. The Biology of Coleoptera. Academic Press, M., Bocak, L., Vogler, A.P., 2007. A comprehensive phylogeny of London. beetles reveals the evolutionary origins of a superradiation. Science Dunn, C.W., Hejnol, A., Matus, D.Q., Pang, K., Browne, W.E., 318, 1913–1916. Smith, S.A., Seaver, E., Rouse, G.W., Obst, M., Edgecombe, G.D., Jervis, M.A., 1998. Functional and evolutionary aspects of mouth- Sørensen, M.V., Haddock, S.H.D., Schmidt-Rhaesa, A., Okusu, part structure in parasitoid wasps. Biol. J. Linn. Soc. 63, 461– A., Kristensen, R.M., Wheeler, W.C., Martindale, M.Q., Giribet, 493. G., 2008. Broad phylogenomic sampling improves resolution of the Jervis, M., Vilhelmsen, L., 2000. The occurrence and evolution of animal tree of life. Nature 452, 745–749. nectar extraction apparatus among Hymenoptera ÔSymphytaÕ. Biol. Eriksson, T., 2003. AutoDecay version 5.0. http://www.bergianska.se/ J. Linn. Soc. 70, 121–146. index_forskning_soft.html Kjer, K.M., 2004. Aligned 18S and insect phylogeny. Syst. Biol. 53, Erwin, T.L., 1997. Biodiversity at its utmost: tropical forest beetles. In: 506–514. Reaka-Kudla, M.L., Wilson, D.E. (Eds.), Biodiversity II: Under- Kristensen, N.P., 1975. The phylogeny of hexapod ‘‘orders’’. A critical standing and Protecting our Biological Resources. Joseph Henry review of recent accounts. J. zool. Syst. Evol. Res. 13, 1–44. Press, Washington, DC, pp. 27–40. Kristensen, N.P., 1981. Phylogeny of the insect orders. Annu. Rev. Farrell, B.D., 1998. ‘‘Inordinate Fondness’’ explained: why are there so Entomol. 26, 135–157. many beetles? Science 281, 555–559. Kristensen, N.P., 1984. The larval head of Agathiphaga (Lepidoptera, Friedrich, F., Beutel, R.G., 2008. The thorax of Zorotypus (Hexapoda, Agathiphagidae) and the lepidopteran groundplan. Syst. Entomol. Zoraptera) and a new nomenclature for the musculature of 9, 63–81. Neoptera. Arthropod Struct. Dev. 37, 29–54. Kristensen, N.P., 1999a. Phylogeny of endopterygote insects, the most Friedrich, F., Beutel, R.G., 2010a. The thoracic morphology of successful lineage of living organisms. Eur. J. Entomol. 96, 237– Nannochorista (Nannochoristidae) and its implications for the 253. phylogeny of Mecoptera and Antliophora. J. Zool. Syst. Evol. Res. Kristensen, N.P., 1999b. 4. The non-glossatan moths. In: Kristensen, 48, 50–74. N.P. (Ed.), Handbook of Zoology, Vol. IV Arthropoda: Insecta, Friedrich, F., Beutel, R.G., 2010b. Good bye Halteria? The evolution Part 35 Lepidoptera, Moths and Butterflies, Vol. 1: Evolution, of the thorax in Holometabola. Cladistics doi: 10.1111/j.1096- Systematics, and Biogeography. De Gruyter, Berlin, New York, 0031.2010.00305.x. pp. 41–49. Friedrich, F., Pohl, H., Hu¨ nefeld, F., Beckmann, F., Herzen, J., Kristensen, N.P., Skalski, A.W., 1999. 2. Phylogeny and paleontology. Beutel, R.G., 2008. SRlCT-based study of external and internal In: Kristensen, N.P. (Ed.), Handbook of Zoology, Vol. IV structures of adults and larvae of Endopterygota (Hexapoda). Arthropoda: Insecta, Part 35 Lepidoptera, Moths and Butterflies. Hasylab Ann. Rep. 2007, Hamburg, pp. 1527–1528. Vol. 1: Evolution, Sytematics, and Biogeography. Handbook of Friedrich, F., Farell, B.D., Beutel, R.G., 2009. The thoracic morphol- Zoology. Vol. IV Arthropoda: Insecta. Part 35. Walter de Gruyter, ogy of Archostemata and the relationships of the extant suborders Berlin, New York, pp. 7–25. of Coleoptera (Hexapoda). Cladistics 25, 1–37. Kukalova´ -Peck, J., Lawrence, J.F., 2004. Use of hind wing characters Giribet, G., Richter, S., Edgecombe, G.D., Wheeler, W., 2005. The in assessing relationships among coleopteran suborders and major position of crustaceans within Arthropoda – evidence from nine endoneopteran lineages. Eur. J. Entomol. 101, 95–144. molecular loci and morphology. Crustac. Issues 16, 307–352. Lawrence, J.F., 1982. Coleoptera. In: Parker, S. (Ed.), Synopsis and Goloboff, P., 1995. NONA Version 1.5. Fundacion e Instituto Miguel Classification of Living Organisms. McGraw-Hill, New York, pp. Lillo, Tucuman. 482–553. Goloboff, P.A., Farris, J.S., Nixon, K.C., 2008. TNT, a free program Lewis, P.O., 2001. A likelihood approach to estimating phylogeny for phylogenetic analysis. Cladistics 24, 774–786. from discrete morphological character data. Syst. Biol. 50, 913– Graybeal, A., 1998. Add taxa or characters to a difficult phylogenetic 925. problem. Syst. Biol. 47, 9–17. Maki, T., 1936. Studies on the skeletal structure, musculature and Grimaldi, D., Engel, M.S., 2005. Evolution of the Insects. Cambridge nervous system of the Alder Chauliodes formosanus Petersen. University Press, Cambridge, UK. Mem. Fac. Sci. Agric. Taihoku Imp. Univ. 16(3), 117–243. Hannemann, H.J., 1956. Die Kopfmuskulatur von Micropteryx Meier, R., Lim, G.S., 2009. Conflict, convergent evolution, and the calthella (L.) (Lep.). Zool. Jb. Anat. 75, 177–206. relative importance of immature and adult characters in endop- Hansen, M., 1997. Evolutionary trends in ‘‘staphyliniform’’ beetles terygote phylogenetics. Annu. Rev. Entomol. 54, 85–104. (Coleoptera). Biol. Skr Kong. Danske Vidensk. Selsk. 48, 1–339. Melzer, R.R., Paulus, H.F., Kristensen, N.P., 1994. The larval eye of Hennig, W., 1966. Phylogenetic Systematics. University of Illinois nannochoristid scorpionflies (Insecta, Mecoptera). Acta Zool. 75, Press, Urbana, IL. 201–208. Hennig, W., 1969. Die Stammesgeschichte der Insekten. Waldemar Mickoleit, G., 1969. Vergleichend anatomische Untersuchungen an der Kramer, Frankfurt a.M. pterothorakalen Pleurotergalmuskulatur der Neuropteria und Hinton, H.E., 1958. The phylogeny of the panorpoid orders. Annu. Mecopteria (Insecta, Holometabola). Zoomorph. 64, 151–178. Rev. Entomol. 3, 181–206. Mickoleit, G., 2009. Die Sperma-Auspreßvorrichtung der Nannocho- Hinton, H.E., 1977. Enabling Mechanisms. Proc. XV Int. Congr. ristidae (Insecta: Mecoptera). Entomol. Gen. 31 ⁄ 2, 193–226. Entomol. Washington, DC, pp. 71–83. Neugart, C., Schneeberg, K., Beutel, R.G., 2009. The morphology of Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference the larval head of Tipulidae (Diptera, Insecta) – the dipteran of phylogeny. Bioinformatics 17, 754–755. groundplan and evolutionary trends. Zool. Anz. 248, 215–235. Hu¨ nefeld, F., Beutel, R.G., 2005. The sperm pumps of Strepsiptera Nylander, J.A.A., Ronquist, F., Huelsenbeck, J.P., Nieves-Aldrey, and Antliophora (Hexapoda). J. Zool. Syst. Evol. Res. 43, 297– J.L., 2004. Bayesian phylogenetic analysis of combined data. Syst. 306. Biol. 53, 47–67. R.G. Beutel et al. / Cladistics 27 (2011) 341–355 355

Pape, T., Bickel, D., Meier, R., (Eds.) 2009. Diptera Diversity: Status, orders inferred from 18S and 28S ribosomal DNA sequences and Challenges, and Tools. Brill Academic Publishers, Leiden. morphology. Syst. Biol. 46, 1–68. Rasnitsyn, A.P., Quicke, D.L.J. (Eds.), 2002. History of Insects. Wiegmann, B.M., Trautwein, M.D., Kim, J.-W., Cassel, B.K., Kluwer Academic Publishers, Dordrecht. Bertone, M.A., Winterton, S.L., Yeates, D.K., 2009. Single-copy von Reumont, B.M., Meusemann, K., Szucsich, N.U., DellÕAmpio, E., nuclear genes resolve the phylogeny of the holometabolous insects. Gowri-Shankar, V., Bartel, D., Simon, S., Letsch, H.O., Stocsits, BMC Biol. 7, 34. R.R., Luan, Y., Wa¨ gele, J.-W., Pass, G., Hadrys, H., Misof, B., Willmann, R., 1981. Das Exoskelett der ma¨ nnlichen Genitalien der 2009. Can comprehensive background knowledge be incorporated Mecoptera (Insecta) I. Morphologie. II. Die phylogenetischen into substitution models to improve phylogenetic analyses? A case Beziehungen der Schnabelfliegen-Familien. Z. zool. Syst. Evol.- study on major arthropod relationships. BMC Evol. Biol. 9, 119. forsch. 19, 96–150. 153–174. Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian Willmann, R., 1987. The phylogenetic system of the Mecoptera. Syst. phylogenetic inference under mixed models. Bioinformatics 19, Entomol. 12, 519–524. 1572–1574. Willmann, R., 1989. Evolution und phylogenetisches System der Savard, J., Tautz, D., Lercher, M., 2006. Genome-wide acceleration of Mecoptera. Abh. Senckenberg. naturforsch. Ges. 544, 1–153. protein evolution in flies (Diptera). BMC Evol. Biol. 6, 7. Wundt, H., 1961. Der Kopf der Larve von Osmylus chrysops L Scotland, R.W., Olmstead, R.G., Bennett, J.R., 2003. Phylogeny (Neuroptera, Planipennia). Zool. Jb. Anat. 79, 557–662. recnstruction: the role of morphology. Syst. Biol. 52, 539–548. Scudder, G.G.E., 1971. Comparative anatomy of insect genitalia. Annu. Rev. Entomol. 16, 379–406. Sorenson, M.D., Franzosa, E.A., 2007. TreeRot, Version 3. Boston Supporting Information University, Boston, MA. Su, K.F.Y., Kutty, S.N., Meier, R., 2008. Morphology versus Additional Supporting Information may be found in molecules: the phylogenetic relationships of Sepsidae (Diptera: the online version of this article: Cyclorrhapha) based on morphology and DNA sequence data from ten genes. Cladistics 24, 902–916. Appendix S1. Taxa examined. Swofford, D.L., 2001. PAUP*: Phylogenetic Analysis Using Parsi- mony and Other Methods, Version 4.0b10. Distributed by Sinauer Appendix S2. Data sources. Associates Inc., Sunderland, MA. Vilhelmsen, L., 1996. The preoral cavity of lower Hymenoptera Appendix S3. Characters used in the phylogenetic (Insecta): comparative morphology and phylogenetic significance. analysis. Zool. Scr. 25, 143–170. Wheeler, Q.D., 2008. Undisciplined thinking – morphology and Appendix S4. Character-state matrix (Winclada). HennigÕs unfinished revolution. Syst. Entomol. 33, 2–7. Wheeler, W.C., Whiting, M., Wheeler, Q.D., Carpenter, J.M., 2001. Appendix S5. List of apomorphies (homoplasious The phylogeny of the extant hexapod orders. Cladistics 17, 113– characters in italics). 169. Whiting, M.F., 2002a. Phylogeny of the holometabolous insect orders: Appendix S6. Results of partitioned Bremer analyses. molecular evidence. XXI International Congress of Entomology, Please note: Wiley-Blackwell is not responsible for the Iguassu Falls, Brazil, August 2000. Zool. Scr. 31, 3–15. Whiting, M.F., 2002b. Mecoptera is paraphyletic: multiple genes and content or functionality of any supplementary materials phylogeny of Mecoptera and Siphonaptera. XXI International supplied by the authors. Any queries (other than missing Congress of Entomology, Iguassu Falls, Brazil, August 2000. Zool. material) should be directed to the corresponding author Scr. 31, 93–104. for the article. Whiting, M.F., Carpenter, J.C., Wheeler, Q.D., Wheeler, W.C., 1997. The Strepsiptera problem: phylogeny of the holometabolous insect