Phylogeny of the Holometabolous Insect Orders: Molecular Evidence

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BlackwellPhylogeny Science Ltd of the holometabolous insect orders: molecular evidence

MICHAEL F. WHITING

Accepted: 5 October 2001 Whiting, M. F. (2002). Phylogeny of the holometabolous insect orders: molecular evidence. — Zoologica Scripta, 31, 3–15. Phylogenetic relationships among the holometabolous insect orders were reconstructed using 18S ribosomal DNA data drawn from a sample of 182 taxa representing all holometabolous insect orders and multiple outgroups. Parsimony analysis supports the monophyly of all holometabolous insect orders except for Coleoptera and Mecoptera. Mecoptera is paraphyletic with respect to Siphonaptera, which is nested within Mecoptera. Coleoptera is scattered as a paraphyletic assemblage across the tree topology. These data support a monophyletic Halteria (Strepsiptera + Diptera), Amphiesmenoptera (Trichoptera + Lepidoptera), Neuropterida (Neuroptera + (Megaloptera + Raphidioptera)), but Antliophora (Halteria + Mecoptera + Siphonaptera) and Mecopterida (Antliophora + Amphiesmenoptera) are paraphyletic. The limitations of using 18S ribosomal DNA as the sole phylogenetic marker for reconstructing insect ordinal relationships are discussed. Michael F. Whiting, Department of Zoology, Brigham Young University, Provo, UT 84602, USA. E-mail: [email protected]

Introduction on a single character associated with the female ovipositor Accounting for more than 80% of insect species and more (Mickoleit 1973; Achtelig 1975). The highly derived order than 50% of all animal species (Wilson 1988; Kristensen Siphonaptera has been associated with Diptera or Mecoptera 1999), Holometabola is the most diverse and successful group based on different character suites (Boudreaux 1979; Hennig of terrestrial organisms. Holometabola comprises 11 insect 1981; Kristensen 1991). The most perplexing question, and orders, four of which — Coleoptera, Hymenoptera, Diptera that which has received the most attention in recent years, and Lepidoptera — account for over 99% of the species has been the placement of Strepsiptera among the other insect diversity of this group. Mecoptera, Strepsiptera, Megaloptera orders. Strepsiptera has been associated with Coleoptera, and Raphidioptera each contain less than 1000 described either within Polyphaga (Crowson 1960) or as sister group species, and Trichoptera, Neuroptera and Siphonaptera to Coleoptera, based on wing morphology and function each contain less than 4000 species. The monophyly of each (Kristensen 1981, 1991; Kathirithamby 1989; Kukalova-Peck insect order is relatively well supported by morphological & Lawrence 1993). Detailed examination of these putative data (Kristensen 1995, 1999; Whiting et al. 1997), with the synapomorphies, however, suggests that they are based on exception of Mecoptera which appears to be paraphyletic mistaken descriptions of strepsipteran wing morphology with respect to Siphonaptera (see Whiting 2002). Apart and function (Kinzelbach 1990; Pix et al. 1993; Whiting 1998b; from Holometabola itself, Amphiesmenoptera (Lepidoptera + Beutel & Haas 2000). There have been a number of reviews Trichoptera) is the only well-established interordinal relation- of phylogenetically informative characters for Holometabola ship, being supported by over 15 synapomorphies (Hennig with their accompanying phylogenetic hypotheses (Kristensen 1981; Kristensen 1997; Whiting et al. 1997). Other postulated 1975, 1981, 1991, 1995; Boudreaux 1979; Hennig 1981). interordinal relationships are based on relatively few morph- Whiting et al. (1997) presented the ﬁrst formal quantitative ological characters or characters of questionable phylogenetic analysis of holometabolan relationships based on a coded utility. For example, no characters support a ﬁrm placement character matrix and also generated molecular sequence data for Hymenoptera, which has been postulated as sister group to from 18S and 28S ribosomal DNA (rDNA) for Holometabola ‘Meronida’ (Mecopterida + Neuropterida) (Boudreaux 1979) and outgroups. The summary topology from the total evidence or to Mecopterida (Kristensen 1991; 1999; Whiting et al. analysis of Whiting et al. (1997) is given in Fig. 1. Kristensen 1997). While a sister group relationship between Coleoptera (1999) presented an excellent review of holometabolan morph- and Neuroptera appears to be widely accepted, it is based ology, and his phylogenetic conclusions largely agree with

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A. (Hennig) B. (Boudreaux)

C. (Whiting) D. (Kristensen)

Fig. 1 Previous hypotheses for holometabolan phylogeny. Hennig (A.) (1981), Boudreaux (B.) (1979) and Kristensen (D.) (1999) are based primarily on morphological data; Whiting et al. (C.) (1997) is based on a combination of morphological and molecular data. Dotted lines refer to poorly supported relationships.

those of Whiting et al. (1997), except for uncertainty as to Mecoptera and Siphonaptera. The Carmean et al. (1992) and whether Strepsiptera should be placed as sister group to Chalwatzis et al. (1996) analyses suggested a paraphyletic Diptera or Coleoptera. Holometabola, and the Pashley et al. (1993) analysis was Given that morphology has led to some ambiguous phylo- unable to test for Holometabola paraphyly. The analysis of genetic relationships within this diverse group, it is not Whiting et al. (1997) supported a monophyletic Holometabola surprising that in the past few years some effort has been and a sister group relationship between Megaloptera and placed on using DNA sequence data to decipher interordinal Raphidioptera. The most intriguing result of these molecular phylogenetic relationships within Holometabola. Carmean analyses, and certainly the most controversial, was the evidence et al. (1992) sequenced a portion of 18S rDNA from 19 taxa presented for a well-supported sister group relationship between representing six holometabolan orders, one hemipteran and Strepsiptera and Diptera ( Whiting & Wheeler 1994; Carmean & one spider outgroup (Fig. 2A). Pashley et al. (1993) used 17 Crespi 1995; Kristensen 1995; 1999; Whiting & Kathirithamby 18S rDNA sequences to represent nine holometabolan orders 1995; Huelsenbeck 1997, 1998; Whiting et al. 1997; Whiting and one hemipteran outgroup, and found a monophyletic 1998a,b). Beyond the question of phylogenetic afﬁnity of a Amphiesmenoptera and Mecopterida, but other relationships remarkable group, this result has been centre stage in debates were unresolved (Fig. 2B). Chalwatzis et al. (1996) sequenced over competing methods of phylogenetic reconstruction and 22 exemplars for 18S rDNA to represent nine holometabolan the role of a homeotic mutation in giving rise to novel mor- orders and four outgroup taxa (Fig. 2C). Whiting et al. (1997) phology in an insect group. A recent summary and re-analysis used 87 exemplars for 18S rDNA and 54 exemplars for 28S of holometabolous relationships based on 18S ribosomal data rDNA to represent all 11 holometabolan orders and 15 out- can be found in Whiting (2001). The analysis in this paper pres- group orders (Fig. 2D). All of these molecular analyses concur ents a broader selection of taxa, particularly from Coleoptera, in supporting Amphiesmenoptera (except for Carmean et al. than in the Whiting (2001) review, and is thus better able to 1992 who omitted these taxa) and a close association between address coleopteran paraphyly based on molecular data.

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Fig. 2 Previous molecular hypotheses for Holometabola based on 18S rDNA including sampled outgroups. Numbers in parentheses refer to the number of exemplars used as terminals.

Materials and methods Tipulidae), taxa under-represented in previous analyses (e.g. 18S rDNA sequences were acquired from GenBank and Neuroptera) or taxa whose phylogenetic position is still con- augmented with sequences generated in the laboratory. Only troversial (e.g. Strepsiptera). This sampling strategy resulted sequences with a length of 1 kb or greater were used in this in 147 ingroup sequences representing all holometabolous analysis, and an attempt was made to represent as many holo- orders and 111 families (Appendix 1). Outgroup taxa were metabolous families as possible. Multiple sequences were used selected from Paraneoptera, the hypothesized sister group to to represent diverse families (e.g. Carabidae, Scarabaeidae, Holometabola (Whiting et al. 1997; Kristensen 1999), and

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Fig. 3 Scheme of alignment for blocked variable regions. Regions that were ambiguously aligned between orders, but unambiguously aligned within orders, were aligned as blocks for each holometabolous insect order (represented as light boxes). Taxa outside of the blocked regions were coded with missing data. These regions were spliced together to create a step-like formation in the total alignment, where the variable blocked regions are ﬂanked by conserved regions. The blocks were combined for Mecoptera and Siphonaptera and excluded for the outgroups.

Polyneoptera (sensu Boudreaux 1979), with a sequence from simultaneously performing alignment (Wheeler 1999), the Ephemeroptera used as the most distant outgroup. This resulted implied alignment option outputs a multiple alignment which in 35 outgroup sequences from 11 orders and 35 families. Of is more optimal than those typically found by other alignment the 182 total sequences included in this analysis, 111 were algorithms, such as MALIGN (Wheeler & Gladstein 1994) or obtained from GenBank and the remaining 71 were gener- Clustal W (Thompson et al. 1994; W. Wheeler, personal ated in the laboratory using the primers and methodology communication, 2000). The implied alignments generated by described in Whiting et al. (1997), except that the entire region POY were used as the aligned matrix for phylogenetic analysis. of 18S was ampliﬁed and sequenced. Roughly two-thirds of Variable alignment regions which appeared ambiguously these sequences consist of the entire region of 18S rDNA and aligned across the insect orders, but relatively conserved the other one-third consist of approximately 1 kb of sequence within each order, were aligned independently within each data. This analysis includes 50 more sequences than in the holometabolous insect order using POY with the parameters Whiting (2001) review and represents the broadest selection as described above. Mecoptera and Siphonaptera were treated of taxa to date for holometabolan phylogeny. as a single order because previous evidence has suggested Sequences were assembled in Sequencher™ 3.1.1 (Genecodes that Mecoptera is paraphyletic with respect to Siphonaptera 1999), and a gross alignment was performed by manually (Schlein 1980; Whiting et al. 1997; Bilinski et al. 1998). These aligning the conserved domains across the taxa. Each con- variable regions were excluded from the outgroups because served domain, and variable regions between the domains, resolution among these taxa is not the focus of this study. Each were removed in sections and entered into the computer region was considered to be an alignment block, and the blocks program POY (Gladstein & Wheeler 1999) to undergo more were assembled into a single matrix by scoring taxa outside of exhaustive alignment. POY was implemented on a dedicated 32 the block with missing values. This produces a blocked align- node parallel cluster using gap cost = 2, change cost = 1, with ment for the variable regions, and each of these blocks was Tree Bisection and Reconnection (TBR) branch swapping on spliced together into a single alignment to form one variable 100 alignments, with the option ‘implied alignment’ implemented. blocked alignment (Fig. 3). The variable blocked regions and Although POY is designed to construct a topology while conserved regions were then assembled into a single alignment,

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with the conserved regions flanking the variable blocked regions. the variable regions, resulted in 4822 trees of length 9353 A theoretical justification for this method of alignment has (CI = 0.43, RI = 0.69), the strict consensus of which is pres- been provided elsewhere (Whiting 2001). The alignment can ented in Fig. 4. This topology supports a monophyletic Neoptera, be found at http://dnasc.byu.edu/~whitinglab/. but a paraphyletic Paraneoptera (Hemiptera + Thysanoptera + Trees were reconstructed under parsimony with gaps treated Psocodea), as Psocodea (Phthiraptera + Psocoptera) is placed as missing data. Because of the large size of the data set, a as the most basal neopteran taxon. Polyneoptera is mono- novel method of tree searching was employed. The program phyletic and placed as sister group to Hemiptera. Within PAUP* 4.0 (Swofford 2000) was utilized using a implementation Polyneoptera, the monophyly of Orthoptera, Blattoidea, of the parsimony ratchet (Nixon 1999) that allows processing Dictyoptera and Embioptera + Phasmatodea is supported, on multiple computers in parallel. The parsimony ratchet but Mantodea is paraphyletic. Hemiptera is monophy- was implemented following the general procedure of Nixon letic, as are the suborders Auchenorrhyncha and Heteroptera. (1999) with the following modifications: (i) individual ratchet Holometabola forms a monophyletic group, although with iterations were parsed to separate machines for computation; only moderate Bremer and bootstrap support values. All (ii) the percentage of reweighted characters was allowed to holometabolan orders are supported as monophyletic groups, randomly vary between 5% and 25% for each iteration; (iii) the except for Mecoptera and Coleoptera. In terms of overall weight assigned to the random character partition was allowed relationships, this analysis divides Holometabola into two to randomly vary from 2 to 10 with each iteration; (iv) the major clades: (Hymenoptera + (Neuropterida + Mecoptera/ central CPU distributed a semi-randomized matrix and topo- Siphonaptera)) and (Amphiesmenoptera + Halteria), with logy to each nodal CPU for computation; (v) the most optimal Coleoptera scattered as a paraphyletic assemblage through- topology from each ratchet iteration was returned to a central out the latter clade. These two groups are supported by low CPU which kept a running tally of all optimal and suboptimal bootstrap and Bremer support values. Antliophora (Diptera + topologies; and (vi) the central CPU then distributed a new Strepsiptera + Mecoptera + Siphonaptera) is paraphyletic, as semi-randomized matrix and optimal topology to the nodal is Mecopterida (Antliophora + Amphiesmenoptera). CPUs for computation. A total of 10 000 ratchet iterations Hymenoptera is supported as a monophyletic group, but were computed on an average of 256 CPUs. The most optimal Symphyta and Apocrita are both paraphyletic, and most intra- topologies were subjected to TBR branch swapping to enu- ordinal relationships are poorly supported. These data support merate the entire set of optimal trees. Because the parsimony a monophyletic Neuropterida, with a sister group relation- ratchet more broadly samples the tree landscape than does ship between Raphidioptera and Megaloptera, in agreement the standard parsimony search technique (Nixon 1999), it is with previous molecular (Whiting et al. 1997) and morpho- likely that this analysis found a better representation of the logical (Kristensen 1999) analyses. However, these results entire set of most parsimonious trees than would a standard differ from the conclusions of Aspöck (2002) who places analysis. Trees were also computed in PAUP* and NONA using Megaloptera as sister group to Neuroptera. Within Neurop- 30 random addition sequences in the former and 100 random terida, Myrmeleontiformia is monophyletic, but it is nested addition sequences in the latter. In all analyses, the parallel within Hemerobiiformia (Aspöck 1995) making the latter implementation of the parsimony ratchet found trees that were group paraphyletic. The monophyly of Hemerobiidae and shorter than any found using random addition sequences, Chrysopidae is also supported in this analysis. Neuroptera is and these analyses were completed in a few hours in contrast notoriously difficult to generate reliable ribosomal sequence to the many days required for standard PAUP* and NONA ana- data for, because the ribosomal genes in these taxa appear to lyses. Bremer support values (Bremer 1994) were calculated be less concerted in their evolution than in other insect groups, in NONA by saving trees up to 10 steps away from the most and they often include large insert regions that are difficult to parsimonious solution. Bootstrapping was performed in PAUP amplify and sequence. The sequences included in this study 4.0* using a full heuristic search with 100 replicates. Trees come primarily from my laboratory, and appear to be authentic were reconstructed with the blocked variable regions included in that the same sequence data can be consistently generated and excluded, and Coleoptera was analysed constrained and from these particular taxa, and the data make sense in the unconstrained as a monophyletic group. light of neuropteran phylogeny. More problematic sequences using previous analyses (e.g. Corydalus and Agulla; Whiting Results et al. 1997) were excluded from this analysis. Indeed, the The alignment of 18S rDNA resulted in 10 conserved regions difficulty of generating reliable sequences from a diversity of (regions aligned across all taxa) and three variable regions neuropteran taxa has made their phylogeny more elusive than which were blocked as described above. Two highly autapo- that of other comparably sized orders. morphic strepsipteran insert regions were excluded from the Mecoptera + Siphonaptera form a relatively well-supported analysis. Phylogenetic analysis of all aligned data, including monophyletic group. The subordination of Siphonaptera

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Ephemerella Psocodea Torridincola Calopteryx Distocupes Archostemata + Myxophaga 100 1 10+ Libellula 2 Hydroscapha 95 Cerastipsocus Clambus 100 6 Valenzuela 1 Platypus 10+ 80 Columbicola 1 Apoderus 54 Menacanthus “Polyneoptera” Meloe 9 2 4 Neohaematopinus 2 Rhipiphorus 95 Stenopelmata 5 Hololepta 55 7 Pterophylla 95 1 Podabrus 10 Paratettix Helochares 4 Melanoplus Oligotoma 1 Coriophorus 2 Diradius Octinodes 2 1 1 Calopteron Timema 1 1 100 Mantis 1 Photuris 55 Reticulitermes 1 Trox 7 Oberea 1 Tenodera 2 Polyphaga 1 61 Gromphadorhina 2 Tetraopes 1 Blaberus 1 Cyphon 2 75 Hemiowoodwardia Apatides Buenoa 1 1 4 100 1 Ptinus Aquarius Xanthopyga 10+ Saldula 175 Heteroptera 1 85 Dynastes 86 Lygus 5 Xyloryctes 91 2 Rhaphigaster 6 Prosapia Acmaeodera 10+ Okanagana 1 1 Trichodes 2 75 Amphotus 2 100 Graphocephalus 9 Spissistilus 2 Eleodes Neoptera 1 90 Olarius 1 Mecomacer 100 3 Prokelisia Auchenorrhyncha Cucujus 10+ 95 Siphanta 1 Oxycraspedus 95 Hysteropterum Trachypachus 1 3 1 80 Haliplus 52 Ips 2 Scolops 1 70 2 8 Suphis Apion Dolerus 1 Polydrusus 3 Hemitaxonus 3 85 Copelatus Adephaga 1 75 Doronomyrmex 4 4 Hygrobia Omophron 5 2 Hartigia Evania Amphizoa Trichoptera

2 2 Megarhyssa Hymenoptera 2 Orectochilus Ophion 3 2 Wormaldia Mesopolobus 100 Oxyethira 2 2 Orussus 80 4 10+ Pycnopsyche Caenochrysis 4 89 Hydropsyche 1 1 Epyris 100 Monobia 2 Oecetis Polistes 10+ Brachycentrus 1 2 Eriocrania 2 1 Apoica 100 Dasymutilla 60 Agathiphaga 55 1 Bareogonalos 1 10+ 75 4 Micropteryx Priocnemus Heterobathmia 4 Lepidoptera 1 1 55 100 2 Campsomeris Amphiesmenoptera Sthenopis 2 Trioxys 10+ 90 Tegeticula 785 Thyridopteryx Megaloptera Prionoxystus Sialis 4 61 75 Platyptilia 100 Agulla 100 Omus 3 100 Cicindela 2 Papilio 10+ Nehga Raphidioptera 10+ Galleria 60 100 Eremochrysa 8 Oxycheila Adephaga Metrius Hemileuca 3 9 Anisochrysa Anthocharis Oliarces 2 Clinidium 1 80 Caenocholax Hyles Lolomyia Ascalapha 4 Mantispa Triozocera Haplogenius Neuroptera 4 100 71 Lymantria 74 100 M. chobauti Tineola Myrmeleon 71 1 60 Micromus 10+ 10+ M. chobauti 2 100 5 1 Hemerobius sp. 1 Crawfordia 1 H. stigmata 80 100 6 100 X. pecki Strepsiptera Boreus 57510+ X. vesparum 5 Caurinus 95 Apteropanorpa 3 95 Elenchus 95 Merope 8 7 Stylops 2 4 Bittacus 100 Simulium 1 66 Chorista Corethrella 80 1 80 Mecoptera 10+ 70 4 Panorpa 1 Ablabesmyia 2 4 6 Brachypanorpa 3 Dixella Nannochorista Tunga 100 100 Lutzomyia Polygenis 75 10+ Phlebotomus Neuropterida Halteria 10+ Epiphragma 1 Megarthroglossus 7 100 Diptera 100 Hystrichopsylla Tanyptera Holometabola Coptopsylla 10+ 100 10+ Holorusia Acanthopsylla 75 10+ Dolichopeza 1 Craneopsylla Siphonaptera 7 Nephrotoma 100 Frontopsylla Chrysops 90 Orchopeas 100 5 94 Laphria 3 Myodopsylla 10+ Mydas 7 70 Mythicomyia 3 66 65 Drosophila 3951 Ornithoica 5 100 Anastrepha 9 Ceratitis

Fig. 4 Strict consensus of 4822 most parsimonious trees (L = 9353, CI = 0.43, RI = 0.69) from the 18S rDNA alignment with blocked variable regions included. Coleoptera and Adephaga are paraphyletic. Numbers above nodes are bootstrap values; numbers below nodes are Bremer support.

within Mecoptera based on 18S rDNA is not a surprise given Archostemata + Myxophaga form the basal-most clade and, the results of previous molecular analyses. However, the although resolution of taxa within Polyphaga is poorly sup- placement of Nannochorista as sister group to the fleas is not ported, the monophyly of this group is supported with a supported in a more extensive molecular and morphological Bremer value of 5. Adephaga is grossly paraphyletic and attaches analysis (Whiting 2002), which argues for a sister group rela- as two paraphyletic assemblages at two positions on the tionship with Boreidae. The monophyly of Siphonaptera topology: basal to Halteria and basal to Amphiesmenoptera. and Ceratophylloidea (Leptopsyllidae + Ceratophyllidae + Maddison et al. (1999) suggested that the taxa Omophron, Ischnopsyllidae) is well supported. However, phylogenetic Metrius, Cicindela, Omus and Clinidium had long branches relationships among fleas are poorly known (Lewis & Lewis for 18S, which may account for their ‘attraction’ (Felsenstein 1985), and so there is no specific morphological hypothesis 1978) to these other lineages. However, exclusion of these with which these molecular data can be readily compared. taxa from this data set still yields a paraphyletic Coleoptera, Within Mecoptera, the monophyly of Panorpomorpha and with Polyphaga and Adephaga placed far apart on different Panorpiini (Choristidae + (Panorpodidae + Panorpidae)) is portions of the overall topology. supported, but this result disagrees with Willmann (1987) in Constraining the monophyly of Coleoptera produces a the placement of Meropeidae as sister group to Bittacidae, topology largely congruent with the unconstrained tree for rather than in Opisthogonomorpha. the non-beetle taxa (Fig. 5). It requires 36 additional steps The second lineage comprises a paraphyletic Coleoptera to constrain Coleoptera as monophyletic, and an increase of and a monophyletic Amphiesmenoptera and Halteria. 0.4% to the overall tree length. Coleoptera is placed as sister

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Fig. 6 Strict consensus of the six most parsimonious trees (L = 9389, CI = 0.41, RI = 0.68) from the 18S rDNA alignment with blocked variable regions included and the Coleoptera forced as monophyletic. Forcing coleopteran monophyly requires 36 additional steps, an increase of 0.4% to the overall tree length. Fig. 5 Summary trees based on the strict consensus trees for parsimony analysis of 18S rDNA. The conserved region analysis excluded the variable blocked regions of the alignment, and produced 19 678 re-analysis of the more extensive Whiting et al. (1997) data trees (L = 7152, CI = 0.37, RI = 0.70). The entire region analysis set by Huelsenbeck (1998), with likelihood methods that generated the topology in Fig. 4. These trees differ in the place- account for rate heterogeneity, could neither support nor ment of Hymenoptera relative to Mecoptera + Siphonaptera, and refute this hypothesized sister group relationship. Hwang et al. the manner in which Coleoptera is paraphyletic across the overall (1998) approached the ‘Strepsiptera problem’ by generating topology. sequence data for a portion of 28S and 5.8S for a small sample of holometabolous taxa (11 exemplars). They found that these group to Amphiesmenoptera + Halteria in the constrained data supported Halteria when analysed via parsimony, but tree, although this is rather poorly supported with low boot- that they did not support Halteria when analysed via maxi- strap and Bremer values. This analysis supports a mono- mum likelihood, and attributed this result to long-branch phyletic Polyphaga (as in the unconstrained tree), but Adephaga attraction. However, as their data in fact supported no inter- is unresolved, as are the relationships among the other two ordinal holometabolous relationships (as indicated by their coleopteran suborders. fully unresolved consensus cladogram for holometabolan As in all molecular analyses to date, Amphiesmenoptera phylogeny), they were unable to retrieve even those groups was well supported and recovered in this analysis with high well supported in other molecular and morphological ana- Bremer and bootstrap values. The monophyly of Lepidoptera lyses. These results add very little towards deciphering was also well supported, although Glossata is paraphyletic holometabolous relationships, much less the phylogenetic with Eriocrania placed as the most basal taxon. The majority position of Strepsiptera. Regardless of what side one happens of lepidopteran taxa sampled have only been sequenced for to take in the Strepsiptera controversy, it is clear that addi- about 1 kb of 18S, and so inferred relationships may change tional data are needed to put the issue to rest. The current with the addition of missing sequence data. Trichoptera was analysis includes two species of what is presumed to be the also well supported as a monophyletic order, although the most primitive strepsipteran genus Mengenilla, although in sampling of caddisfly taxa was rather sparse. this analysis they are not placed as the basal-most strep- The controversial group Halteria (Strepsiptera + Diptera) sipteran taxon. Within Diptera, Brachycera, Cyclorrhapha was supported in this analysis. The group was first proposed and Tipulidae are all well-supported monophyletic groups, by Whiting & Wheeler (1994) and subsequently attributed but Nematocera is paraphyletic. to long-branch attraction (Felsenstein 1978; Carmean & When the variable blocked alignment regions are excluded Crespi 1995; Huelsenbeck 1997). I have argued elsewhere from the analysis and only the conserved regions are that this relationship is most congruent with morphological retained, the same general topology of interordinal relation- data, and that it should not be surprising to find sister taxa ships is produced, with the exception that Hymenoptera with elevated substitution rates (Whiting 1998a,b; Sidall & and Mecoptera + Siphonaptera switch positions on the tree Whiting 1999). Indeed, despite earlier claims that this is the (Fig. 6). Coleoptera is still grossly paraphyletic with the classic case of long-branch attraction (Huelsenbeck 1997), suborders attaching in the same position as in the entire

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ticularly at the higher levels (reviewed in Hillis 1998), the difficulty of adequately sampling Holometabola has received little attention. Holometabola encompasses what is the most speciose and arguably the most diverse group of organisms on Earth, and it is a challenge to adequately sample the diversity of this clade. This analysis used 147 ingroup sequences to represent Holometabola. While this is an improvement over earlier studies, it must be acknowledged that the current number of samples equates to one sequenced species representing the diversity of about 58 000 species (Wilson 1988). Thus one might anticipate that inadequate taxon sampling Fig. 7 Summary tree for holometabolan phylogeny based only on 18S may lead to unusual results in the phylogeny, and this may be rDNA. Dotted lines refer to portions of the topology poorly supported the case in the topologies generated above. by the molecular data. The importance of thorough taxon sampling in phylogenetic estimation is perhaps best illustrated by the status of analysis. Removing these blocks reduces resolution within Coleoptera as based on 18S rDNA studies. In the first such Lepidoptera, Trichoptera and Polyphaga, and changes rela- study, Coleoptera was represented by a single sequence tionships within Strepsiptera (Mengenilla is the most basal making it impossible to test for beetle monophyly (Carmean group) and Mecoptera (Apteropanorpa is the most basal et al. 1992). In a second study, Coleoptera was represented mecopteran taxa, excluding Boreidae). Because the overall by two sequences from a single polyphagan superfamily, relationships are the same between the entire and conserved and resulted in Coleoptera forming a monophyletic group analysis, these results are not particularly sensitive to the (Chalwatzis et al. 1996). It now appears that this presumably inclusion/exclusion of blocked variable regions. correct answer was an artifactual result of including only two exemplars of a single superfamily. Indeed, this analysis was a Discussion relatively weak test of beetle monophyly because the diversity This analysis includes more than twice the number of taxa of the order was not characterized as broadly as it might have than the Whiting et al. (1997) analysis, and 50 more taxa than been with one exemplar from each of the major suborders. the Whiting (2001) analysis, yet the results are very similar to Two 18S rDNA studies have done an admirable job of rep- these other analyses. 18S rDNA sequence data do a reason- resenting Adephaga and Polyphaga, although neither study ably good job of supporting the monophyly of each holo- specifically tested for coleopteran monophyly, and they lacked metabolous insect order (with the exception of Coleoptera, as the proper outgroup selection to do so. In the study of Adephaga, discussed below), but do a relatively poor job of supporting the tree was rooted only to Neuropterida (represented by five most interordinal relationships, particularly those at the deeper sequences; Maddison et al. 1999) on the assumption that this nodes of the phylogeny. Amphiesmenoptera, Halteria and sister group relationship was well supported by morphology, Siphonaptera + Mecoptera are well supported (Bremer > 4, although it is based on a single character suite which may or bootstrap > 95), Raphidioptera + Megaloptera and Neuropterida may not be reliable (Mickoleit 1973; Whiting et al. 1997). In are moderately supported (Bremer > 3, bootstrap > 60), but the study of Polyphaga, the tree was rooted to other beetle other interordinal relationships are poorly supported. Con- taxa from within Polyphaga (Farrell 1998), and so beetle mono- trary to morphological data, there is no evidence from 18S rDNA phyly could not be tested. However, as demonstrated above to support the monophyly of Antliophora or Mecopterida. (in agreement with Whiting et al. 1997 and Caterino et al. These results also suggest that Siphonaptera is nested within 2002), when a wide range of beetle and outgroup taxa are Mecoptera, although the exact position of the fleas relative included, the disturbing result is a paraphyletic Coleoptera, to Mecoptera is not well supported with these data. In some generally with Polyphaga forming one clade and Adephaga cases, the 18S rDNA data elucidate the same basic pattern of forming another, but each placed apart on the topology. While relationships within each order as supported by other data I agree with Hennig (1981) that ‘the Coleoptera are as well (e.g. in Lepidoptera, Strepsiptera, Diptera, Siphonaptera and founded a monophyletic group that we could ever hope to Mecoptera), although in two cases the results directly con- find’ (p. 300), the point is that the current 18S rDNA data do tradict other data (Hymenoptera and Coleoptera). Fig. 7. not actually support a monophyletic Coleoptera, although These results highlight some of the difficulties associated under some sampling strategies and in some analyses they with inferring higher level phylogenetic relationships from have appeared to do so. This lack of support may be due molecular data. While it is generally recognized that taxon in large part to the difficulty of adequately representing sampling plays a critical role in phylogeny estimation, par- coleopteran diversity in a molecular phylogenetic study, at

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least with 18S rDNA. It is tempting to think that the inclusion Aspöck, U. (1995). Neue hypothesen zum System der Neuropterida. of additional taxa would remedy this problem, and it may do Mitteilungen der Deutschen Gesellschaft fur Allgemeine und Angewandte just that, but it is also possible that Coleoptera are so diverse Entomologie, 10, 633–636. Aspöck, U. (2002). Phylogeny of the Neuropterida (Insecta: Holo- that it does not matter how many taxa are sampled, the group metabola). Zoologica Scripta, 31, 51–56. will always be paraphyletic when relationships are inferred Beutel, R. G. & Haas, F. (2000). Phylogenetic relationships of the from 18S rDNA. Notice, however, that other insect orders suborders of Coleoptera (Insecta). Cladistics, 16, 103–142. which are also very large (Lepidoptera and Hymenoptera) Bilinski, S., Bünnig, J. & Simiczyjew, B. (1998). The ovaries of Mecoptera: are supported as monophyletic with the current 18S rDNA basic similarities and one exception to the rule. Folia Histochemica data, so one cannot simply conclude that large and diverse et Cytobiologica, 36, 189–195. insect groups cannot be supported as monophyletic with 18S Boudreaux, H. B. (1979). Arthropod Phylogeny with Special Reference to Insects. New York: John Wiley & Sons. rDNA data. Bremer, K. (1994). Branch support and tree stability. Cladistics, 10, One of the frustrations of current insect ordinal system- 295–304. atics is that the information recovered from molecular data Carmean, D. & Crespi, B. J. (1995). Do long branches attract flies? is more redundant with the information from other sources Nature, 373, 666. than we would prefer. While, on the one hand, it is comfort- Carmean, D., Kimsey, L. S. & Berbee, M. L. (1992). 18S rDNA sequences ing that the molecular data recover the monophyly of most and holometabolous insects. Molecular Phylogenetics and Evolution, insect orders, on the other hand these monophyletic groups 1, 270–278. Caterino, M. S., Shull, V. L., Hammond, P. M. & Vogler, A. P. were already well supported and the monophyly of most of (2002). Basal relationships of Coleoptera inferred from 18S rDNA them has never really been in doubt. It is the interordinal sequences. Zoologica Scripta, 31, 41–49. relationships that we are interested in, and they seem to be Chalwatzis, N., Hauf, J., Peer, Y. V. D., Kinzelbach, R. & Zimmerman, F. K. the most difficult to extract from the current molecular data (1996). 18S ribosomal RNA genes of insects: primary structure of with any degree of confidence. Perhaps the greatest problem the genes and molecular phylogeny of the Holometabola. Annals in this study, and that of many other higher level phylogenetic of the Entomological Society of America, 89, 788–803. studies, is the reliance on a single marker for phylogenetic Crowson, R. A. (1960). The phylogeny of Coleoptera. Annual Review of Entomology, 5, 111–134. inference. Systematics seems to have a constant vibrato of the Farrell, B. D. (1998). ‘Inordinate fondness’ explained: why are there superiority of one character system over another, but if the so many beetles? Science, 281, 555–558. history of systematics has taught us only one thing, it is that Felsenstein, J. (1978). Cases in which parsimony or compatibility methods single character systems are nearly guaranteed to fail, at least will be positively misleading. Systematic Zoology, 27, 401–410. in some portion of the topology. This is true whether one Genecodes (1999). Sequencher, Version 3.1.1. Ann Arbor, MI: Gene- uses morphology, molecules or developmental data to infer codes Co. phylogeny. We should not be surprised to find that the 18S Gladstein, D. S. & Wheeler, W. C. (1999). POY : Phylogeny Reconstruc- tion Via Direct Optimization of DNA Data, Version 2.0. New York: rDNA data do a good job on one portion of the topology, but American Museum of Natural History. are rather ill-behaved on other portions; why should they per- Hennig, W. (1981). Insect Phylogeny. New York: Academic Press. form differently from any other character system? The future Hillis, D. M. (1998). Taxonomic sampling, phylogenetic accuracy, of insect molecular systematics lies not only in increasing the and investigator bias. Systematic Biology, 47 (1), 3–8. taxon size for a particular marker, but increasing the range of Huelsenbeck, J. P. (1997). Is the Felsenstein zone a fly trap? System- markers used in phylogenetic inference. The combination of atic Biology, 46, 69–74. careful taxon sampling and careful analyses will undoubtedly Huelsenbeck, J. P. (1998). Systematic bias in phylogenetic analysis: is the Strepsiptera problem solved? Systematic Biology, 47, 519–537. lead to greater insights into the evolution of the most diverse Hwang, U. W., Kim, W., Tautz, D. & Friedrich, M. (1998). Molecular group of organisms on Earth, the Holometabola. phylogenetics at the Felsenstein zone: approaching the Strepsiptera problem using 5.8S and 28S rDNA sequences. Molecular Phylo- Acknowledgements genetics and Evolution, 9, 470–480. I thank Paige Humphreys, Alison Whiting, Taylor Maxwell Kathirithamby, J. (1989). Review of the order Strepsiptera. Systematic and Matthew Gruwell for assistance in generating the sequence Entomology, 14, 41–92. data, and Matthew Terry, Heath Ogden and Jason Cryan for Kinzelbach, R. K. (1990). The systematic position of Strepsiptera comments on the manuscript. This work was supported by (Insecta). American Entomologist, 36, 292–303. Kristensen, N. P. (1975). The phylogeny of hexapod ‘orders’. A critical NSF grants DEB-9615269 and DEB-9806349, and NSF review of recent accounts. Zeitschrift für die Zoologische Systematik CAREER award DEB-9983195. und Evolutionsforschung, 13, 1–44. Kristensen, N. P. (1981). Phylogeny of insect orders. Annual Review References of Entomology, 26, 135–157. Achtelig, M. (1975). Die Abdomenbasis der Neuropteroidea (Insecta, Kristensen, N. P. (1991). Phylogeny of extant hexapods. In CSIRO Holometabola). Zoomorphologie, 82, 201–242. (Ed.) The Insects of Australia: a Textbook for Students and Research

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Workers, 2nd edn (pp. 125–140). Melbourne: CSIRO and Melbourne (*and Other Methods), Version 4.0b4. Sunderland, MA: Sinauer University Press. Associates. Kristensen, N. P. (1995). Fourty [sic] years’ insect phylogenetic sys- Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). Clustal W: tematics. Zoologische Beiträge Neue Folge, 36, 83–124. Improving the sensitivity of progressive multiple sequence align- Kristensen, N. P. (1997). Early evolution of the Lepidoptera + ment through sequence weighting, positions speciﬁc gap penalties Trichoptera lineage: phylogeny and the ecological scenario. Mémoires and weight matrix choice. Nucleic Acids Research, 22, 4673–4680. du Muséum National d’Histoire Naturelle, 173, 253–271. Wheeler, W. (1999). Fixed character states and the optimization of Kristensen, N. P. (1999). Phylogeny of endopterygote insects, the molecular sequence data. Cladistics, 15, 379–386. most successful lineage of living organisms. European Journal of Wheeler, W. C. & Gladstein, D. L. (1994). MALIGN, Version 1.93. Entomology, 96, 237–253. New York: American Museum of Natural History. Kukalova-Peck, J. & Lawrence, J. F. (1993). Evolution of the hind Whiting, M. F. (1998a). Long-branch distraction and the Strepsiptera. wing in Coleoptera. Canadian Journal of Entomology, 125, 181–258. Systematic Biology, 47, 134–138. Lewis, R. E. & Lewis, J. H. (1985). Notes on the geographical dis- Whiting, M. F. (1998b). Phylogenetic position of the Strepsiptera: tribution and host preferences in the order Siphonaptera. Journal review of molecular and morphological evidence. International Journal of Medical Entomology, 22 (2), 134–152. of Insect Morphology and Embryology, 27, 53–60. Maddison, D. R., Baker, M. D. & Ober, K. A. (1999). Phylogeny of Whiting, M. F. (2001). Phylogeny of the holometabolous insect Carabid beetles as inferred from 18S ribosomal DNA (Coleoptera: orders based on 18S ribosomal data: when bad things happen to Carabidae). Systematic Entomology, 24, 103–138. good data. In R. DeSalle, G. Giribet & W. C. Wheeler (Eds) Mickoleit, G. (1973). Uber den ovipositor der Neuropteroidea Molecular Systematics and Evolution: Theory and Practice, in press. und Coleoptera und seine phylogenetische Bedeutung (Insecta, Whiting, M. F. (2002). Mecoptera is paraphyletic: multiple genes Holometabola). Zeitscrift für Morphological Tiere, 74, 37–64. and phylogeny of Mecoptera and Siphonaptera. Zoologica Scripta, Nixon, K. C. (1999). The parsimony ratchet, a new method for rapid 31, 93–104. parsimony analysis. Cladistics, 15 (4), 407–414. Whiting, M. F., Carpenter, J. C., Wheeler, Q. D. & Wheeler, W. C. Pashley, D. P., McPheron, B. A. & Zimmer, E. A. (1993). Systematics (1997). The Strepsiptera problem: phylogeny of the holometa- of holometabolous insect orders based on 18S ribosomal RNA. bolous insect orders inferred from 18S and 28S ribosomal DNA Molecular Phylogenetics and Evolution, 2, 132–142. sequences and morphology. Systematic Biology, 46, 1–68. Pix, W., Nalbach, G. & Zeil, J. (1993). Strepsipteran forewings are Whiting, M. F. & Kathirithamby, J. (1995). Strepsiptera do not share haltere-like organs of equilibrium. Naturwissenschaften, 80, 371– hind-wing venational synapomorphies with the Coleoptera. Journal 374. of the New York Entomological Society, 103, 1–14. Schlein, Y. (1980). Morphological similarities between the skeletal Whiting, M. F. & Wheeler, W. C. (1994). Insect homeotic transforma- structures of Siphonaptera and Mecoptera. In: Traub, R. and tion. Nature, 368, 696. Starcke, H., eds. Proceedings of the International Conference on Fleas Willmann, R. (1987). The phylogenetic system of the Mecoptera. (pp. 359–367). Rotterdam: A. A. Balkerma. Systematic Entomology, 12, 519–524. Sidall, M. E. & Whiting, M. F. (1999). Long-branch abstractions. Wilson, E. O. (1988). The current state of biological diversity. Cladistics, 15, 9–24. In E. O. Wilson (Ed.) Biodiversity (pp. 3–18). Washington DC: Swofford, D. L. (2000). PAUP*: Phylogenetic Analysis Using Parsimony National Academy Press.

Appendix 1 List of taxa used in the analysis with 18S sequence accession numbers.

Order Family Name 18S

Ephemeroptera Ephemerellidae Ephemerella sp. Walsh 1863 U65107 Odonata Calopterygidae Calopteryx maculata (Beauvois 1805) U65108 Odonata Libellulidae Libellula pulchella Drury 1773 U65109 Embioptera Oligotomidae Oligotoma saundersii (Westwood 1837) U65117 Embioptera Teratembiidae Diradius vandykei (Ross 1944) AF423802 Phasmida Timematidae Timema knulli Strohecker 1951 AF423806 Orthoptera Tettigoniidae Pterophylla camellifolia (Fabricius 1775) AF423804 Orthoptera Stenopelmatidae Stenopelmatus sp. Burmeister 1838 AF423792 Orthoptera Acrididae Melanoplus sp. Stål 1873 AF423803 Orthoptera Tetrigidae Paratettix toltecus (Saussure 1861) AF423791 Blattodea Blaberidae Blaberus sp. Serville 1831 U65112 Blattodea Blaberidae Gromphadorhina portentosa (Burmeister 1838) AF423763 Mantodea Mantidae Tenodera aridifolia (Stoll 1813) AF423805 Mantodea Mantidae Mantis religiosa (L. 1758) U65113 Isoptera Rhinotermitidae Reticulitermis tibialis Banks 1920 AF423782 Phthiraptera Philopteridae Columbicola columbae (L. 1758) AF423794 Phthiraptera Menoponidae Menacanthus sp. Neumann 1912 AF423796 Phthiraptera Polyplacidae Neohaematopinus sciuri (Mjöberg 1910) AF423798

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Appendix 1 Continued

Order Family Name 18S

Psocodea Psocidae Cerastipsocus venosus (Burmeister 1839) U65118 Psocodea Caeciliusidae Valenzuela sp. Mockford 1979 AF423793 Hemiptera Cicadidae Okanagana utahensis Distant 1914 U06478 Hemiptera Cercopidae Prosapia plagiata (Say 1823) U16264 Hemiptera Cicadellidae Graphocephalus atropunctata (Signoret 1854) U15213 Hemiptera Membracidae Spissistilus festinus (Say 1823) U06477 Hemiptera Delphacidae Prokelisia marginata Osborn 1914 U09207 Hemiptera Cixiidae Olarius hesperinus Van Duzee 1917 U15215 Hemiptera Dictyopharidae Scolops fumida (Uhler 1891) U15216 Hemiptera Issidae Hysteropterum severini Caldwell & DeLong 1948 U15214 Hemiptera Flatidae Siphanta acuta (Walker 1851) U06481 Hemiptera Peloridiidae Hemiowoodwardia wilsoni (Say 1825) AF131198 Hemiptera Gerridae Aquarius remigis (Say 1823) U15691 Hemiptera Saldidae Saldula pallipes (Fabricius 1794) U65121 Hemiptera Notonectidae Buenoa sp. Kirkaldy 1904 U65120 Hemiptera Lygaeidae Lygus lineolaris (Palisot 1832) U65122 Hemiptera Pentatomidae Rhaphigaster nebulosa (Poda 1865) X89495 Coleoptera Cupedidae Distocupes sp. Neboiss 1984 AF201421 Coleoptera Torrindicolidae Torridincola sp. Spangler 1934 AF201420 Coleoptera Hydroscaphidae Hydroscapha natans LeConte 1874 AF012525 Coleoptera Trachypachidae Trachypachus gibbsii LeConte 1861 AF002808 Coleoptera Carabidae Cicindela s. sedecimpunctata Klug 1834 AF012518 Coleoptera Carabidae Oxycheila nigroaenea Bates 1914 AF201393 Coleoptera Carabidae Metrius contractus Eschscholtz 1829 AF012515 Coleoptera Carabidae Clinidium calcaratum LeConte 1870 AF012521 Coleoptera Carabidae Omophron obliteratum Horn 1870 AF012513 Coleoptera Carabidae Omus californicus Eschscholtz 1829 AF012519 Coleoptera Haliplidae Haliplus sp. Latreille 1802 AF199516 Coleoptera Hygrobiidae Hygrobia sp. Latreille 1817 AF199523 Coleoptera Amphizoidae Amphizoa sp. LeConte 1853 AF199520 Coleoptera Noteridae Suphis inflatus (LeConte 1863) AF012523 Coleoptera Dytiscidae Copelatus chevrolati Aube 1838 AF012524 Coleoptera Gyrinidae Orectochilus sp. Lacordaire 1873 AF199513 Coleoptera Hydrophilidae Helochares lividus Mulsant 1844 AF201418 Coleoptera Histeridae Hololepta sp. Paykull 1811 AF423765 Coleoptera Staphylinidae Xanthopyga cacti Horn 1868 AF002810 Coleoptera Scirtidae Cyphon hilaris Klausnitzer 1976 AF201419 Coleoptera Clambidae Clambus arnetti Endrody-Younga 1981 AF012526 Coleoptera Trogidae Trox sp. Fabricius (1775) AF423774 Coleoptera Scarabaeidae Dynastes granti Horn 1870 AF002809 Coleoptera Scarabaeidae Xylorcytes faunus Hope 1837 U65127 Coleoptera Buprestidae Acmaeodera sp. Eschscholtz 1829 AF423771 Coleoptera Elateridae Octinodes sp. Candeze 1863 U65128 Coleoptera Elateridae Cardiophorus sp. Eschscholtz 1829 AF423776 Coleoptera Lycidae Calopteron sp. Guerin-Meneville 1830 AF423764 Coleoptera Lampyridae Photuris pennsylvanica De Geer 1774 U65129 Coleoptera Cantharidae Podabrus sp. Westwood 1838 AF423770 Coleoptera Bostrichidae Apatides sp. Casey 1898 AF423766 Coleoptera Anobiidae Ptinus sp. L. 1767 AF423772 Coleoptera Cleridae Trichodes ornatus Say (1823) AF423775 Coleoptera Nitidulidae Amphotus sp. Erichson 1843 AF423768 Coleoptera Cucujidae Cucujus clavipes Fabricius 1777 AF423767 Coleoptera Rhipiphoridae Rhipiphorus fasciatus (Say 1823) U65130 Coleoptera Meloidae Meloe proscarabaeus (Linnaeus 1758) X77786 Coleoptera Tenebrionidae Eleodes sulcipennis Blaisdell 1909 AF423769 Coleoptera Cerambycidae Tetraopes tetropthalmus (Forster 1771) U65131 Coleoptera Cerambycidae Oberea sp. Mulsant 1839 AF423773 Coleoptera Nemonychidae Mecomacer sp. Kuschel 1954 AF250069

Holometabolan phylogeny • M. F. Whiting

Appendix 1 Continued

Order Family Name 18S

Coleoptera Belidae Oxycraspedus cornutus Zimmerman 1994 AF250068 Coleoptera Attelabidae Apoderus giraffa Suffrian 1870 AF250065 Coleoptera Brentidae Apion sp. Herbst 1797 AF250060 Coleoptera Curculionidae Polydrusus sericeus (Schaller 1783) AF250086 Coleoptera Platypodidae Platypus sp. Herbst 1793 AF250077 Coleoptera Scolytidae Ips grandicollis (Eichhoff 1868) AF250074 Megaloptera Sialidae Sialis sp. Latreille 1802 X89497 Raphidioptera Raphidiidae Agulla adnixa (Hagen 1861) AF286301 Raphidioptera Inoceliidae Nehga inflata (Hagen 1861) AF286272 Neuroptera Ithonidae Oliarces clara Banks 1908 AF012527 Neuroptera Berothidae Lomamymia texana (Banks 1897) U65134 Neuroptera Mantispidae Mantispa pulchella (Banks 1912) U65135 Neuroptera Hemerobiidae Hemerobius stigmata Stephens 1835 U65136 Neuroptera Hemerobiidae Micromus sp. Rambur 1842 AF423789 Neuroptera Hemerobiidae Hemerobius sp. L. 1758 AF423790 Neuroptera Chrysopidae Anisochrysa carnea Brauer 1851 X89482 Neuroptera Chrysopidae Eremochrysa tibialis Banks 1950 AF423788 Neuroptera Ascalaphidae Haplogenius appendiculatus (Fabricius 1793) AF423787 Neuroptera Myrmeleontidae Myrmeleon immaculatus De Geer 1773 U65137 Hymenoptera Cephidae Hartigia cressonii (Kirby 1882) L10173 Hymenoptera Orussidae Orussus thoracicus Ashmead 1898 L10174 Hymenoptera Tenthredinidae Hemitaxonus sp. Ashmead 1898 U65150 Hymenoptera Tenthredinidae Dolerus sp. Panzer 1801 AF423781 Hymenoptera Trigonalyidae Bareogonalos canadensis (Harrington 1896) L10176 Hymenoptera Evaniidae Evania appendigaster (Linnaeus 1758) L10175 Hymenoptera Ichneumonidae Ophion sp. Fabricius 1798 U65151 Hymenoptera Ichneumonidae Megarhyssa sp. Ashmead 1900 AF423779 Hymenoptera Braconidae Trioxys pallidus (Haliday 1833) AJ009351 Hymenoptera Pteromalidae Mesopolobus sp. Westwood 1833 L10177 Hymenoptera Bethylidae Epyris sepulchralis Evans 1969 L10180 Hymenoptera Chrysididae Caenochrysis doriae (Gribodo 1874) L10179 Hymenoptera Pompilidae Priocnemus oregana Banks 1933 L10181 Hymenoptera Mutillidae Dasymutilla gloriosa (Saussure 1868) U65152 Hymenoptera Scoliidae Campsomeris sp. Guerin-Meneville 1838 AF423780 Hymenoptera Vespidae Apoica sp. (Fabricius 1775) U65153 Hymenoptera Vespidae Monobia quadridens (L. 1763) U65154 Hymenoptera Formicidae Doronomyrmex kutteri Wheeler 1906 X73274 Lepidoptera Micropterigidae Micropterix calthella (L. 1761) AF136883, AF136863 Lepidoptera Agathiphagidae Agathiphaga queenslandensis (Grote 1865) AF136884, AF136864 Lepidoptera Heterobathmiidae Heterobathmia pseuderiocrania (Peck 1818) AF136887, AF136867 Lepidoptera Eriocraniidae Eriocrania semipurpurella (Stephens 1834) AF136886, AF136866 Lepidoptera Hepialidae Sthenopis quadriguttatus (Grote 1864) AF136891, AF136871 Lepidoptera Prodoxidae Tegeticula yuccasella (Riley 1872) AF136889, AF136869 Lepidoptera Psychidae Thyridopteryx ephemeraeformis (Haworth 1803) AF136894, AF136874 Lepidoptera Tineidae Tineola bisselliella (Hummel 1823) AF136893, AF136873 Lepidoptera Cossidae Prionoxystus robiniae (Peck 1818) AF423783 Lepidoptera Pterophoridae Platyptilia sp. Hübner 1825 AF423784 Lepidoptera Pyralidae Galleria mellonella (L. 1758) AF286298 Lepidoptera Papilionidae Papilio troilus L. 1758 AF286299 Lepidoptera Pieridae Anthocharis sara Lucas 1852 AF423785 Lepidoptera Saturniidae Hemileuca sp. Walker 1855 AF286273 Lepidoptera Sphingidae Hyles lineata (Fabricius 1775) AF423786 Lepidoptera Lymantriidae Lymantria dispar (L. 1758) AF136892, AF136872 Lepidoptera Noctuidae Ascalapha odourata (L. 1758) U65140 Trichoptera Philopotamidae Wormaldia moesta (Banks 1914) AF136881, AF136861 Trichoptera Brachycentridae Brachycentrus nigrosoma (Banks 1905) AF136880, AF136860 Trichoptera Limnephilidae Pycnopsyche lepida (Hagen 1861) AF286292 Trichoptera Limnephilidae Hydropsyche sp. Pictet 1834 AF286291

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M. F. Whiting • Holometabolan phylogeny

Appendix 1 Continued

Order Family Name 18S

Trichoptera Hydroptilidae Oxyethira dualis Morton 1905 AF423801 Trichoptera Leptoceridae Oecetis avara Banks 1895 AF286300 Siphonaptera Pulicidae Tunga monositus Barnes & Radovsky 1969 AF286279 Siphonaptera Rhopalopsyllidae Polygenis pradoi (Wagner 1937) AF286277 Siphonaptera Hystricopsyllidae Hystrichopsylla t. talpae (Curtis 1826) AF286281 Siphonaptera Coptopsyllidae Coptopsylla africana Wagner 1932 AF286275 Siphonaptera Pygiopsyllidae Acanthopsylla r. rothschildi (Rainbow 1805) AF286283 Siphonaptera Stephanocircidae Craneopsylla minerva wolffheugeli (Rothschild 1909) AF286286 Siphonaptera Ctenophthalimidae Megarthroglossus divisus (Baker 1898) AF286276 Siphonaptera Ischnopsyllidae Myodopsylla palposa (Rothschild 1904) AF286282 Siphonaptera Leptopsyllidae Frontopsylla nakagawai Liu, Wu, & Chang 1986 AF286280 Siphonaptera Ceratophyllidae Orchopeas sexdentatus (Baker 1904) AF286274 Mecoptera Nannochoristidae Nannochorista dipteroides Tillyard 1917 AF334799 Mecoptera Boreidae Boreus colouradensis Byers 1955 AF286285 Mecoptera Boreidae Caurinus dectes Russell 1979 AF286288 Mecoptera Meropeidae Merope tuber Newman 1838 AF286287 Mecoptera Bittacidae Bittacus strigosus Hagen 1861 AF286290 Mecoptera Apteropanorpidae Apteropanorpa evansi Yeates & Byers 1999 AF286284 Mecoptera Choristidae Chorista australis Klug 1838 AF286289 Mecoptera Panorpodidae Brachypanorpa carolinensis Banks 1905 AF286296 Mecoptera Panorpidae Panorpa takenouchii Miyake 1908 AF286278 Diptera Tipulidae Tanyptera dorsalis (Walker 1848) AF286295 Diptera Tipulidae Epiphragma fasciapenne (Say 1823) AF286294 Diptera Tipulidae Holorusia rubiginosa Loew 1863 AF423778 Diptera Tipulidae Dolichopeza subalbipes (Johnson 1909) AF286297 Diptera Tipulidae Nephrotoma altissima (Osten Sacken 1877) U48379 Diptera Chaoboridae Corethrella wirthi Stone 1968 U49736 Diptera Dixidae Dixella cornuta (Johannsen 1923) U48381 Diptera Chironomidae Ablabesmyia rhamphe Sublette 1964 U48384 Diptera Simuliidae Simulium vittatum Zetterstedt 1838 U48383 Diptera Psychodidae Lutzomyia shannoni (Dyar 1929) U48382 Diptera Psychodidae Phlebotumus papatasi Franca 1924 AF145187 Diptera Tabanidae Chrysops niger Macquart 1838 AF073889 Diptera Asilidae Laphria sp. Meigen 1803 AF286293 Diptera Mydidae Mydas clavatus (Drury 1773) AF423777 Diptera Bombyliidae Mythicomyia atra Cresson 1915 U65158 Diptera Tephritidae Ceratitis capitata (Wiedemann 1824) AF096450 Diptera Tephritidae Anastrepha sp. (Schiner 1868) U01248, U01249 AF114472, AF187101 Diptera Drosophilidae Drosophila melanogaster Meigen 1830 M21017 Diptera Hippoboscidae Ornithoica vicina (Walker 1849) AF073888 Strepsiptera Mengenillidae Mengenilla chobauti Hofeneder 1910 X89441 Strepsiptera Mengenillidae Mengenilla chobauti. 1910 AF423800 Strepsiptera Corioxenidae Triozocera mexicana Pierce 1909 U65159 Strepsiptera Myrmecolacidae Caenocholax fenyesi Pierce 1909 U65160 Strepsiptera Elenchidae Elenchus japonica Esaki & Hashimoto 1931 U65162 Strepsiptera Stylopidae Xenos vesparum Rossius 1793 X77784 Strepsiptera Stylopidae Stylops melittae Pierce 1909 X89440 Strepsiptera Stylopidae Xenos peckii Kirby 1813 U65164 Strepsiptera Stylopidae Crawfordia n.sp. U65163