The Pupae of Culicomorpha—Morphology and a New Phylogenetic Tree

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ZOOTAXA

3396

Art Borkent

Research Associate, Royal British Columbia Museum, American Museum of Natural History and Instituto Nacional de Biodiversidad, 691-8th Ave. SE, Salmon Arm, British Columbia, V1E 2C2, Canada. (e-mail: [email protected])

Magnolia Press Auckland, New Zealand

Accepted by J.K. Moulton: 22 Mar. 2012; published: 23 Jul. 2012 Art Borkent The Pupae of Culicomorpha—Morphology and a New Phylogenetic Tree (Zootaxa 3396) 98 pp.; 30 cm. 23 Jul. 2012 ISBN 978-1-86977-957-3 (paperback) ISBN 978-1-86977-958-0 (Online edition)

FIRST PUBLISHED IN 2012 BY Magnolia Press P.O. Box 41-383 Auckland 1346 New Zealand e-mail: [email protected] http://www.mapress.com/zootaxa/

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ISSN 1175-5326 (Print edition) ISSN 1175-5334 (Online edition)

Abstract ...... 3 Introduction ...... 4 Materials And Methods ...... 5 Acknowledgments ...... 6 Glossary of the Structures of the Pupae of Culicomorpha ...... 6 Adult Emergence ...... 21 Pupal Key to the Families of Culicomorpha ...... 22 Description of Ptychopteridae and Families of Culicomorpha ...... 23 Ptychopteridae ...... 23 Culicomorpha ...... 24 Chironomidae ...... 24 Ceratopogonidae ...... 26 Thaumaleidae ...... 27 Simuliidae ...... 28 Dixidae ...... 30 Corethrellidae ...... 31 Chaoboridae ...... 32 Culicidae ...... 33 Phylogenetic Analysis ...... 34 Eggs ...... 35 Larvae ...... 36 Pupae ...... 39 Adults ...... 48 Misinterpreted or Questionable Synapomorphies ...... 52 Conflicting Synapomorphies ...... 55 Phylogenetic Conclusions ...... 55 Bionomic Divergence ...... 60 References ...... 63

ABSTRACT

The pupae of each of the families of the Culicomorpha are described and, for the first time, their structures homologized. A glossary provides a standard set of terms to be applied to each structure, including a common chaetotaxy. A cladistic analysis incorporates information from each life stage, including a number of new features discovered from the pupal stage, to provide a new phylogenetic hypothesis, as well as indicating autapomorphies for each family. Analysis included states for one egg, 21 larval, 33 pupal, and 37 adult characters. The Chironomidae is the sister group of all remaining Culicomorpha, the Ceratopogonidae is the sister group of Thaumaleidae + Simuliidae and these three are newly recognized as members of the re-defined superfamily Simulioidea. The superfamily Culicoidea are the sister group of the Simulioidea and include, as previous work has already demonstrated, the Dixidae as the sister group of Corethrellidae + Chaoboridae + Culicidae. Corethrellidae is the sister group of Chaoboridae + Culicidae. The superfamily Chironomoidea now includes only Chironomidae. Analysis of the fossil record shows that the Chironomidae (and the Culicomorpha) originated in the Triassic and both Simulioidea and Culicoidea were present by 176 million years ago in the Jurassic. Phylogenetic patterns are used to interpret bionomic features such as differences in the nature of blood-feeding by adult females, daytime or nighttime feeding by adult females, and occurrence of immature stages in aquatic habitats. Chironomidae do not feed on blood as adults and have likely diversified by invading virtually all aquatic habitats as larvae. Its sister group is more than twice as diverse and feeding on vertebrate blood is strongly correlated with high diversification within the Simulioidea + Culicoidea (likely because a reliable source of protein was available to dispersing females since the Triassic from terrestrial vertebrates). Families with blood-feeding females have larger numbers of species than do those without this behaviour. Each family in the Simulioidea + Culicoidea have specialized larval habitats or specialized habits, largely in aquatic habitats where Chironomidae are either not, or are marginally present, suggesting a level of competitive exclusion by the Chironomidae.

Key Words: Chironomidae, Ceratopogonidae, Thaumaleidae, Simuliidae, Dixidae, Corethrellidae, Chaoboridae, Culicidae, aquatic, phylogeny, pupal homologies, key, glossary, blood-feeding, diversification, egg, larva, pupa, adult.

One of the joys but also one of the great challenges of being an entomologist is the remarkably vast remaining ignorance regarding our cherished organisms. Diptera is a case in point. Largely due to the remarkable contributions of the three volumes of the Manual of Nearctic Diptera (McAlpine et al. 1981; McAlpine et al. 1987; McAlpine & Wood 1989), we have a consistent set of morphological terms applied to the larvae and adults of the order, based on careful study of homologous structures. The pupae of Diptera, however, are another matter. They were not included in that massive effort (other than within some family treatments) and this stage has been largely ignored by subsequent major treatises (e.g. Papp & Darvas 1997, 1998, 2000a, 2000b; Brown et al. 2009). As such, there has never been a concerted effort to homologize the morphological details of pupae at the familial level; nor do we have a good key to the pupae of the families. In this paper, I have carved out a slice of that diversity and interpret the pupae of the Culicomorpha. At present, there is a great disparity in the vocabulary applied to the pupal structures of the included families. Researchers of the most diverse families or groups of Culicomorpha, namely the Simuliidae, Chironomidae, Ceratopogonidae and the superfamily Culicoidea have each developed independent sets of terms. The homologies and associated terms shared by Culicidae, Chaoboridae, Corethrellidae and Dixidae are primarily due to the historical inclusion of all four in the Culicidae and their subsequent treatment by Belkin and his associates (e.g. Belkin et al. 1970), who described the pupae of the latter three families as part of their primary studies of Culicidae. This study originated, in part, from my continuing quest to better understand the phylogenetic relationships between the genera of Ceratopogonidae. For the past number of years I have gathered and studied the immature stages of this family as a basis for a comprehensive analysis. An important barrier to the phylogenetic interpretation of pupal features was the question of outgroup comparisons with other families of Culicomorpha. Therefore I examined material of each of the other families of Culicomorpha in an attempt to interpret homologies. As a consequence, I provide a single set of terms for the pupae of Culicomorpha, based on my understanding of their homology and also provide a new key for their identification to the family level. As I studied the pupae of other culicomorph families, it rapidly became clear there were significant morphological differences between the families and this provided the impetus to study even a broader outgroup. This broader survey resulted in the discovery of new and important synapomorphies grouping various families within the infraorder that challenge earlier conclusions regarding relationships. When Wood & Borkent (1989) published their study of the phylogenetic relationships between the families of nematocerous Diptera they wrote that "The monophyly of the Culicomorpha, along with the relationships between its component families, is one of the least contentious issues in the phylogenetic interpretation of the nematocerous Diptera." It is true that the composition of the Culicoidea and the relationships between its component families, Dixidae, Corethrellidae, Chaoboridae and Culicidae, has remained stable and appears secure. Some studies of the relationships between the remaining families, Thaumaleidae, Simuliidae, Chironomidae and Ceratopogonidae traditionally placed in the Chironomoidea, have confirmed the arrangement by Wood & Borkent (1989) (Oosterbroek & Courtney 1995; Borkent & McKeever 1990; Beckenbach & Borkent 2003; Sinclair et al. 2007; Borkent et al. 2008; Woodley et al. 2009). However, these were based on only a few features and largely included the previous synapomorphies proposed by Wood & Borkent (1989). Other studies, some based solely on molecular information, have come to significantly different conclusions (Pawlowski et al. 1996; Miller et al. 1997; Sæther 2000; Blagoderov et al. 2007; Bertone et al. 2008; Cranston et al. 2012). Most recently, Wiegmann et al. (2011) incorporated both molecular and morphological data in an analysis that resulted in yet another unique arrangement of the families. The latter study, dominated by molecular data, was based on a very small sample of exemplars, and failed to explicitly document the morphological evidence for any node, so that the results cannot be further appraised using cladistic argumentation (i.e. by reexamination of proposed homologies). A strong motivation for writing this paper, therefore, was to provide further character states and reappraise previous hypotheses in order to better understand the phylogenetic relationships between the families of the Culicomorpha. Aside from the intrinsic value of reexamining a group presently in flux, it is important to remember that these families include the most medically important families of Diptera. As a group, the Culicidae, Ceratopogonidae and Simuliidae have more vectors of various human and animal diseases than any other group of insects (Marquardt 2005; Adler 2009). Further to this, the infraorder, with 18,103 described extant species (Fig. 30; see references in figure caption), includes highly diverse families of aquatic Diptera, occupying virtually every non-marine aquatic habitat on the planet. In as much as phylogenetic relationships provide a vital basis for understanding all features of organisms, the continuing study of the phylogeny of the families of Culicomorpha will provide a better understanding of this biologically important and remarkably diverse group of organisms. In

4 · Zootaxa 3396 © 2012 Magnolia Press BORKENT this paper, I propose a novel phylogenetic arrangement of the families, based on numbers of previously unknown pupal features and also including all previously proposed synapomorphies known from every other stage.

MATERIALS AND METHODS

Specimens were examined, studied, measured and drawn using a Wild M3 dissecting microscope and a Zeiss Jenaval compound microscope. Photomicrographs were taken with a Nikon CoolPix 995 through these microscopes. Some photographs were modified in Adobe Photoshop 7.0 and plates were constructed using Word Publisher 2007. To determine pupal homologies between the families of Culicomorpha, original material of each family was examined in glycerine and with two or more pupae lying side by side under both dissecting and compound microscopes, as well as specimens mounted on microscope slides using the method described by Borkent & Spinelli (2007). For each family I studied both whole pupae and their exuviae. For most families I also studied whole pupae with pharate adults, using the position of structures of developing adults to better interpret the homologies of the overlying pupal structures. To determine comparable terms used by modern workers in the different families I especially referred to Belkin et al. (1970), Harbach & Knight (1980) and Ogawa (2007) for Culicidae, Chaoboridae, Corethrellidae and Dixidae; Sæther (1980) and Coffman (1986) for Chironomidae; Crosskey (1990) and Adler et al. (2004) for Simuliidae; Peterson et al. (1989), Sinclair (1992, 1996, 2000), Sinclair &Saigusa (2002) for Thaumaleidae; and Borkent & Craig (2004) for Ceratopogonidae. The terms previously used for Ceratopogonidae vary considerably and will be discussed further by Borkent (in prep.) in a work describing the pupae of all genera of Ceratopogonidae. For outgroup comparisons I examined pupae of the following taxa, arranged according to the phyletic sequence presented in Woodley et al. (2009). In addition, I have listed some useful references for each family that include descriptions of pupae.

Mecoptera: Nannochoristidae: Nannochorista philpotti Tillyard; Pilgrim (1972). Siphonaptera: Parapsyllus longicornis (Enderlein), Xenopsylla cheopis (Rothschild), Ctenocephalides felis (Bouché); Brauns (1954). Tipulidae: Nephrotoma suturalis (Loew); Brauns (1954), Gelhaus & Young (1991). Nymphomyiidae: Nymphomyia walkeri Ide; Courtney (1994a). Deuterophlebiidae: Deuterophlebia coloradensis Pennak, D. inyoensis Kennedy; Courtney (1990, 1994b). Blephariceridae: Blepharicera cherokea Hogue, Blepharicera sp., Agathon elegantulus von Röder, Edwardsina confusa Zwick; Hogue (1981), Zwick (1977). Axymyiidae: Axymyia furcata McAtee; Wood (1981); Wihlm et al. (2012). Pachyneuridae: Pachyneura oculata Krivosheina & Mamaev; Cramptonomyia spenceri Alexander, Haruka elegans Okada. Bibionidae: Bibio albipennis Say, Plecia nearctica Hardy; Sutou (2006). Bolitophilidae: Bolitophila (Cliopisa) sp. Ditomyiidae: Symmerus brevicornis Okada; Munroe (1974). Keroplatidae: Arachnocampa flava Harrison; Matile (1990). Mycetophilidae: Leptomorphus nebulosus (Walker); Brauns (1954). Sciaridae: Bradysia sp., Sciara coprophila Linter; Madwar (1937). Cecidomyiidae: Asphondylia aucubae Yukawa & Ohsaki, 'Mayetiola' thujae Hedlin, Oligotrophus nezu Kikuti, Rhopalomyia yomogicola (Matsumura), Mayetiola destructor (Say); Brauns (1954), Yukawa (1971). Psychodidae: Lutzomyia longipalpis (Lutz & Neiva); Psychoda sp.; Brauns (1954), Hanson (1968). Trichoceridae: Trichocera bifurcata Nakamura & Saigusa; Nakamura & Saigusa (1997). Canthyloscelidae: Peterson & Cook (1981). Anisopodidae: Sylvicola sp., Sylvicola fenestralis Scopoli; Peterson (1981). Tanyderidae: none examined as no specimens are present in any public museum; Alexander (1930), Knight (1964), Wood (1952). Ptychopteridae: specimens as listed below; Alexander (1981). Rhagionidae: Mackey & Brown (1977). Unidentified orthorrhaphous Brachyceran pupa: three from Mt Burnaby, BC, Canada.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 5 The matrix of characters was constructed using Mesquite 2.74 (Maddison & Maddison 2011) for use in the parsimony analysis. The analysis was performed using TNT 1.1 (Goloboff et al. 2003). A heuristic search using parsimony was run with 1000 replicates, saving 100 trees per replicate. The MaxTrees limit was set to 50,000 trees. Tree Bisection Reconnection (TBR) branch swapping was used for the search and branches were collapsed when the maximum length was zero. Characters were treated as unordered in the analysis. Bremer support (Bremer 1994) and bootstrap (Felsenstein 1985) values were calculated using TNT. Suboptimal trees with 1–20 extra steps were used to calculate Bremer support values. Bootstrap values were calculated using the same parameters as in the heuristic search.

ACKNOWLEDGMENTS

Foremost I thank my travelling companion and wife Annette for her incredible support of this study, for her finances, love, and enthusiasm for new discoveries. This project was also supported in part by NSF-DEB-0334948 "AToL: Building the Dipteran Tree: Cooperative Research in Phylogenetics and Bioinformatics of True Flies (Insecta: Diptera)" awarded to B. Wiegmann. While working on this project I very much appreciated the advice of a number of colleagues, particularly Doug Craig (University of Alberta) and Doug Currie (Royal Ontario Museum) on Simuliidae, Ralph Harbach (Natural History Museum) on Culicidae and Pete Cranston (Australian National University) on those hyperdiverse Chironomidae. Morphological and systematic studies depend on specimens and in this case my colleagues helped out with material that was often difficult and time consuming to collect and rear. As such my special thanks goes to the following who sent or lent me specimens that formed much of the basis of my investigation—Greg Courtney (Iowa State University), Doug Craig (University of Alberta), Toyohei Saigusa (Fukuoka, Japan), Chris Borkent (McGill University), Peter Cranston, Robb Bennett (Victoria, BC), Bradley Sinclair (Canadian National Collection), Scott Fitzgerald (Oregon State University), Brian Ma (Simon Fraser University), Junichi Yukawa (Kyushu University), J. Kevin Moulton (University of Tennessee), Joshua Ogawa (California Department of Public Health), Doug Currie (Royal Ontario Museum), Maurizio Cocchi and Irene Raffaelli (Grosseto, Italy), Patrick Ashe (Dublin, Ireland), Andrew Norton (Colorado State University), and Arthur Forer (York University). Roger Crosskey (Natural History Museum, London) kindly loaned me his original photographs of Simulimima grandis Kalugina and Elena Lukashevich and D. Shcherbakov (Russian Academy of Sciences) graciously provided additional photographs that made some structures more apparent. I sincerely thank the following for reviewing this manuscript, or portions of it, before submission to this journal. They provided important suggestions that improved the manuscript substantially: Ralph Harbach, Elena Lukashevich, Jeff Cumming, Doug Currie, Ole Sæther, Peter Cranston, Bill Grogan, Larry Hribar and Patrick Ashe. My sincere gratitude also goes to Bradley Sinclair and an anonymous reviewer who gave detailed critiques of the manuscript that led to substantial improvements. Chris Borkent gave much appreciated help with the parsimony analysis. The Willi Hennig Society provided free access to TNT (Goloboff et al. 2003).

Glossary of the Structures of the Pupae of Culicomorpha

This glossary is primarily for the recognition and explanation of terms used for the descriptions of the pupae of Culicomorpha, although most will apply to all pupae of nematocerous Diptera and orthorrhaphous Brachycera. It is hoped that this glossary will form the basis for a better understanding of all Diptera pupae. Terms are grouped by major body region, head, thorax and abdomen and within each body region there follows an anterior to posterior arrangement of structures. Preferred terms appear in boldface; synonyms for terms in each family of Culicomorpha are given in Table 1. Terms rely heavily on the excellent glossary for adults by Cumming & Wood (2009). In large measure, only the external form of the pupa is described, although for the sake of clarity, underlying features are sometimes discussed. For example, the term 'palpus' here refers only to the external structure of the palpus and not to any interior pupal modifications associated with the developing adult palpus.

6 · Zootaxa 3396 © 2012 Magnolia Press BORKENT TABLE 1. Proposed pupal homologies between Ptychopteridae and families of Culicomorpha as used by other workers in each family. Ptychopteridae have never been described in sufficient detail to include them here but their anatomy is described in the text and figures.

Proposed Chironomidae Ceratopogonidae Thaumaleidae Simuliidae Simuliidae Culicoidea Terms for Culicomorpha Authority Saether (1980); Borkent and Craig Peterson et al. Adler et al. Crosskey Harbach and Wiederholm (2004) (1989) (2004) (1990) Knight (1980) (1986) General body spicules spinules spicules shagreen shagreen microtu- bercles (in part)

muscle mark muscle mark Head head head head head head eye ocular field eye eye compound eye, lateralia antenna antenna antennal antenna antenna sheath dorsal apotome frontal operculum frontoclypeal cephalic head plate dorsal apotome apotome apotome plate dorsolateral posteriolateral pronotum cephalic genae sclerite face clypeus clypeus clypeus labrum labrum labral labrum labrum sheath mandible mandible mandible lacinia maxilla maxilla palpus palpus palp maxillary palpus labium labium labium labium Sensilla dorsal apotome frontal seta anteromedial dorsolateral cephalic trichomes sensilla (dorsal seta trichomes apotomals) dorsolateral not named; anterodorsal 2 setae seta 5-CT, cephalic posterior absent in sclerite sensilla frontal setae of Corethrellidae Brundin 1966; and prefrons of Chaoboridae Cranston et al. 1987 clypeal/labral ventromedial sensilla (clypeal/ labrals) ocular sensilla postorbital 1, ventrolateral setae 1-CT, 2- (oculars) postorbital 2, CT, 3-CT vertical antennal sensilla (antennals) Cephalothorax cephalothorax cephalothorax cephalothorax

Continued on next page...

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 7 TABLE 1. Continued Proposed Chironomidae Ceratopogonidae Thaumaleidae Simuliidae Simuliidae Culicoidea Terms for Culicomorpha Thorax thorax thorax thorax thorax thorax thorax ecdysial suture ecdysial line eclosion slit dorsal ecdysial line prothoracic extension mesonotum scutum mesothorax mesonotum mesosternum respiratory thoracic horn respiratory organ respiratory gill gill trumpet organ horn outer surface reticulate area of respiratory organ plastron, filter plastron plate pores spiracular pits plastron plastron atrial wall, filter apparatus, or apparatus pores basal spiralled felt chamber tracheoid area trachea distal tracheal respiratory felt tube atrium chamber wing wing wing sheath wing wing mesothoracic sheath wing leg (fore-, leg (fore-, mid-, leg sheath leg sheath leg (fore-, leg mid-, hind) hind) (I, II, III) mid-, hind) metathorax metathorax postscutel- metanotum + lar bridge metathoracic wing Sensilla anteromedials median dorsomedial precorneal (3 thoracic wiry sensilla seta 4-CT antepronotals setae) trichomes ("usually (3 setae); (4–7) six") precorneals (3 setae) anterolaterals lateral dorsolaterals setae 6-CT, 7- antepronotals CT (3 setae) dorsals dorsocentrals dorsals i–v dorsocentral trichomes setae 8-CT, 9- (2–5 setae) setae (dc 1–4) CT prealars prealar (1 seta) supraalar supraalar dorsal vi "x"—only in Dixidae, Corethrellidae metathoracics metanotals (3 metathoracic setae 3 dorsal setae setae 10-CT, 11- setae) CT, 12-CT Abdomen abdomen abdomen abdomen abdomen abdomen abdomen tergite tergite tergite tergite tergite tergite tergite sternite sternite sternite sternite sternite sternite sternite membrane conjunctive membrane membrane intersegmental membrane spiracle, spiracle rudimentary rudimentary spiracle spiracle Continued on next page...

8 · Zootaxa 3396 © 2012 Magnolia Press BORKENT TABLE 1. Continued Proposed Chironomidae Ceratopogonidae Thaumaleidae Simuliidae Simuliidae Culicoidea Terms for Culicomorpha genital lobe genital sac genital sac genital lobe terminal anal lobe (both apicolateral caudal hook terminal terminal or paddle process lateral sides process spine tail hook combined) Sensilla - D-1-IV O seta dorsal seta 0-IV segment 4 D-2-IV D1 dasm i seta seta seta 2-IV D-3-IV dasm ii seta seta seta 3-IV D-4-IV unnamed dpm ii D-5-IV D5 dpm i dorsal seta 2 recurved tergal hook hook D-6-IV D4 recurved tergal hook seta 1-IV hook D-7-IV dpm iii abdominal puncture D-8-IV D3 dpm iv recurved tergal hook seta 5-IV hook D-9-IV D2 dpm v dorsal seta 1 recurved tergal hook seta 4-IV hook

L-1-IV L1 lasm lateral seta—L1 seta 8-IV

L-2-IV L2 lpm i lateral set—L3 seta seta seta 6-IV L-3-IV L3 lpm ii seta seta seta 9-IV L-4-IV L4 lpm iii seta seta seta 7-IV V-1-IV unnamed unnamed seta abdominal puncture or seta 14-IV V-2-IV O seta ventral vasm ventral seta V-3-IV V1 seta seta V-4-IV V3 recurved sternal hook hook V-5-IV V5 vn iii recurved sternal hook seta 10-IV hook V-6-IV V4 vn ii seta seta 11-IV V-7-IV V2 vn i Sensilla - D-1-IX L seta segment 9 D-2-IX D-3-IX D-4-IX anal macroseta seta 1-IX D-5-IX anal macroseta D-6-IX anal macroseta L-1-IX L-2-IX L-3-IX L-4-IX V-1-IX seta 2-IX V-2-IX

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 9 The terms used for orientation assume that all pupae lie with the long axis of their thorax and abdomen in a horizontal position, regardless of the position of the pupa in life. Therefore, the underside of the thorax and abdomen are described as ventral. The pupal head is somewhat modified and is variable in position but is mostly tucked at least partially under the front of the thorax. To provide consistent description, the top of the developing adult head is taken as anterior, even if in some pupae this area is anterodorsal or nearly dorsal. Mouthparts are directed posteriorly (with the exception of the palpus of Chironomidae, which is directed laterally) and mouthparts are best seen in a ventral view. In the following descriptions, Simulioidea is newly recognized as including Ceratopogonidae, Thaumaleidae and Ceratopogonidae (Fig. 28A).

Integument and Sensilla In some taxa, the integument has protrusions with various shapes. Elongated swellings that are continuous with surrounding cuticle are referred to as tubercles. They may or may not bear sensilla. Other swellings that are elongate are called ridges or crenulations. Spicules are various minute projections that lack innervation. In addition, the surface of the cuticle may have short bumps and dense but short protrusions that are referred to as shagreen. Setae are innervated sensilla and have sockets. Campaniform sensilla are innervated sensilla that form an area of thin cuticle that are, in Culicomorpha pupae, circular. Borkent & Craig (2004) referred to these as pores. Sensilla in a given area are basically numbered in an anterior to posterior, medial to lateral sequence, with those on the abdomen given additional abbreviations as explained below. Numbering assumes the highest number of sensilla for a particular body area and I have attempted to give the same sensillum number to what may be homologous sensilla in different taxa (in many instances, homologs were unclear and I applied sensilla numbers in a somewhat arbitrary manner). As such, some numbering for a given taxon does not follow an anterior to posterior, medial to lateral sequence because probable homologs have different arrangements. In nearly all instances, it is clear that groups of sensilla are homologous between families, even if an exact one to one homology of a given sensillum is uncertain (because sensilla clearly have moved positions evolutionarily). Even so, each sensillum is named, recognizing that specific homologies will likely be changed in the future. These specific homologies need further testing through broader comparisons with more taxa as well as more detailed morphological and developmental studies. Craig (2005) proposed convincing homologies of larval cephalic sensilla between Simuliidae and Culicidae by using the position of sensilla and by following their innervation. A homologous chaetotaxy of larvae and pupae of Culicidae has been determined similarly by following innervations from the larva to the pupal stage by Biasas & Pagayon (1949), Belkin (1952, 1960) and others cited by Harbach & Knight (1980). It seems likely that larval and pupal homologies of specific or at least groups of sensilla will be achievable using this methodology. In the meantime, the position of pupal sensilla is used to propose interfamilial homologies. Readers unfamiliar with the evolution of sensilla should be aware that campaniform sensilla are known to change form into trichoid sensilla and vice versa. As such, both are treated as significant. In some groups with shagreen (e.g. thaumaleids), it is difficult to see campaniform sensilla and at least some of these may have been overlooked. Future SEM studies would be invaluable in this regard.

Abbreviations for sensilla: Head— CL-1-H to CL-3-H: clypeal/labral DA-1-H, DA-2-H: dorsal apotomal DL-1-H to DL-3-H: dorsolateral cephalic sclerite sensilla O-1-H to O-4-H: oculars A-1-H, A-2-H: antennals Thorax— AM-1-T to AM-6-T: anteromedials AL-1-T to AL3-T: anterolaterals D-1-T to D-7-T: dorsals MT-1-T to MT-7-T: metathoracics

10 · Zootaxa 3396 © 2012 Magnolia Press BORKENT PA-1-T to PA-3-T: prealars SA-1-T to SA-2-T: supralars Abdomen (not all listed; Arabic numerals refer to sensilla numbers, Roman numerals to segment numbers) – D-1-IV: dorsal (here of fourth abdominal segment) L-1-IV: lateral (here of fourth abdominal segment) V-1-IV: ventral (here of fourth abdominal segment)

Habitus Culicomorpha pupae are strikingly diverse in overall body form (Figs. 1A–I). Generally, the cephalothorax is enlarged dorsoventrally and the abdomen may be slightly to strongly tapered posteriorly. Nearly all have a pair of well developed respiratory organs (absent only in some Chironomidae). Live pupae may be elongate, with the abdomen directed posteriorly (some Chironomidae, nearly all Simulioidea, Corethrellidae, some Chaoboridae) or with the abdomen strongly curved under the cephalothorax (Dixidae, some Chaoboridae, all Culicidae).

Head The segments of the head are fused into a single head capsule and bear posteriorly directed mouthparts (Figs. 3B– C, 12A–D, 13A–D) (other than the palpi of Chironomidae, which are directed laterally, Figs. 11B, 18C–D). In full face view, the head capsule is somewhat rounded to somewhat squared in outline. In dorsal view of the thorax, the relative position of the head varies from being visible (dorsal portion) (Fig. 3A) to entirely tucked ventrally under the thorax (Figs. 1H–I). As such, the pupal cuticle over the dorsum of the developing adult head is generally placed anteriorly or anterodorsally. This anterior or anterodorsal portion of the pupal head is composed of three sclerites (Figs. 2A–C, 4A–B): the medial dorsal apotome and two lateral anterolateral cephalic sclerites. The dorsal apotome extends dorsally to either the anterior margin of the thorax, and therefore separates the anterolateral cephalic sclerites medially or only separates them partially, so that the anterolateral cephalic sclerites abut medially and together form the border with the anterior margin of the thorax. These three head sclerites are important components of adult emergence, described separately below. Culicid workers have identified the anterolateral cephalic sclerite as the pronotum but in this family also the anterolateral cephalic sclerite is situated over the dorsum of the pharate adult eye. In previous literature (e.g. Belkin et al. 1970; Ogawa 2007), sensillum 5-CT of Culicidae (as identified by Harbach & Knight 1980) is on this sclerite (and therefore an anterolateral cephalic sclerite sensillum) but 5-CT of Chaoboridae actually is on the thorax and an anteromedial sensillum (hence the 5-CT of Culicidae and Chaoboridae as recognized previously are not homologous). Chaoboridae and Corethrellidae lack anterolateral cephalic sclerite sensilla. In the Chaoboridae genera Eucorethra Underwood and Mochlonyx Loew the dorsal apotome and much of the cephalic sclerites are tucked under the anterior margin of the thorax and therefore only the anterior portions of the cephalic sclerites are evident in whole pupae of these two genera. The dorsal apotome is either ventrally or posteroventrally hinged or simply continuous with the anterior margin of the generally poorly delineated face (Figs. 2C, 11B, 12C), defined as the area from the dorsal apotome to the anterior margin of the clypeus (with the anterior tentorial pits marking its anterolateral margins) and lying between the bases of the antennae. In taxa in which the dorsal apotome is hinged, the anterior margin of the face is evident (sometimes only after ecdysis). In some taxa, the dorsal apotome and face are continuous, without any distinction between the two (e.g. Ptychopteridae, Fig. 11A; Simuliidae, Fig. 12B). Similarly, the posterior margin of the face may be distinct (other than the presence of the anterior tentorial pits) because the anterior margin of the clypeus is evident (e.g. many Chironomidae, Fig. 11B). In many Ceratopogonidae (e.g. species of Culicoides Latreille) the clypeus bulges anteroventrally and the anterior margin of this bulge marks the posterior margin of the face (Fig. 3B). The lateral margins of the face are defined partially by the basal, posteromedial portion of the antenna but otherwise are not distinguishable from the area covering the developing adult eyes (in Culicomorpha; in Ptychopteridae the face is laterally defined, Fig. 11A). Indeed, for the whole pupae of many taxa there is no defining demarcation between the face, eyes, clypeus (other than the anterior tentorial pits at its anterolateral margins) and labrum of the whole pupa and these all form a single undifferentiated surface that can only be defined by the underlying pharate adult structures. In many taxa, at least some further demarcation is possible in exuviae. The base of each antenna (Figs. 2B–C, 4A–B, 11B) lies on either the ventrolateral margin of the dorsal apotome or the anterolateral margin of the face. The scape and pedicel are separate, although closely appressed in most taxa;

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 11 in some they appear to form a single basal swelling of the antenna. The flagellum extends first posterolaterally along the lateral margin of the eye (in at least some taxa, over the developing adult eye) and then either terminates before the posterior margin of the head (Simuliidae and Thaumaleidae) or extends further posteriorly (all other taxa) beside the lateral margin of the mesonotum and then beside or beneath the anterior margin of wing. The pupal eye (Figs. 2B–C, 4A–B) covers the area of the developing adult eye and is defined anteriorly by the posterior margin of the antennal base (or the entire antenna in Simuliidae and Thaumaleidae). It is poorly defined medially. The eye abuts laterally against the antenna and foreleg (most taxa), against the antenna, foreleg and ventromedial sclerite (some Ceratopogonidae), or against just the thorax and prothoracic extension (Simuliidae, Thaumaleidae). Posteriorly the eye abuts against the lateral margin of the base of the palpus or there is no distinction between the eye and the base of the palpus. The clypeus (Figs. 11B, 12B, D) lies posterior to the face but in most taxa there are no distinguishing features indicating the clypeus and it is continuous with the anterior face, the lateral eyes, and the posterior labrum (as indicated by the underlying pharate adult structures). In Culicidae (Fig. 13D), and some Ceratopogonidae (Fig. 3C), Chaoboridae (Fig. 13C) and Chironomidae (Fig. 11B), and at least some Chironomidae (Fig. 11B), there is either a border or an anterior bulging of the clypeus to indicate its anterior margin and there may be a border separating it from the lateral eye (Fig. 11B). Similarly, in a few taxa the anterior point of the lateral margin of the labrum marks the border between the clypeus and labrum (e.g. Simuliidae, Fig. 12B; Ceratopogonidae, Fig. 12D) but in other taxa there is no congruence between the two (e.g. Chironomidae, Fig. 11B; Culicidae, Fig. 13D). The mouthparts extend posteriorly, with nearly all lying anterior to the base of the foreleg (forecoxae). Only those of Culicidae (Figs. 13D, 20D) extend beyond the forecoxae to the apex of the wings and then recurve anterodorsally between the margins of the wings. The labrum (Figs. 11B, 12A–D, 13A–D) is a consistent feature of all Culicomorpha pupae. It forms a prominent medial part of the mouthparts and is either rounded or narrowed posteriorly. In taxa other than Archaeochlus (Chironomidae), its apex abuts the labium (in some there is a question as to whether a small sclerite representing the hypopharynx lies between the apex of the labrum and the labium). In Culicidae (Figs. 13D, 20D) the labrum is greatly elongate and extends to a point between the apices of the wings and then recurves anterodorsally between the margins of the wings. The mandible (Figs. 11B, 12A–D, 13A–D) is elongate and present along the posterolateral margin of the labrum of most taxa. The apices of the two mandibles are nearly always separated medially (they meet medially in Archaeochlus Brundin (Chironomidae)). The mandible is absent only in Dasyhelea Kieffer (Ceratopogonidae) and in derived lineages of some Chironomidae (e.g. some Chironominae). In Culicidae it is greatly elongated and extends to a point between the apices of the wings and then recurves anterodorsally between the margins of the wings. I couldn't be certain of their identity in Dixidae (Fig. 13A), although there are structures there which likely represent the mandible and maxilla. The lacinia (Figs. 11B, 12B–D, 13D) is present in most taxa with biting mouthparts as adult females (present in both male and female pupae) and is generally mostly ventral to the mandible, with either just the apex or the lateral portion of the apical portion visible. In higher lineages of Ceratopogonidae (Fig. 12D) and Corethrellidae (Fig. 13B), the lacinia lies entirely dorsal to the mandible and is not visible externally. In Culicidae (Fig. 20D), the lacinia is greatly elongate, lies lateral to the mandible and extends to a point between the apices of the wings and then recurves anterodorsally between the margins of the wings. Each palpus is either directed laterally (Chironomidae) (Figs. 11B, 18C–D) or posteriorly, posteromedially, or medially (all other taxa) (Figs. 12A–D, 13A–D). In Dixidae the palpi either abut or overlap medially (Fig. 13A). In Chironomidae, the palpus lies between the eye and foreleg (femur). In some other Culicomorpha, the palpus abuts the eye and foreleg laterally and posteriorly and medially abuts the lateral margin of the labium. In Simuliidae, Thaumaleidae and most Ceratopogonidae a lateral portion of the palpus additionally abuts the prothoracic ventromedial extension or ventromedial sclerite (Figs. 12A–D). In Culicidae, the palpus abuts the eye, foreleg and maxilla and the pharate adult palpus uniquely forms a midlength curve near the base of the lacinia (Fig. 13D). The labium (Figs. 11B, 12A–D, 13A–C) forms the posteromedial margin of the mouthparts in most Culicomorpha (other than the Culicidae with their extremely long mouthparts where the apex of the labium lies ventral to abdominal sternites 1 or 2). Sensilla on the head of the pupa (Figs. 3A–C, 11B, 12A–D, 13A–D) vary in number, position, size and form but are either setae (some strongly modified) or campaniform sensilla (Table 2). They are found on five areas and

12 · Zootaxa 3396 © 2012 Magnolia Press BORKENT are named accordingly. The dorsal apotomals are located on the dorsal apotome. Dorsal apotomal 1 is generally stout and on a raised tubercle (in all but Culicoidea, where it is absent). Dorsal apotomal 2 is present in some Simulioidea as a campaniform sensillum (Figs.12A–C). Dorsolateral cephalic sclerite sensilla are absent only in Corethrellidae, others have one seta and some have 1–2 additional sensilla. There are 0–3 clypeal/labral sensilla (absent in all Culicoidea) located on the clypeal/ labral area. Ocular sensilla are present in the area lateral to the clypeus and labrum and covering the lateral and ventral portion of the pharate adult eye. In Ptychopteridae there are three setae and one campaniform sensillum (Fig. 11A); in Culicomorpha there 0–4 sensilla (Figs. 11B, 12A–D, 13A–D). Antennal sensilla are present as a pair of campaniform sensilla on the antennal pedicel of at least Ptychopteridae and at least most Ceratopogonidae (Fig. 4B). They are homologous to a pair of well developed setae on the pedicel of Nannochorista philpotti. I have not surveyed these throughout the families of Culicomorpha because they are difficult to locate consistently.

Thorax The thorax forms the widest and largest part of the pupa (Figs. 1A–I, 2A–C). Dorsally and anterolaterally, most of the thorax is a single unit which is composed of a combined pro- and mesothorax (here called the mesonotum). Each of the two respiratory organs, rarely absent (only some Chironomidae), is on the anterolateral part of the mesonotum. The metathorax is present as a dorsal sclerite, generally with posterolateral extensions (containing the developing halters). The fore- and midlegs are positioned ventrally between the anterior margins of the wings and the hind leg is mostly under the wing. In many taxa most of the apex of the midleg is hidden ventrally by the foreleg and additionally in some taxa by the wing (e.g. Fig. 19A–C). These structures are described more fully below. The mesonotum (Figs. 2A–B, 14B–D, 15A–D, 16A–B), comprising most of the dorsal and anterolateral region of the thorax is actually a composite of the prothorax, scutum, scutellum and postnotum (as evidenced by the underlying pharate adult structures). It forms one undivided unit but may bear transverse ridges, various crenulations, or tubercles. Medially there is either a longitudinal ecdysial suture (sometimes as a raised keel) (Figs. 4A–B, 6A–F), which splits medially upon adult emergence. Posterolaterally the mesonotum abuts the base of the wing (there may be no distinction between the mesonotum and posterior portion of the wing base), and posteriorly it abuts the metathorax. In some taxa, the mesonotum projects posteromedially (sometimes partially or completely dividing the metathorax) (Figs. 17A–B); this projection accommodates the developing elongate scutellar setae of the pharate adult. There appears to be a relationship between the relative length of the scutellar setae of the pharate adult and the relative length of the posteromedial projection of the pupal mesonotum so that when the posteriorly directed mesonotum divides the metathorax medially, the pharate setae are proportionally more elongate (e.g. many Forcipomyia Meigen (Ceratopogonidae)). The prothoracic extension (Figs. 2B–C, 7E–F, 8A–E) is a unique feature of Simuliidae, Thaumaleidae and some Ceratopogonidae (it is secondarily lost in a few genera of Ceratopogonidae) (see character 33). In Simuliidae and Thaumaleidae, the prothorax is partially identified by a suture that extends laterally from a point anterolateral of the base of the respiratory organ. The prothorax forms a narrow ventral and then ventromedial band that extends to the lateral base of the palpus. In most Ceratopogonidae, this extension is separated from the dorsolateral portion of the thorax by the overlying antenna and externally forms a separate sclerite. In early lineages of Ceratopogonidae, the prothoracic extension is small or absent. In other ceratopogonids the sclerite is short (laterally) and abuts either only the antenna or the palpus. These modifications will be discussed further in another paper (in prep.). The mesosternum (Figs. 2C, 18A–D, 19B–C, 20A–C) forms a medially divided ventral sclerite posterior to the base of the forelegs and between the anterolateral to posteroventral part of the forelegs. It is sometimes also divided into anterior and posterior parts by a suture (giving the whole a four-part appearance). In some, such as Chironomidae, the anterior part overlies the ventrum of the katepisternum in pharate adults and posterior part overlies the trochanters and coxae of the developing adult midlegs. In others, such as Ceratopogonidae, the mesosternum overlays only the trochanters and coxae of the pharate adult midlegs. In Simuliidae, the anterior part is fused with the base of the foreleg (lying over the forecoxae) (Figs. 8A, 19C). The mesosternum is not visible in Culicidae because of the elongate mouthparts that cover this area. Further study of this area is warranted, both for a better understanding of homologies and possible additional synapomorphies.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 13 TABLE 2. Proposed pupal homologies of sensilla between Ptychopteridae and families of Culicomorpha. Specific names for each sensillum in a given group are provided in the descriptions and figures. Ptychopteridae have a very large number of sensilla on abdominal segments 2–8 (indicated in gray), making it impossible to suggest homologies with those of Cu- licomorpha. Abbreviations: cmp. sen. = campaniform sensillum(a); abd. = abdominal. For abdominal segments, D = dorsal, L = lateral, V = ventral, Latin numerals give sensilla number, Roman numerals refer to segment number.

Sensilla Proposed Ptychopteridae: Chironomidae: Ceratopogonidae: Thaumaleidae Simuliidae: Culicoidea: Term for all Ptychoptera and Archaeochlus all genera Prosimulium + all families Culicomorpha Bittacomorpha Parasimulium (in part) Head dorsal apotome 1 seta at least 1 seta 0–4 sensilla 1 seta 1 seta, 1 cmp. sen. absent sensilla (dorsal apotomals) dorsolateral 1 seta, 1 cmp. sen. 1 seta, 2 cmp. 1–3 sensilla 2 setae 3 setae 0–1 seta cephalic sclerite in Ptychoptera; at sen. sensilla least 1 seta in Bit- tacomorpha clypeal/labral 1 seta, 2 cmp. sen. 1 seta on edge 0–2 sensilla 3 tiny cmp. 2 setae, cmp. sen. absent sensilla in Ptychoptera; at of clypeus sen. (clypeal/labrals) least 1 seta in Bit- tacomorpha ocular sensilla 3 seta, 1 cmp. sen. 3 setae 1–4 sensilla 1 seta, 1 cmp. 1 seta, 2 cmp. sen. 0–3 setae (oculars) in Ptychoptera; 4 sen. setae in Bittaco- morpha antennal sen- 2 cmp. sen. absent 0–2 cmp. sen. absent absent absent silla (antennals) Thorax anteromedials 0—dirty speci- 4 setae, 1 cmp. 0–6 sensilla 2–4 setae 6 setae seta mens sen. anterolaterals 2 setae in Bittaco- 2 setae 0–3 sensilla 3 setae 2 setae 1–2 setae morpha; at least 1 in Ptychoptera dorsals 5 setae, 2 cmp. 2 setae 3–5 sensilla 4 setae, 1 5 setae, 2 cmp. sen. 2–3 setae sen., in one grp cmp. sen. prealars absent absent absent absent 1 seta (Parasimu- absent lium); 3 setae (Pros- imulium) supraalars absent 1 seta, 1 cmp. 0–2 bare 1 seta 0–1 seta or sen. cmp. sen. metathoracics 6 setae (3 lateral, 3 setae, 1 cmp. 0–3 sensilla 3 setae, 1 5 setae, 2 cmp. sen. 1–3 setae, 3 more medial) sen. cmp. sen. or 1 0–1 cmp. seta sen. Abd. D-1-IV very large number very small seta seta or absent (?) small seta seta seta segment of sensilla on seg- 4 ment D-2-IV seta seta seta seta seta D-3-IV absent 0–1 seta seta seta (absent in absent Parasimulium) D-4-IV cmp. sen. cmp. sen. absent absent absent D-5-IV seta 0–1 seta thick seta thick recurved seta 0–1 seta D-6-IV seta absent absent thick recurved seta seta D-7-IV absent cmp. sen. cmp. sen. 0-1 cmp. sen.; seta in cmp. sen. Parasimulium D-8-IV seta seta or cmp. sen. seta thick recurved seta seta or absent D-9-IV seta seta thick seta thick recurved seta seta L-1-IV seta seta or absent seta seta (on sternite); seta absent in Parasimu- lium

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14 · Zootaxa 3396 © 2012 Magnolia Press BORKENT TABLE 2. Continued Sensilla Proposed Ptychopteridae: Chironomidae: Ceratopogonidae: Thaumaleidae Simuliidae: Culicoidea: Term for all Ptychoptera and Archaeochlus all genera Prosimulium + all families Culicomorpha Bittacomorpha Parasimulium (in part) L-2-IV seta seta seta seta seta L-3-IV seta 0–1 seta seta seta seta L-4-IV seta 0–1 seta seta seta; absent in seta Parasimulium V-1-IV seta 0–1 cmp. sen. seta seta on membrane seta or cmp. sen. V-2-IV seta 0–1 cmp. sen. seta absent absent V-3-IV seta absent absent seta; absent in 0-1 seta Parasimulium V-4-IV seta absent absent thick recurved seta; absent simple seta in Parasimulium V-5-IV seta seta seta thick recurved seta; seta slender in Parasimu- lium V-6-IV seta seta seta seta; absent Parasimu- seta lium V-7-IV seta 0–1 seta seta seta absent Abd. D-1-IX absent seta 0–1 seta seta seta 0–1 seta segment 9 D-2-IX absent seta 0–1 cmp. sen. absent absent absent D-3-IX absent seta 0–1 cmp. sen. absent absent absent D-4-IX absent seta 0–1 seta seta seta 0–1 seta D-5-IX absent cmp. sen. cmp. sen. cmp. sen. absent absent D-6-IX absent cmp. sen. cmp. sen. cmp. sen. absent absent L-1-IX absent absent absent absent seta absent L-2-IX absent absent absent absent seta absent L-3-IX absent absent absent absent seta absent L-4-IX absent absent absent absent seta absent V-1-IX absent absent absent seta seta 0-1 seta V-2-IX absent absent absent cmp. sen. absent absent

Each of the two respiratory organs (Figs. 2A–C, 21A–L), absent only in some Chironomidae, is situated on the anterolateral portion of the mesonotum, near its anterior edge in the Simulioidea (Figs. 14C–D, 15A–B) and more posterior in other Culicomorpha (Figs. 1A–B, F–I, 14B, 15C–D), and each has at least some basal articulation. The illustrations of Chaoboridae and Culicidae (Figs. 16A–B) make the respiratory organs appear to be near the anterior edge of the thorax but this is due to the strong anteroventral curvature of the thorax (see Figs. 1H–I). The respiratory organs are markedly variable in form, being either a single, undivided unit, contorted, or a multibranched structure. To obtain oxygen they may have pores, a multiporous filter apparatus, or no direct openings at all (and function as a physical gill). It is difficult or impossible using a light microscope to distinguish between a filter apparatus (with fine perforations allowing oxygen to pass) and a plastron as a sculpted surface. Consequently , it is assumed that taxa that obtain oxygen at the surface have a filter apparatus. The base is articulated to allow some movement of the respiratory organ; in some groups, the apices of some head or (more commonly) thoracic setae abut the respiratory organ and almost certainly are used to determine the position of the respiratory organ. In most groups, the inner tracheal tube (Fig. 21C) of the respiratory organ is distinct and separate from the outer wall. Basally the tracheal tube may be continuous with the trachae in the thorax itself, and have similar spirals, but it is separate in some Chironomidae (Coffman 1979). Basally the tracheal tube often bears the similar spirals of the thoracic trachae but apically the tracheal tube is generally either plain or has a different reticulate pattern. A separate tracheal tube is not present in some taxa (e.g. Fig. 21L). The outer surface of the respiratory organ may be smooth, bear spicules or plates, or form a plastron.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 15 The legs are positioned ventrally on the body (Figs. 2B–C, 18A–D, 19A–C, 20A–D) and, like the wings, are variably appressed against the body—from loosely so (many Chironomidae, Culicidae) to tightly fused with the rest of the thorax (all Simulioidea). In nearly all taxa, they do not extend beyond the apex of the wings; there are a few exceptions in the Chironomidae (Brundin (1966: 428–432; Murray & Ashe 1981) (Figs. 18B–D), and this is discussed further below under the key to families and characters 43–45. The base of the foreleg lies posterior to the labium where it overlays the developing adult coxa and/or trochanter and/or base of the femur. In the figures I simply refer to this area as the forecoxa. In Simuliidae, the base of the foreleg (overlying the pharate adult coxae and trochanters) is fused with the anterior portion of the mesosternum (overlying the pharate adult katepisternum). The forefemur is directed anterolaterally from the forecoxa (this ensheaths the developing adult femur) where it abuts the antenna (most taxa) or lateral margin of the thorax (Thaumaleidae and Simuliidae because the antennae are restricted to the head in these two groups). In Simuliidae and Dixidae, the forefemur lies under the palpus and is not evident externally or is present only as a very narrow band. The remainder of the foreleg is then directed posteromedially (this turning point marks the developing adult femur-tibia junction). In most taxa, the two forelegs meet medially before the apex of wings and terminate either at the wing apex or significantly anterior to the wing apex. In the strongly modified Culicidae, the forelegs do not meet apically but go underneath (in ventral view) the midlegs. In many taxa there is a medial protrusion along this posteroventrally directed portion of the foreleg, which indicates the developing adult tibial spurs or elongate setae or bristles at the apex of the tibia (Figs. 18A, 19A). As such, it marks the junction of the apex of the tibia and base of the first tarsomere of the developing adult. The pharate adult midleg originates under the mesosternum of the pupa. Most of the pupal midleg is only visible lateral to the anterolateral/ posteromedial portion of the foreleg where it ensheaths the midtibia and, in some taxa, a portion of the first or subsequent tarsomeres. Laterally, this elongate portion of the midleg abuts the anterior margin of the wing and, in taxa with an elongate antenna, the apical portion of the antenna. In many taxa the subapical portion of the midleg lies dorsal to the apical portion of the foreleg and additionally in some by the wing (and is thus hidden ventrally). The very apex of the midleg is visible in only some taxa, at the apex of the wing. In Culicidae, the midleg curves anterodorsally between the apices of the wings and lies more anteriorly. A similar condition is present in some Chironomidae. The bulk of the hind leg lies under the wing with the femur directed anterolaterally, the tibia folded back posteriorly, the apical portion of the tibia and tarsomere 1 or tarsomeres 1 and 2 bent anterolaterally, and then, upon reaching the posterior margin of the wing, either tarsomeres 2–5 or tarsomeres 3–5 bend posteromedially to lie along the hind margin of wing. In some taxa, this portion lying along the wing margin is visible externally (in others it remains obscured under the wing hind margin). Otherwise only the apex of the hind leg is visible externally. As noted elsewhere, there are a few exceptions to the S-shaped hind leg of the Culicomorpha (e.g., some Chironomidae with a straight hind leg). The wing (Figs. 2A–C) originates on the posterolateral margin of the mesonotum and extends posteroventrally as a broad structure to the posteroventral margin of the thorax. The two wings are separated ventrally by the forelegs and, in some, the midlegs (or just the labrum, mandible, and midleg in Culicidae) and meet medially only in one genus of Ceratopogonidae (Paryphoconus Enderlein). In some Chironomidae there are modifications of the wing border (called "pearl row" by chironomid workers) but this is unadorned in other Culicomorpha other than a short subterminal or terminal lobe (also present in some Chironomidae, where it is called a "nose") that accommodates the elongate setae on the wing apex of the developing adult. The wings are variably appressed against the body from loosely appressed to tightly fused against the surrounding sclerites. The metathorax (Figs. 2A–B, 17A–H) lies between the mesonotum and the first abdominal segment and may be divided medially by a posteromedial projection of the mesonotum (as discussed above). In Corethrellidae, the metathorax is represented medially by a narrow transverse band that is separated laterally by membrane from the larger lateral portion of the metathorax. In most taxa, the metathorax extends posterolaterally along the lateral margin of abdominal tergite 1, where it lies over the developing adult halter (Fig. 17C). In some Chironomidae and all Simuliidae (Figs. 8B, 27F), the metathorax has no posterolateral extension and the developing adult halter lies under the base of the wing (see characters 36–37). In other Chironomidae, there is a lateral or slightly posterolateral extension, with the apex directed under the wing. A variety of sensilla are on the thorax of the pupa (Figs. 3A, C, 14B–D, 15A–D, 16A–B), and these vary in number, position, size and form. Setae (simple or bifurcated) and campaniform sensilla are present (Table 2).

16 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Thoracic sensilla are found on six areas and are named accordingly (but see below). The 0–6 anteromedial sensilla are found from near the base of the respiratory organ to the anterior margin of the mesonotum (anterior or anteromedial to the base of the respiratory organ). The 2–3 anterolateral sensilla are on the anterolateral corner of the mesonotum (anterolateral to the base of the respiratory organ), and are generally in a tight group and on a small lobe or projection. The area posteromedial to the base of the respiratory organs bears 2–7 dorsal sensilla. D-1-T – D-5-T form a similar pattern in Simulioidea (Figs. 3A, 14C–D, 15A), with four setae and one campaniform sensillum (see character 39). The pattern in the early lineage simuliid Parasimulium Malloch (Fig. 15B) differs but this needs to be re-examined when better material becomes available (the specimen studied was crushed). Sensillum D-5-T is apparently missing in Austroconops Wirth & Lee but it could easily be D-4-T that is missing. In addition, D-5-T is consider to be situated posteromedially in most other Ceratopogonidae (Fig. 3A)—an arbitrary decision, as it could easily be D-4-T or some other rearrangement of the dorsal setae that give the consistent pattern in most Ceratopogonidae. There are only 2–3 dorsals in Culicoidea (but see below). The prealar sensilla are anterodorsal to the base of the wing and found only in Simuliidae; Sæther (1980) reports the presence of one prealar sensillum in Chironomidae. The 0–2 supraalar sensilla are medial to the base of the wing and posterolateral to the dorsal sensilla. When present, SA-1-T is a seta and SA-2-T is a campaniform sensillum. There are 0-7 metathoracic sensilla (Figs. 17A-H), with most groups having 3 setae and possibly a campaniform sensillum. Only Simuliidae have a large number with 5 setae and 2 campaniform sensilla (Fig. 17D). Although all sensilla were identified for each family (Figs. 3A, 14B–D, 15A–D, 16A–B), the placement of the following sensilla remained particularly uncertain: - homology between dorsals of Ptychopteridae (Fig. 14A) with specific mesothoracic setae of Culicomorpha. Although Simuliidae share the same number of sensilla (with 5 setae and 2 campaniform sensilla), it is possible that some of the anteromedials or supralars in other families are homologous to some dorsals in Ptychopteridae. - identity of D-5-T, D-6-T and D-7-T of Simuliidae (Fig. 15A). They may well be homologous to some or all of the anteromedials of Chironomidae (Fig. 14B), or, alternatively, some anteromedials of Chironomidae may be dorsals. - one of the dorsals of Chaoboridae may actually be a supraalar. If so Dixidae, Corethrellidae and Chaoboridae would have a total of two dorsals and one supraalar, with a loss of one sensillum in Culicidae. - the thorax of the Thaumaleidae has abundant shagreen and there are undetected campaniform sensilla likely present, especially in the area of D-6-T, D-7-T, and SA-1-T of Simuliidae. A SEM study would be invaluable to search for further homologs and potential synapomorphies..

Abdomen The abdomen is composed of nine discrete segments with the last (ninth) segment bearing the genital lobes, and including the developing adult genitalia and proctiger (elements of segment 10 such as the cercus) (Figs. 2A–C). For most taxa, each of segments 1–8 appear either square or as a wide rectangle from a dorsoventral view. Segment 9 is variable in shape (Figs. 25B–G, 26A–E)—short and wide to long and narrow and is very reduced in Chaoboridae and Culicidae (other than the terminal processes). The first abdominal tergite of Simuliidae extends posterolaterally along the lateral margin of segment 2 (to accommodate the elongate setae of the first abdominal tergite of the developing adult) (Figs. 8B, 17D, 27F). In some taxa, tergites 1 and 2 are fused or tightly appressed, but always with a clear suture between them. Sternites 1 and 2 are not visible externally because they are covered ventrally by the wings and legs. In most taxa, if the wings and legs are removed, sternites 1 and 2 are represented by thin cuticle; they are better developed in the Culicidae. As such, sternite 3 is the first sternite visible in ventral view. It is somewhat difficult and sometimes impossible to distinguish abdominal tergites or sternites from the pleuron on each of the abdominal segments for most taxa. Some Simuliidae have some abdominal segments with lateral membrane between the tergite and sternite that serves to distinguish the pleuron. Some Chironomidae (including Archaeochlus), and all Dixidae and Thaumaleidae have a somewhat squared abdomen in cross-section in which the lateral portion might be identified as a pleuron, even though the integument is undifferentiated dorsoventrally (i.e. no separation of tergite, pleuron or sternite dorsoventrally). Ceratopogonidae tend to have abdominal segments that are nearly circular in cross-section, with no longitudinal divisions; as such the lateral setae are more or less grouped on the laterally rounded margin of each abdominal segment (Fig. 2B). Many Chironomidae and all Corethrellidae, Chaoboridae and Culicidae have dorsoventrally compressed abdominal

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 17 segments (Figs. 1B, G–I) in which the tergite and sternite meet laterally and the named lateral setae are mostly ventral but occasionally dorsal (Figs. 24A–C). In some taxa, each of segments 2–8 has a spiracular scar (Figs. 23C, 24A–C) which indicates the underlying developing pharate adult spiracle. Thaumaleidae are the only Culicomorpha with open spiracles, present on abdominal segments 2–7, 3–7, 4–7 or 5–7 (depending on the species) (Figs. 10D, 22C). The integument has a variety of ornamentation. Some taxa have elongate tubercles (only in the Forcipomyiinae (Ceratopogonidae)) or short tubercles, especially for some lateral sensilla, but generally the cuticle is more or less flat with a variety of sensilla (innervated) or unarticulated (not innervated) protruberances called spicules, shagreen, or spines (depending on which family or taxon is being described). There are other names for cuticular projections used by workers in specific families (e.g. Culicidae—Harbach & Knight 1980). All pupae have muscle plaques (Fig. 5B) which are bare circular or oval patches on the abdomen indicating the previous attachment of larval muscles as the pharate pupa developed (allowing for movement of the larva until emergence of the pupa). Similar marks are present on the bodies of adult nematocerous Diptera, orthorrhaphous Brachycera, Empidoidea and Apystomyiidae (Trautwein et al. 2010) where pupal muscles were previously attached to their developing adult bodies (called 'plaques'; Stoffolano et al. 1988). In this paper, a new system of numbering the abdominal sensilla of Culicomorpha is provided, recognizing three basic groups of sensilla as dorsal, lateral and ventral (Figs. 2A–C, 5A–B, 22A–C, 23A–C, 24A–C). Within each group the individual sensilla are identified by their overall position, D, L, and V to indicate dorsal, lateral and ventral, followed by a sensillum number as a Arabic numeral and the number of the abdominal segment number as a Roman numeral. The dorsal sensilla on segment 4 are therefore called D-1-IV, D-2-IV, D-3-IV, etc. Within each of these three groups, sensilla are numbered more or less anteriorly to posteriorly and then, for dorsal and ventral sensilla, generally medially to laterally. This was not done in some instances because of different positions of what are probably homologous sensilla in different taxa. Although it appears that there are three basic groups of sensilla in all Culicomorpha, with segment IV having 6–9 dorsal, 4 lateral, and 3–7 ventral sensilla, the specific homologies of a given sensillum between different families is often difficult or presently impossible to determine with certainty—further ontological and morphological study will be necessary to better understand specific homologies. The chaetotaxy of tergite 1 (Figs. 17A–H) is based on comparisons with the fourth tergite (Figs. 5A–B, 22A– C, 23A–C, 24A–C), as well as between taxa. There are uncertain homologies within each group (dorsal or lateral) regarding, especially, homologies between taxa. Regardless, there are some patterns. Chironomidae, Ceratopogonidae, Corethrellidae, Chaoboridae and Culicidae have five dorsal sensilla, all of which are setae. Within this group of five, D-2-I, D-8-I, and D-9-I are consistently identified. The fourth and fifth are identified as either D-5-I and D-6-I in Chironomidae (Fig. 17A), D-3-I and D-4-I in Ceratopogonidae and Simuliidae (Figs. 17B, D), or D-3-I and D-6-I in Culicoidea (Figs. 17E-H) because of probable serial homologies with sensilla on tergite 4. Simuliidae have these five setae and an additional campaniform sensillum (Fig. 17D). Thaumaleidae and Dixidae have only three dorsal setae. Chironomidae and Simulioidea have only one lateral sensillum which is always a seta, Corethrellidae have none, and other Culicoidea have three lateral sensilla, all as setae. The identification of some sensilla in the Culicoidea differs from that proposed by Belkin et al. (1970) because of more likely homologies with other families, as well as evidence based on comparisons between abdominal segments of a given taxon. The chaototaxy of segment 4 (Figs. 2A–C, 5A–B, 22A–C, 23A–C, 24A–C) is described for each of the families and the choice of this segment as representative requires some explanation. Belkin (1950, 1952, 1953, 1962, 1968), Belkin & McDonald (1955), and Belkin, et al. (1970) (and numerous other papers on Culicidae) provide detailed descriptions of the pupal abdominal chaetotaxy of the Culicoidea and a single numbering system for sensilla. Belkin used the second abdominal segment as the basis to determine homologies between larvae and pupae and between taxa because he considered this segment to be the most representative and most easily homologized. Further to this, because the second segment appeared to have the greatest number of sensilla and to be the most conservative in the placement of sensilla (i.e. most easily homologized between taxa), he considered its chaetotaxy to also be most representative of the serial homology between the abdominal segments. In contrast, the chaetotaxy of only the first and fourth abdominal segments are described in this publication. The description of the first tergite complemented the description of the metathorax and its lateral modifications. Otherwise, only the fourth segment is fully described. There are several reasons for this. First, the fourth segment is the first abdominal segment that consistently appears to be uninfluenced by the ventral legs and wings of the

18 · Zootaxa 3396 © 2012 Magnolia Press BORKENT pupa and looks similar to segments 5–7. As such, the fourth abdominal sternite has a somewhat different chaetotaxy from that of sternite 3 in some taxa and, in most Culicomorpha, significantly different from sternites 1– 2. Second, this study was initiated, in part, as an outgroup exercise to better understand my continuing investigation of Ceratopogonidae, and in this family it is standard to describe primarily the fourth abdominal segment. Third, although the tenacity and thoroughness of the descriptions of the pupae of Culicoidea by other workers should be admired, in which every sensilla of the body is illustrated and described, for the purposes of this study it would be too time-consuming to do the same for each taxon here. The initial goals of this study were to find outgroup features for the Ceratopogonidae and to better understand the phylogeny of the Culicomorpha. Some synapomorphies may have been missed that would be discovered through a more thorough comparison of the chaetotaxy of segments 2, 3, and 5–8 but other workers will need to investigate this. Although it is presently difficult to provide strong evidence of which specific sensilla on segment 4 are homologous within each of the three groups (dorsal, lateral, ventral), there are certain landmarks and features suggesting some homologies. The following are some patterns (Figs. 5A–B, 22A–C, 23A–C, 24A–C): - dorsal sensilla. There are six dorsal setae and one campaniform sensillum in Chironomidae, Thaumaleidae, Dixidae, Chaoboridae and Culicidae. Ceratopogonidae (most) have an extra campaniform sensillum. Simuliidae have an extra dorsal seta and Corethrellidae have one less seta. This strongly suggests the basic pattern of the Culicomorpha is the presence of six dorsal setae and one campaniform sensillum. However, based on position, I have not recognized these all as homologous (see Figs. 22A–C, 23A–C, 24A–C). - dorsal sensillum D-1-IV. This minute seta is present in all Culicomorpha with at least some secondary losses in some Ceratopogonidae (although the very small size may mean they have been overlooked). - dorsal sensilla D-2-IV, D-3-IV. Although the specific homologies of the dorsal setae are difficult to determine with precision, these two setae form a close pair in most Simulioidea (Figs. 22B–C, 23A), with subsequent losses in many Ceratopogonidae and in Parasimulium (Fig. 23B; where what is identified here as D-7-IV may actually be D-2-IV and D-2-IV actually D-3-IV, with the pair therefore still present). - dorsal sensillum D-4-IV. This campaniform sensillum is located posteriorly in some Ceratopogonidae (Fig. 5A). - sensillum D-7-IV. This is consistently a campaniform sensillum in all taxa other than Chironomidae, although there is a possibility that the chironomid D-4-IV is actually homologous to D-7-IV and not to the campaniform sensillum D-4-IV of Austroconops (Ceratopogonidae). Sensillum D-7-IV is either uniquely a small seta in Parasimulium (Fig. 23B) or is missing (see comment above regarding dorsal sensilla D-2-IV, D-3-IV). - lateral sensilla. Four laterally placed setae are always present in Culicomorpha, with L-1-IV generally placed anteriorly and L-2-IV, L-3-IV, and L-4-IV generally arranged dorsally to ventrally (Fig. 5B). Sensillum L-3-IV forms a thick spine on the posterolateral margin of segment 4 in a number of taxa (e.g. some Chironomidae, Fig. 22A; Thaumaleidae, Fig. 22C; Culicidae, Fig. 24C). - ventral sensilla. Sensilla V-1-IV and/or V-2-IV are present in all Culicomorpha, often as minute setae. Sensilla V- 5-IV, V-6-IV, and V-7-IV are present in Chironomidae and Simulioidea as a transverse row, whereas only V-5- IV and V-6-IV are present in Culicoidea. Chironomidae and Simuliidae have additional ventral setae (V-3-IV, V-4-IV) situated more anteriorly in Chironomidae, or just V-3-IV more anteriorly and V-4-IV as part of the posterior transverse row in Simuliidae.

In spite of the laudable efforts to homologize the chaetotaxy of the different families of Culicoidea by Belkin and his associates, the present study suggests some differences or at least questionable homologies. These differences are primarily concerned with the dorsal and lateral setae as well as the serial homology between the first and fourth tergites. Some difficulties in homology are compounded by the fact that Dixidae have somewhat squared abdomens in cross-section, in which the lateral setae are clearly on the lateral portion of each segment. In Corethrellidae, Chaoboridae, and Culicidae, the abdomen is dorsoventrally compressed, so that there is no clear lateral portion other than the very edge. As such, some of the lateral setae in these three families are actually on the ventral surface of the segments (other than one being dorsal in Culicidae). In addition, I attempted to homologize the Culicoidea with those of other Culicomorpha with only partial success and this has resulted in some different interpretations for the various families of Culicoidea than proposed by Belkin's work. Segment 9 (Figs. 2A–C, 25A–G, 26A–E) is the last abdominal segment and bears the paired male genital lobes or, in the females of some taxa, a short medial genital lobe and cerci. In nearly all Culicomorpha with the lobes, they are situated on the ventral surface of this terminal segment (they are dorsal only in one group of

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 19 subgenera of Forcipomyia in the Ceratopogonidae). Each of the paired genital lobes of males contains the developing gonostylus and in some, the apex or whole of the gonocoxite. In both males and females there may be additional small ventral lobes that ensheath the pharate adult cercus. Although identified as segment 9 here, this segment is a fusion of primitive segments 9 and 10. In the Mecopteran Nannochorista philpotti (Nannochoristidae), there are 10 distinct abdominal segments (Pilgrim 1972). In this species segment 9 encloses the developing gonocoxite and gonostylus in males (there is no distinct lobe in females) and segment 10 contains the developing male and female cerci (pers. obs.). In Ceratopogonidae, the cerci of both males and females develop at the base of the terminal processes, which are therefore almost certainly homologous to segment 10 of Nannochorista philpotti (which is also apically bilobed in females to accommodate the elongate cerci). As such, segment 9 of Culicomorpha pupae is a fused composite of segment 9 (bearing the genital lobes) and segment 10 (at least the terminal processes and perhaps more) seen in Mecoptera. The shape and vestiture of segment 9 is strikingly variable in the Culicomorpha and reflects its importance in pupal locomotion for either swimming or pushing against substrate, and in the case of many Chironomidae, for moving water through their pupal chambers for aeration. Segment 9 of Culicomorpha have a pair of terminal processes that are either lobes, conical projections, flanges, dorsoventrally flattened paddles, or in the case of Chaoboridae and Culicidae, articulated membranous paddles with supporting ribs. The terminal processes of Chaoboridae and Culicidae require further comment. I consider these distinctive structures to be homologous to the terminal processes of other Culicomorpha for several reasons. Although apparently articulated against the posterolateral margin of segment 8, the base of the paddle in Culicidae is actually in the membrane between segment 8 and a very reduced segment 9 (represented by the cercus and perhaps a small band of cuticle). In Chaoboridae, the base of segment 9 appears to be absent. The base of the terminal process of other Culicomorpha encloses the developing male or female cercus. In Culicidae, the adult male segment 10 and in Culicidae and Chaoboridae the adult female cerci develop in two lobes situated between the bases of the articulated paddles (labeled here as cerci). The paddles are likely a modified portion of the terminal process (so the paddle and cercus together are the full homolog of the terminal process of other Culicomorpha). The presence of what are likely homologous sensilla in other Culicomorpha also indicates the articulated paddle is a true homolog of the terminal process of other Culicomorpha. Finally, the pupal paddles of Chaoboridae and Culicidae develop inside the larval respiratory siphon (lost in Chaoborus Lichtenstein), which is located on larval abdominal segment 8 (but is likely fused segments 8 and 9). In some Chironomidae, the terminal process develops in the procercus (Cranston et al. 1987:314) (likely homologs of the culicid siphon or at least a portion of it). This evidence suggests the pupal paddle of Chaoboridae and Culicidae is homologous to the terminal process of other Culicomorpha. There remains a great need to study the homology and sequence of development of segments 8–10 in larvae, pupae and adults in nematocerous Diptera. At present there are some serious inconsistencies in interpretation. Segment 9 of Culicomorpha always has sensilla that may appear as setae (some modified into hooks or spines) or campaniform sensilla (the latter often difficult to see, especially if there is thick shagreen present). In addition many Chironomidae have elongate lateral fringes of non-innervated hair-like spicules (i.e. not sensilla). The homology of these sensilla is uncertain across the Culicomorpha, and I am not confident of the names of specific sensilla of some of the families. In particular, it may be that some dorsal sensilla have become ventral, as is nearly certainly the case of D-4-IX in Chaoboridae (Fig. 26C) (moving from a dorsal, nearly apical position, as is present in Corethrellidae and Culicidae, to a ventral position in Chaoboridae). However, there are some patterns that appear consistent: - sensillum D-1-IX. In most families, there is a strong, more basal dorsal seta (D-1-IX). In Archaeochlus (Chironomidae), there are two associated setae (D-2-IX, D-3-IX) that appear in many other Chironomidae. In the Ceratopogonidae, only species of Leptoconops Skuse have a strong D-1-IX, and also more than one sensillum in this area of segment 9 (as campaniform sensilla); other Culicomorpha never have more than one. - lateral sensilla. Simuliidae are the only Culicomorpha with lateral sensilla, all as setae (Fig. 25G) (Adler et al. 2004, figs. 599–601). - sensillum at the base of the terminal process. The terminal process has three sensilla (D-4-IX, D-5-IX, D-6-IX), which are reduced to two campaniform sensilla in most Chironomidae and Thaumaleidae. Most Ceratopogonidae have two campaniform sensilla (species of Leptoconops have three campaniform sensilla on the base of the terminal process). Simuliidae and most Culicoidea (not Dixidae) have a single dorsal seta or spine (Figs. 25F, 26B–D). Thaumaleidae, Simuliidae and Culicoidea are the only Culicomorpha with ventral sensilla (as setae or spines).

There are two quite different modes of adult emergence in Culicomorpha. In most (perhaps all) Chironomidae and all Culicoidea (Figs. 6C, 7C–D, 9A–F, 10A–B), the top of the head folds open and the thoracic ecdysial suture splits wide open to form a "platform" from which the adult emerges (generally at the water surface). This form of emergence is also present in Ptychopteridae (Fig. 7B) and at least partially in Nannochoristidae (see character 25). The splitting of the head capsule happens as follows. As the dorsal apotome splits dorsally, it hinges outward and forward. The dorsolateral cephalic sclerites split medially (and from the dorsal apotome) and, with the bases of the antennae, fold ventrolaterally and then medially, forming a longitudinal crease along the lateral margin at the base of the dorsal apotome and face. The sides of the head (largely the covering of the pharate adult eyes) also fold ventrolaterally and then medially, forming a longitudinal crease along the lateral margin of the clypeus/labrum. There is also a transverse fold in the area between the clypeus and face that is bent during emergence. In short, this looks like a central inward "crumpling" of the whole ventral portion of the head, as if a blow has been struck at the junction where the clypeus borders the face of the pupa, with the surrounding parts directed partially ventrally. It appears likely that this folded appearance of the ventral view of the head is caused or facilitated by the inward pull on the tentoria (near the center of the folding) by the emerging adult. In pupal exuviae of Ptychopteridae (Figs. 7A–B) and some Chironomidae, this crumpled appearance of the head can be "popped" back into the original appearance of the whole pupa by pushing the interior of the clypeus/labrum-face junction with forceps. At the same time, the mesonotum splits medially along the ecdysial suture, with the anteromedial portion of each side additionally bending further outward along a more or less transverse to anterolateral crease (not visible in whole pupae). The medial end of the crease is about a quarter to a third of the length of the ecdysial suture (measuring from the anterior margin of the thorax). This form of emergence leaves an exuviae with the various parts of the head and the margins of the thorax spread out, in a position that often makes it difficult to determine some structures clearly evident in whole pupae. Adult emergence in Simulioidea differs significantly, in that the pupal exuviae retains the external features of the whole pupa, although some parts are somewhat separated. In Ceratopogonidae (Fig. 6A–B, D, E–F), there are three head sclerites that fold outward like an opening of a three petal flower: the dorsal apotome and the two lateral dorsolateral cephalic sclerites. The dorsal apotome separates from the dorsolateral cephalic sclerites and, in some taxa, the very anteromedial margin of the thorax, to hinge forward and outward. The two dorsolateral cephalic sclerites separate medially and fold outwardly. At the same time, the mesonotal ecdysial suture splits medially to near the anterior margin of the metathorax, with the two halves of the mesonotum each moving laterally as an uncreased unit. The adult emerges through this opening. Upon complete emergence, the various parts of the exuviae largely spring back to their original positions and thus look rather similar to a whole pupa. Adult emergence of Ceratopogonidae must therefore be more like slipping out of a tight-fitting glove, compared to the permanent 'snap' opening of the pupae of Chironomidae and Culicoidea. Thaumaleidae and Simuliidae have a similar "tight fit" emergence, with the mesonotal ecdysial suture splitting medially to near the anterior margin of the metathorax. However, the escape from the head differs somewhat from each other and from that in Ceratopogonidae. In Thaumaleidae (Figs. 6E, C–E), the large dorsolateral sclerites separate and hinge outwardly and the entire ventrum of the rest of the head capsule, including the dorsal apodeme separates as a unit, either remaining very loosely attached to the thorax with thin membrane or separating entirely. The dorsolateral sclerites are loosely attached to the thorax and/or the head with membrane after emergence. Indeed, it is difficult to keep the head capsule with the remainder of the body of the pupal exuviae of many specimens of Thaumaleidae. In Simuliidae (Figs. 6F, 8A–B), the dorsolateral sclerites are reduced in many species and also differ in the two sexes (probably because of the enlarged eyes of males). In females the dorsolateral sclerite is permanently fused laterally to the antenna as a triangular sclerite (Fig. 6F). In males, the sclerite is a longitudinal and narrow strip of cuticle partially fused laterally to the antenna and partially continuous laterally with the eye. The dorsal apotome is exceptionally large and splits laterally from the dorsolateral cephalic sclerites and, at least in some species, the base of the antennae, affording a large opening for the emerging adult head. At the same time, the entire head tilts forward ventrally from its permanent attachment to the forecoxae and mesosternum (which moves somewhat dorsally on emergence).

The pupae of Culicomorpha are the only Diptera with the hind leg curved in a S-shaped position, tucked under the wing and with its apical portion (tarsus) directed medially, posteriorly or posteroventrally and lying along the posterolateral and apical margin of the wing (Figs. 1I, 18A–C). This feature is absent only in three genera of Chironomidae: Harrisonina Freeman, Lopescladius Oliveira and Stictocladius Edwards (Brundin 1966; Lopescladius described as Cordites, in litt.), where the hind leg lies parallel to the midleg (Fig. 18D). To make this paper more inclusive, diagnostic features for Lopescladius and Stictocladius are given so they can be identified. Harrisonina, a monotypic genus restricted to southern Africa, cannot yet be diagnosed as a member of the infraorder.

Lopescladius—the only Diptera with the hind leg parallel to the midleg (not S-shaped), with legs firmly appressed against each other, and segment 9 with paired, elongate narrow (digitiform) lobes, each with a moveable base and bearing 2–3 elongate and stout apical setae (Fig. 25C). The genus is restricted to the Nearctic and Neotropical Regions. Stictocladius—the only Diptera with the hind leg directed posteriorly (not S-shaped), legs not firmly appressed against each other (more or less loose) and segment 9 with paired, firmly placed, elongate narrow (digitiform) lobes, each bearing 2–3 elongate and stout setae (as in Fig. 25D). The genus occurs in the Nearctic and Neotropical Regions (Patagonia and Andes), Australia and New Zealand (Cranston & Sæther 2010, Sæther & Cranston in press).

An additional distinctive feature of Culicomorpha, and in conjunction with the presence of the curved S- shaped hind leg, is that the apices of the two hind legs (each directed posterolaterally along the posterodorsal margin of a wing) generally either meet medially or abut (or very nearly abut) the apices of the posteriorly directed midlegs; there are exceptions in the highly modified Culicidae where the apices abut the midleg subapically, and in those rare Chironomidae noted above, and a few other genera of Chironomidae ("Lepidopodites", Paratrichocladius Santos-Abreu, Nanocladius Kieffer , Paraheptagyia Brundin, Buchonomyia Fittkau; Brundin 1966 (as Syncricotopus Brundin, Microcricotopus Goetghebuer); Murray & Ashe 1981) (Figs. 18B–D). In other nematocerous Diptera and Brachycera, the hind leg is either entirely parallel to the midleg or lies dorsal to it, and the apex of the hind leg does not abut that of the midleg. There are previous keys to the families of the pupae of the Culicomorpha (Malloch 1917; Brauns 1954; Hennig 1950; Wirth & Stone 1956; Merritt & Webb 2008). The following is organized differently, incorporates some of the features previously used, and adds further character states.

1. Apex of abdomen with characteristic pair of paddles (Figs. 10E–F, 26D–E), each with an articulated base and with at least one medial, elongate rib supporting membrane (in many there are additional lateral, thickened margins) ...... 2 - Apex of abdomen either blunt or with pair of projections (Figs. 25B–G, 26A–C), each of which is fixed basally (except in the Chironomidae genus Lasiodiamesa Kieffer), with or without membrane, and lacking supporting ribs (including Lasiodiamesa) ...... 3 2. Labrum, mandibles, maxillae elongate (Figs. 10B, 13D, 20D), extending posteriorly between separated forelegs to apex of wings and then curving anterodorsally along margin of wing ...... Culicidae - Labrum, mandibles, maxillae short (Figs. 9F, 13C), not extending posteriorly beyond forecoxae; apices of forelegs abutting ...... Chaoboridae 3. Antennae restricted to head (Figs. 6E–F, 8A–E, 12A–B), with posteromedially directed palpus (Figs. 12A–B); with well developed prothoracic extension (Figs. 8B, D, 12A–B) ...... 4 - Antennae extending posteriorly, with apex posterior to head (Figs. 2B–C, 12C. 18C–D) or, if restricted to head (some Chirono- midae), then with laterally directed palpus (Figs. 11B, 18C–D) and without prothoracic extension ...... 5 4. Respiratory organ with at least three branches (Fig. 21G); abdomen without open spiracles (Fig. 10C); pupa in silk cocoon (Fig. 1E) ...... Simuliidae - Respiratory organ undivided, more or less cylindrical (Fig. 21F); abdomen with open spiracles on at least segments 5–7 (Fig. 10D); pupa without cocoon (Fig. 1D) ...... Thaumaleidae 5 Palpus directed laterally (Figs. 11B, 18C–D); respiratory organ either multibranched (Fig. 21D), undivided (Fig. 21C) or absent; terminal process with (Figs. 25B–D) or without at least one elongate lateral seta (some with many setae or cuticular extensions as a marginal fringe), if elongate seta absent, then either without respiratory organ or respiratory organ without exterior opening...... Chironomidae - Palpus directed posteriorly, posteromedially or anteromedially (Figs. 12C–D, 13A–B); terminal process with or without a single elongate lateral seta (some with additional small sensilla) (Figs. 25E, 26A–C); respiratory organ present and undivided,

22 · Zootaxa 3396 © 2012 Magnolia Press BORKENT either with pores (Figs. 3C, 21E) or with terminal funnel-shaped opening (Figs. 21H–I), or as a flattened almost disc-shaped structure (Fig. 21J) ...... 6 6. Respiratory organ (Figs. 3C, 21E) with at least some apical pores (many with additional subapical pores); exuviae with face like a mask (Figs. 6B, D, 7E,F), retaining the original shape of the whole pupa ...... Ceratopogonidae - Respiratory organ with a broad, open apex (Fig. 21I), or flattened, almost disc shaped (Fig. 21J) and with peripheral pores (or at least pore-like structures) (peruviana species group of Corethrella Coquillett); exuviae (Figs. 9A–D) with face reconfigured, with the ocular lobes folded longitudinally against the dorsal apotome and with a sharp transverse bend between the dorsal apotome and clypeus ...... 7 7 Palpus directed posteromedially (Fig. 13b), with apices well separated medially; metanotum (Fig. 17F) divided medially by posteriorly projecting mesonotum...... Corethrellidae - Palpus directed anteromedially (Fig. 13A), with apices abutting or overlapping medially; metanotum continuous medially, undivided (Fig. 17E) ...... Dixidae

Description of Ptychopteridae and Families of Culicomorpha

In this section, the pupa of each of the families of Culicomorpha is described as well as that of Ptychopteridae as an exemplar of the outgroup. Interpretation of various features is given in the section on phylogenetic relationships. As noted in the introduction, there have been no previous efforts to homologize pupal structures in the various families of Culicomorpha (or of Diptera pupae as a whole), other than the most obvious of parts (e.g. antenna, wing, etc.). Aside from these obvious parts that require no further discussion, the glossary above provides the basis for description. Further discussion of character states is given under "Phylogenetic Relationships". Table 1 provides synonyms for the different pupal structures as used by modern researchers and the terms used in this study. Terms which refer to specialized structures or modifications in a given family are not included. Although specimens were examined for various characters of a variety of species in different genera of families, a complete survey of the variation within each family was not attempted. Rather, I have concentrated on exemplars of early lineages within each family, or, failing that, taxa that are generally considered "primitive" within a given family. For example, although Culicidae are divided into two subfamilies, the Anophelinae have numerous plesiomorphic features within the family. As such special attention was paid to the character states of species of Anopheles Meigen. The same was true for the other families. Because cladistic relationships within Dixidae are entirely unknown, specimens were picked arbitrarily based on availability; relationships between genera of Thaumaleidae are unpublished (see below). As such, the descriptions of the families below do not, in some instances, describe the variation in a given family; this is especially true in the very diverse Chironomidae. The description of the Ceratopogonidae, however, is based on an extensive study of all available genera (Borkent, in prep.). Finally, the literature that described pupae of Chironomidae and Culicidae was surveyed to better understand the pupae of those diverse families. For other families, all (or nearly all) previously published descriptions were examined.

Ptychopteridae (Figs. 7A–B, 11A, 14A, 21A–B, 25A)

Description: (sensilla listed in Table 2). Body (Fig. 21A) elongate, tubular, with cephalothorax not larger dorsoventrally than abdomen, apical portion of abdomen directed posteriorly in life; mouthparts and thoracic appendages loosely fused to each other and/or remainder of cephalothorax. Head (Figs. 7A–B, 11A): without tubercles other than on dorsal apotome and dorsolateral sclerite, setae otherwise arising from flat cuticle; dorsal apotome slender, ventrally abutting anterior margin of clypeus; anterolateral cephalic sclerite well developed, each side broadly abutting medially, fused anterolaterally to orbital portion of head capsule in exuviae; antenna elongate, apex extending posteriorly to lie between wing margin and midleg; mouthparts somewhat elongate, restricted to area anterior to forecoxae / trochanters, except for labella which lie between forecoxae / trochanters; apex of labrum truncate; mandible present, maxilla absent; palpus directed obliquely anterolaterally; labella divided medially; exuviae with head entirely detached anteriorly, attached with membrane to forecoxae / trochanters, head capsule (Fig. 7B) folded longitudinally with each ocular half folded along margin of dorsal apotome and anterolateral margin of clypeus/labrum, and clypeus folded forward; sensilla: setae arising from flat cuticle; dorsal

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 23 apotomals—1 seta on short tubercle; anterolateral cephalic sclerite with 1 seta, 1 campaniform sensillum in Ptychoptera Meigen, at least 1 seta in Bittacomorpha Westwood; clypeals—1 seta, 2 campaniform sensilla in Ptychoptera, at least 1 seta in Bittacomorpha; oculars—3 setae, 1 campaniform sensillum in Ptychoptera, 4 setae in Bittacomorpha; antennals—2 campaniform sensilla. Thorax (Figs. 11A, 14A): prothorax without ventrolateral extension; respiratory organs (Figs. 21A–B) unequal, each elongate, undivided, more or less tubular, with numerous annulations, pores scattered along length and a group concentrated at apex, thoracic trachea extending into base of respiratory organ and close to external surface of respiratory organ, basal posteromedial apodeme not apparent but not studied carefully; legs (Fig. 11A) extending posteriorly, with tarsi extending posteriorly beyond wing apex and either lying beside one another (Ptychoptera) or with foretarsus lying ventral to midtarsus; anterior portion of midleg (other than protruding tarsus) extending only partway to wing apex (so that wing apex covers portion of midleg); metathorax (Fig. 14A) well developed, undivided medially, extending posterolaterally beyond anterolateral margin of abdominal tergite 2; sensilla: setae, non-bifurcated, with anterolateral arising from low tubercle, dorsals arising from single low tubercle; anteromedials—absent; anterolaterals—at least 1 seta in Ptychoptera, 2 setae in Bittacomorpha; dorsals—5 setae, 2 campaniform sensilla, close to each other; prealar— absent; metathoracics—6 setae. Abdomen: circular in cross-section; tergite 1 (Fig. 14A) without posterolateral extension; segments 2–8 with pleural membrane; segment 9 (Fig. 25A) with anterior dorsomedial fleshy lobe; terminal process a short or long, apically tapered or rounded lobe bearing basal, laterally or anterolaterally projecting prong or short lobe with 2–3 apical tubercles; sensilla: segments 1–8 with large number of setae, most (all?) bifurcated, each arising from short, generally bifid tubercle; setae on tergites, sternites arranged in transverse rows on sclerotized bands, those on pleurites arranged in 3 longitudinal rows. Segment 9 without sensilla. Discussion: The specimens examined here were somewhat dirty (especially those of Bittacomorpha), certainly because of the detritus-filled habitat these species live in. As such, there is a possibility that some campaniform sensilla and perhaps some broken setae were not observed. Each of abdominal segments 2–7 of Ptychoptera pupae has numerous transverse rows of setae, each on a short raised tubercle, and the pleural areas of segment 2–8 have longitudinal rows of similar setae. The abdominal segments of Bittacomorpha are not distinguishable from one another and have numerous transverse rows of setae with each row arising from a narrow sclerotized band of cuticle, as well as three longitudinal rows of setae on the pleural areas. As such it was not possible to homologize the abdominal setae of segments 2–8 of Ptychopteridae with those of Culicomorpha. Alexander (1981) referred to the smaller of the two respiratory organs as "degenerate", but it has a number of open pores that indicate it is functional. Material examined: Two pupal exuviae of Ptychoptera sp., Tsuruyashiki Kobayashi, Miyazaki Pref., Kyushu, Japan; pupa of Ptychoptera sp., Iowa Lakeside, Dickinson Co., Iowa, USA; 3 pupae of Bittacomorpha sp., Silver Lake Fen, Dickinson Co., Iowa, USA.

Culicomorpha

Chironomidae (Figs. 1A–B, 4A, 7C–D, 11B, 14B, 17A, 18A–D, 21C, 22A, 25B–D)

Description: (sensilla listed in Table 2). Body (Figs. 1A–B) with cephalothorax larger dorsoventrally than abdomen, apical portion of abdomen directed posteriorly to entirely curved under cephalothorax in life; antenna, head loosely attached to thorax, mouthparts and thoracic appendages loosely attached to each other and/or remainder of cephalothorax. Head (Figs. 4A, 11B, 18C–D): with or without tubercles; dorsal apotome somewhat T-shaped, dorsally wide, ventrally narrow, fused to face; anterolateral cephalic sclerite well developed, each side broadly abutting medially, attached to head and, at least in some, by folded membrane to anterior margin of thorax in exuviae; antenna elongate, extending well posterior of margin of head, in most (especially males) extending posteriorly beyond anterior margin of wing (either under wing or along its anterior margin); mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum rounded; mandible present or absent (absent in some derived lineages, e.g. some Chironominae; present in many in which adult females have reduced, non- functional mandibles); maxilla present (in Archaeochlus, Austrochlus Cranston; i.e. those with biting mouthparts as

24 · Zootaxa 3396 © 2012 Magnolia Press BORKENT adult females) or absent (all other Chironomidae); palpus directed laterally, apices of palpi separated medially; labella not divided medially; exuviae (Figs. 7C–D) with head detached dorsally, partially attached with membrane laterally to thorax and posteriorly to forecoxae / trochanters, distorted from original shape, folded longitudinally with each antennal base and ocular half folded along margin of dorsal apotome and with sharp bend between dorsal apotome and clypeus (dorsal apotome folded forward); sensilla (mostly as in Archaeochlus): dorsal apotomal absent or as a seta arising from flat cuticle or from tubercle of various size; other setae arising from flat cuticle; anterolateral cephalic sclerite with 1 seta, 1–2 campaniform sensilla; clypeals—absent (most Chironomidae) or 1 seta (e.g. Archaeochlus) on dorsal margin of clypeus; oculars —3 setae; antennals—absent. Thorax (Fig. 14B): without notable tubercles, at most with very slight bumps; prothorax without ventrolateral extension; respiratory organ (Figs. 21C–D) absent or multibranched and without plastron or variably shaped tube with apical plastron or filter apparatus, tracheal tube well separated from (early lineages) or appressed against external surface of respiratory organ, base supporting respiratory organ without short posteromedial apodeme; legs (Figs. 18A–D), foreleg in nearly all taxa extended to apex of wing and in some extended beyond wing or curving dorsally along wing margin; apex of foreleg ventromedial or ventral to apex of midleg in most taxa (except for a few derived genera); midleg extending to near apex of wing (except for a few genera extending beyond wing); apical portion of hind leg visible in lateral view along posterior margin of wing or entirely covered by wing, apex abutting apex of midleg (except for a few genera abutting subapically); metathorax (Fig. 17A) divided medially by mesothorax, extending laterally under wing, in some partially visible posterolaterally with underlying halter pointed posterolaterally but mostly under wing; sensilla (in Archaeochlus): setae, non-bifurcated, arising from flat cuticle or on short tubercle; anteromedials—4 setae, 1 campaniform sensillum anteromedial or medial to base of respiratory organ; anterolaterals—2 setae on anterolateral margin of thorax; dorsals—2 setae; prealar—absent; supraala—1 seta, 1 campaniform sensillum; metathoracics—3 setae, 1 campaniform sensillum, all on 1 short tubercle. Abdomen: segments either circular in cross-section or somewhat flattened dorsoventrally, without membranous pleura; segments without open spiracles but some taxa with segments 2–7 with lateral spiracular scar; tergite 1 (Fig. 17A) with anterolateral margin loosely under wing, without posterolateral extension; terminal process (Figs. 25B–D) moderately elongate, variously shaped, in most Podonominae more or less conical or lobe- like, other subfamilies dorsoventrally flat, nearly all bearing more than 1 marginal or apical seta, in Archaeochlus apex posterodorsally curved and somewhat hook-like; sensilla (in Archaeochlus): setae, non-bifurcated, arising from flat cuticle or short, stout tubercles; tergite 1 with 6 setae; segment 4 (Fig. 22A) with D-1-IV, D-2-IV, D-5-IV, D-6-IV, D-7-IV setae, D-4-IV a campaniform sensillum, L-1-IV, L-2-IV, L-3-IV, L-4-IV setae with L-1-IV, L-3-IV each on tubercles, V-1-IV, V-2-IV, V-3-IV, V-4-IV, V-5-IV, V-6-IV, V-7-IV setae, with V-5-IV on tubercle. Segment 9 with 6 dorsal sensilla: 3 setae at midlength, 1 seta, 2 campaniform sensilla on terminal process. Discussion: The pupae of Chironomidae are extremely variable and much of the description is restricted to the features of a few early lineages of Chironomidae and particularly those of Archaeochlus (especially the chaetotaxy). However, other Podonominae and representatives of a few other subfamilies (especially Buchonomyiinae) were also studied in some detail. The description therefore covers all of the Chironomidae in some aspects but is far more restrictive for some features, especially details of the chaetotaxy. This description will provide a basis for making further comparisons of other Chironomidae to those of other Culicomorpha. Nearly all Chironomidae have their legs arranged as is typical of other Culicomorpha (with the hind leg S- shaped and under the wing) (Fig. 18A). A few genera of Chironomidae have more elongate legs (Figs. 18B–D) and three genera do not have the hind leg S-shaped (Fig. 18D). These are discussed in the introduction to the key to families above. The pupa of Buchonomyia thienemanni Fittkau differs in significant ways from Archaeochlus. It has elongate fore- and midlegs (Fig. 18B) and lacks respiratory organs. Five ocular setae, 1 dorsal (D–T), and 1 supralar and 2 supralar campaniform sensilla are present. There may be 2 distinctive prealar setae, or perhaps these are disassociated anteromedial setae (especially considering the respiratory organ is absent in this species). The literature describing pupae of Chironomidae is very large and as such this study is generally restricted to Brundin (1966), Sæther (1980) and Wiederholm (1986). Material examined: 1 pupa of Parochlus sp. Enderlein, Remarkables Stream, South Island, New Zealand; 1 pupa of Parochlus araucanus Brundin (?), Fundo Flor del Lago, Villarica, Araucania, Chile; 1 pupal exuviae of Parochlus ohakunensis (Freeman), Remarkables, Alta Tarn, South Island, New Zealand; 2 pupae of Archaeochlus bicirratus Brundin, Quadia's Nek, Lesotho; 1 pupa of Archaeochlus biko Cranston, Edward & Colless, Matchless

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 25 River, Namibia; 5 pupal exuviae of Austrochlus parabrundini Cranston, Edward & Colless, Mt. Augustus, WA, Australia; 1 pupa, 3 pupal exuviae of Harrisonina petricola Freeman, Komga, Eastern Cape, South Africa; 1 pupal exuviae of Stictocladius sp. Mt. Field National Park, entrance creek, Tasmania, Australia; 1 pupal exuviae of Stictocladius sp., Gibraltar Falls, ACT, Australia; 8 pupal exuviae of Stictocladius uniserialis (Freeman), Bindi Ck., Tambo, VIC, Australia; 1 pupa, 2 pupal exuviae of Psectrotanypus sp. Kieffer sp., 5.6 km NW Enderby, BC, Canada; 4 pupal exuviae of Buchonomyia thienemanni, Flesk River, (White Bridge) Killarney, Co. Kerry, Ireland, Irish Grid ref. V987904.

Ceratopogonidae (Figs. 1C, 2A–C, 3A–C, 4B, 5A–B, 6A–B, D, 7E–F, 12C–D, 14C, 17B, 19A, 21E, 22B, 25E)

Description: (sensilla listed in Table 2). Body (Figs. 1C, 2A–C) with cephalothorax somewhat larger dorsoventrally than abdomen, apical portion of abdomen nearly always directed posteriorly in life (only known exception is Dasyhelea ampullariae Macfie with abomen curved entirely under the cephalothorax, Borkent pers. obs. (as in Fig. 1B)); slender; mouthparts and thoracic appendages fused to each other and/or remainder of cephalothorax. Head (Fig. 3A–C, 4B, 6A–B, D, 12C–D, ): with or without tubercles; dorsal apotome shape various, in early lineages T-shaped with slender (ventral) base, in derived lineages broader and either continuous with face or with transverse fold lying between bases of antennae (with further modifications in higher groups); anterolateral cephalic sclerite well developed, broadly meeting medially in early lineages, divided medially by dorsal apotome in derived lineages, articulated with membrane to margin of antenna and anterior margin of mesonotum in exuviae of early lineages, fused to anterior margin of thorax in derived lineages; antenna elongate, apex extending posteriorly to various points along anterior margin of wing; mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum narrowly to broadly rounded; mandible present or absent; maxilla present (most of those with biting adult females) or absent (most of those with non-biting adult females), when present with apicomedial portion of maxilla extending slightly posteriorly from under mandible or lying completely ventral to mandible; palpus directed posteriorly or posteromedially, apices of palpi separated medially; labella divided or not divided medially; exuviae (Figs. 7E–F) with head entirely detached dorsally, fused posteriorly to forecoxae / trochanters, not distorted from original shape; sensilla: setae arising from flat cuticle or from tubercle on dorsal apotome in some taxa; dorsal apotomals—0–3 setae, 0–3 campaniform sensilla; anterolateral cephalic sclerite with 1–2 setae, 0–1 campaniform sensilla; clypeals—0–2 setae, 0–1 campaniform sensilla in variable positions on labrum; oculars—0–2 seta, 0–2 campaniform sensilla; antennals—2 campaniform sensilla. Thorax (Fig. 14C): with or without tubercles; prothorax with or without ventrolateral extension abutting against lateral margin of palpus (not visible in Forcipomyiinae); respiratory organ (Figs. 3A–C, 21E) tubular, of varying shape, surface with or without annulations, with or without spicules, tracheal tube with spirals, at least basally, extending into respiratory organ, well separated from external surface of respiratory organ, with pores at apex of respiratory organ and some taxa with additional pores distributed along length, with or without short to long posteromedial apodeme at base of respiratory organ; legs (Fig. 19A), foreleg extending partway to or near apex of wing; apex of foreleg anterior to ventral to apex of midleg; apex of midportion of midleg well anterior to apex of wing (Fig. 19A); apical portion of hind leg visible in lateral view along posterior margin of wing or not visible and under wing, apex abutting apex of midleg; metathorax (Fig. 17B) either narrow to broad band undivided medially or divided medially by mesothorax, extending posterolaterally from halfway length of abdominal tergite 1 (early lineages) to posterior to anterolateral margin of abdominal tergite 2, apex separate from to broadly abutting hind leg; sensilla: setae, non-bifurcated or bifurcated, arising from flat cuticle or from tubercles; anteromedial— 0–2 setae, 0–2 (6 in Leptoconops) campaniform sensilla anteromedial, anterior, or anterolateral to base of respiratory organ; anterolaterals—3 setae on anterolateral margin of thorax; dorsals—2–4 setae, 1 campaniform sensillum; prealar – absent; supraala—1 seta in Austroconops only, 0–1 campaniform sensillum; metathoracics—0–2 setae, 1–3 campaniform sensilla. Abdomen: circular in cross-section; with or without tubercles; segments without open spiracles but some taxa with segments 2–7 with lateral spiracular scar; tergite 1 (Fig. 17B) without posterolateral extension; segments without pleural membrane; terminal process (Fig. 25E) short to elongate, dorsally curved and hook-like, lobe-like, or conical; sensilla: setae, bifurcated or non-bifurcated, arising from flat cuticle or tubercles; tergite 1 with 2–8 setae, 1–4 campaniform sensilla; segment 4 (Fig. 22B) with D-1-IV a minute seta (absent in some?), D-2-IV, D-3-IV each a seta (D-3-IV a campaniform sensillum or absent in

26 · Zootaxa 3396 © 2012 Magnolia Press BORKENT some), D-4-IV a campaniform sensillum or absent, D-5-IV, D-8-IV, D-9-IV each a seta (D-5-IV, D-8-IV, 9-IV absent in some), D-7-IV a campaniform sensillum, laterals L-1-IV, L-2-IV, L-3-IV, L-4-IV setae (L-1-IV anterior to others in most taxa; L-1-IV, L-3-IV, L-4-IV absent in some), V-1-IV, V-2-IV each a campaniform sensillum or absent, V-5-IV, V-6-IV, V-7-IV setae (V-7-IV absent in some). Segment 9 generally with 2 campaniform sensilla on apicolateral process, Leptoconops with D-1-IX at midlength of segment and further campaniform sensilla, some Dasyhelea with 2 setae, 2 campaniform sensilla. Discussion: The pupae of Ceratopogonidae are highly variable and their variation will be the subject of another paper (Borkent, in prep.). Borkent & Craig (2004) described the pupa of an early lineage of Ceratopogonidae, Austroconops mcmillani Wirth & Lee, in detail and this forms a good basis for understanding most plesiomorphic features within the family. Of course, there are some features of this taxon that are derived, as discussed by Borkent & Craig (2004). Further analysis will be provided by Borkent (in prep.). Borkent & Craig (2004) misinterpreted some aspects of the pupa of Austroconops mcmillani: on the thorax they missed supraalar 2 and on the fourth abdominal segment they missed D-1-IV, thought V-2-IV was a campaniform sensillum when there is a minute seta present, and considered the closely abutting D-2-IV and D-3- IV as a single seta. Material examined: All known genera and numerous species within many of those genera, to be listed by Borkent (in prep.).

Thaumaleidae (Figs. 1D, 6E, 8C–E, 10D, 12A, 14D, 17C, 19B, 21F, 22C, 25F)

Description: (sensilla listed in Table 2). Body (Fig. 1D) with cephalothorax not or only slightly larger dorsoventrally than abdomen, apical portion of abdomen directed posteriorly in life; mouthparts and thoracic appendages fused to each other and/or remainder of cephalothorax. Head (Fig. 12A): without tubercles; dorsal apotome broad, ventrally completely fused to face; anterolateral cephalic sclerite well developed, each side broadly abutting medially, loosely attached by membrane to thorax in exuviae, easily detached entirely; antenna short, apex not extending to posterior margin of head; mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum slightly narrowed; mandible present, maxilla absent; palpus directed posteromedially, apices of palpi separated medially; labella not divided medially; exuviae (Figs. 6E, 8C–E) with head entirely detached dorsally, in most completely detached, in a few attached posteriorly with membrane to forecoxae / trochanters, not distorted from original shape; sensilla: dorsal apotomal 1 arising from short tubercle, other setae arising from flat cuticle; dorsal apotomals—1 seta; anterolateral cephalic sclerite with 2 setae; clypeals—3 campaniform sensilla, 2 on labrum, 1 farther anterior; oculars—1 seta, 1 campaniform sensillum; antennals—absent. Thorax (Fig. 14D): with dorsal crenulations; prothorax with ventrolateral extension abutting lateral margin of palpus; respiratory organ (Fig. 21F) undivided, more or less tubular, surface with pattern of fine bumps, tracheal tube extending into respiratory organ, well separated from external surface of respiratory organ, with apical plastron possibly surrounded by pores, short tubercle supporting respiratory organ with short posteromedial apodeme; legs (Fig. 19B), foreleg extending to apex of wing; apex of foreleg ventromedial to apex of midleg; apex of midportion of midleg well anterior to apex of wing; apical portion of hind leg visible in lateral view along posterior margin of wing, apex abutting apex of midleg; metathorax (Fig. 17C) a broad band, undivided medially by mesothorax, extending posterolaterally past anterolateral margin of abdominal tergite 2 and apex narrowly abutting hind leg; sensilla: setae, non-bifurcated or with some foliaceous (only in some Austrothaumalea Tonnoir), arising from flat cuticle or from tubercles or ridges; anteromedials—2–4 setae anterior to base of respiratory organ; anterolaterals— 3 setae on short tubercle on anterolateral margin of thorax; dorsals—4 setae, 1 campaniform sensillum; prealar— absent; supraalar—absent; metathoracics—1 seta (Trichothaumalea Edwards) or 3 setae, 1 campaniform sensillum (Androprosopa Mik). Abdomen (Fig. 10D): segments with dorsal portion flat and distinctly angled with lateral portion, without membranous pleura; with rounded tubercles; segments 2–7, 3–7, 4–7 or 5–7 with lateral open spiracle; tergite 1 (Fig. 17C) without posterolateral extension; terminal process (Fig. 25F) moderately elongate, dorsally curved, hook-like (Androprosopa) or absent (e.g. Trichothaumalea); sensilla: setae, non-bifurcated, arising from flat cuticle, rounded tubercles or ridges; tergite 1 with 1 seta or 3 setae, 1 campaniform sensillum; segment 4 (Fig. 22C) with D-1-IV, D-2-IV, D-3-IV,D-5-IV, D-8-IV, D-9-IV setae (in some Androprosopa D-9-IV absent), D-

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 27 7-IV a campaniform sensillum, L-1-4-IV setae with L-1-IV on anterolateral margin of segment, V-1-IV, V-2-IV, V- 5-IV, V-6-IV, V-7-IV setae. Segment 9 with 1-2 more anterior dorsal setae, D-1-IX, D-2-IX (present or absent), 3 dorsal sensilla near base of terminal process D-4-IX, D-5-IX, D-6-IX, either 1 seta, 2 sensilla campaniform sensilla (e.g. A. linsayorum (Arnaud & Boussy)) or 2 setae, 1 sensillum campaniforma (e.g. A. striata (Okada)); 1 ventral seta, 1 campaniform sensillum, V-1-IX, V-2-IX near base of terminal process. Discussion: There are few detailed descriptions of pupae of Thaumaleidae. I particularly consulted Peterson et al. (1989), Gillespie et al. (1994), McLellan (1983, 1988), Sinclair (1992, 1996, 2000), and Sinclair & Saigusa (2002). In the above description, the respiratory organ is consider to have an apical plastron. However, there is some uncertainty about this because a ring of small apical pores may be present. I was unable to resolve this with my microscopes and a SEM study of this structure would be invaluable. There have been no published cladograms depicting the relationships between the genera of Thaumaleidae. However, B. Sinclair (pers. comm.) has a working hypothesis indicating that Thaumalea + Androprosopa are the sister group of all other Thaumaleidae and that within the latter assemblage, Trichothaumalea is the sister group of the remaining members of the family. As such, I studied Androprosopa and Trichothaumalea. Most preserved Thaumaleidae pupae have the head partially detached posteroventrally, reflecting the subsequent condition in the exuviae, where the head capsule is only very loosely attached and easily detaches entirely. There may be more sensilla on the body of Thaumaleidae than shown here because the species examined were covered with dense shagreen and undetected small setae or campaniform sensilla may be hidden among them. A SEM study would help to confirm the presence of additional sensilla. Material examined: 1 pupa, 1 pupal exuviae of Androprosopa americana (Bezzi), Owen Sound, Ontario, Canada; 2 pupal exuviae of Androprosopa striata, Tenninkyo, Japan; 1 pupa, 3 pupal exuviae of Androprosopa linsayorum, 1 mi. E. Hwy. 34, Mary's Peak, Benton Co., Oregon, USA ; 1 pupal exuviae of Trichothaumalea pluvialis (Dyar & Shannon), 3 km. E. Sicamous, BC, Canada.

Simuliidae (Figs. 1E, 6F, 8A–B, 10C, 12B, 15A–B, 17D, 19C, 21G, 23A–B, 25F, 27A–F)

Description: (sensilla listed in Table 2). Body (Fig. 1E) uniform across family, with cephalothorax somewhat larger dorsoventrally than abdomen, apical portion of abdomen directed posteriorly in life; mouthparts and thoracic appendages fused to each other and/or remainder of cephalothorax. Head (Fig. 12B): without tubercles; dorsal apotome broad, ventrally completely fused to face; anterolateral cephalic sclerite small, each side broadly separated medially by dorsal apotome, triangular and fused laterally to antenna in female exuviae, elongate and slender and fused laterally to antenna and eye in male exuviae; antenna short, apex not extending to posterior margin of head or just posteriorly; mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum broadly rounded; mandible, maxilla present, with apicomedial portion of maxilla extending slightly posterior from under mandible; palpus directed posteromedially, apices of palpi separated medially; labella not divided medially; exuviae (Figs. 6F, 8A–B) with head entirely detached dorsally, fused posteriorly to forecoxae / trochanters, not distorted from original shape; sensilla: setae arising from flat cuticle; dorsal apotomals—in some Austrosimulium Tonnoir 2 setae, in Prosimulium Roubaud 1 seta and 1 campaniform sensilla displaced posteriorly, situated posteromedially from base of antenna; anterolateral cephalic sclerite with 3 setae; clypeals—2 setae, 1 campaniform sensillum near anterior margin of labrum; oculars—1 seta, 2 campaniform sensilla; antennals— absent. Thorax (Figs. 15A–B): without tubercles or with 2 large tubercles at base of respiratory organ (Crozetia Davies); prothorax with ventrolateral extension abutting lateral margin of palpus; respiratory organ (Fig. 21G) with at least three divisions (many species with many more), with single basal opening (called basal fenestra), surface with annulations, separate tracheal tube extending only short distance into base of respiratory organ (Parasimulium) or not at all (all other Simuliidae) and in only Parasimulium this portion attached to one wall of respiratory organ, with surface plastron on remainder of respiratory organ (not on elongate base of Parasimulium), with short posteromedial apodeme at base of respiratory organ; legs (Fig. 19C), foreleg extending to apex of wing; apex of foreleg ventral to apex of midleg; apex of midportion of midleg well anterior to apex of wing; apical portion of hind leg visible in lateral view along posterior margin of wing, apex abutting apex of fore-, midleg;

28 · Zootaxa 3396 © 2012 Magnolia Press BORKENT metathorax (Fig. 17D) either a narrow band but undivided medially, or divided medially by mesothorax, not extending posterolaterally past anterolateral margin of abdominal tergite 1 (so that adult halter develops under wing); sensilla (based primarily on Prosimulium): setae, non-bifurcated or with some bifurcated, arising from flat cuticle; anteromedials—6 setae anterior to base of respiratory organ; anterolaterals—2 setae just anterolateral of respiratory organ (close to anteromedials); dorsals—5 setae, 2 campaniform sensilla; prealar—1 or 3 setae; supraalar 1 seta; metathoracics—5 setae, 2 campaniform sensilla. Abdomen (Fig. 10C): circular in cross-section; without tubercles; segments without open spiracles but in some taxa segments 2–7 with lateral spiracular scar; tergite 1 (Fig. 17D) with posterolateral extension abutting or nearly abutting projecting portion of hind leg along posterior margin of wing; tergites 3–4 with at least 1 sensillum modified into recurved hook (generally with 4 of these recurved); with at least sternites 5–7 each with at least 1 sensillum modified into recurved hook; segments 4– 8 with pleural membrane; terminal process (Fig. 10C, 25G) short, dorsally curved, hook-like; sensilla (based on Parasimulium and Prosimulium): setae, non-bifurcated, arising from flat cuticle or recurved hooks each with slightly raised base; tergite 1 with 6 setae, 1 campaniform sensillum; segment 4 (Figs. 23A–B) with D-1-IV a minute seta, D-2-IV, D-3-IV each an elongate seta (D-3-IV absent in Parasimulium), D-5-IV, D-6-IV, D-8-IV, D-9- IV each a strong recurved hook-like seta, D-7-IV a campaniform sensillum, or a small seta in Parasimulium, L-1- IV, L-2-IV, L-3-IV, L-4-IV, setae (L-1-IV, L-4-IV absent in Parasimulium but L-1-VII, L-2-VII, L-3-VII present on segment 7), V-1-IV, V-3-IV, V-5-IV, V-6-IV, V-7-IV setae, V-5-IV a strong recurved hook-like seta (only V-1-IV, V- 5-IV, V-7-IV present, with V-5-IV a simple seta in Parasimulium but V-5-VI, V-5-VII, V-5-VIII hook-like). Segment 9 with D-1-IX, D-4-IX, L-1-IX, L-2-IX, L-3-IX, L-4-IX, V-1-IX, all setae. Discussion: Crosskey (1990) and Adler et al. (2004) described the pupae of Simuliidae, provided significant further details concerning the morphology of the pupal respiratory organ and discussed a few other features of the pupae of Simuliidae. Douglas A. Craig provided further important information on Austrosimulium and Crozetia. The respiratory organ of Simuliidae is uniquely modified. The outer surface of the structure serves as a plastron (except for the elongate base of Parasimulium) and the inside of the respiratory organ is filled with water (via a basal hole which tears through very thin cuticle at the base of the respiratory organ called the basal fenestra). Oxygen diffuses from the surrounding water into the surface plastron and is drawn toward the base of the respiratory organ. In Parasimulium oxygen is then taken in by the short tracheal tube present in the basal "stalk" of the respiratory organ, and in other Simuliidae, by the tracheal tube near the very base of the respiratory organ. It appears that the apical plastron present in some other Culicomorpha has been modified to cover most, or all of the surface of the respiratory organ of Simuliidae, leaving the interior to be filled with liquid. In most other Culicomorpha (other than some Culicidae, Chaoboridae and Chironomidae), the trachael tube is separate from the outer surface of the respiratory organ and the intervening space is filled with fluid (presumably haemolymph). The genus Parasimulium, as the sister group of all remaining Simuliidae (Adler et al. 2004), is of particular significance. Unfortunately, there is little material available for study. Reexamination of parts of a slide mounted specimen (ROM) allowed interpretation of a portion of the right side of the thorax (dorsum, including the respiratory organ) and of the abdomen posterior to segment 2. The portion of the thorax bearing the respiratory organ was torn and compressed but the reconstructed view provides at least the relative positions of sensilla (Fig. 15B). At least a portion of the anterolateral cephalic sclerite was also present and bore two sensilla, one less than the number present on Prosimulium; however, it is possible that the condition in Parasimulium was misinterpreted (because of damage to the specimen). Similarly, the abdomen was strongly flattened so the drawing in Fig. 23B is a reconstruction. It will be valuable to obtain fresh material of Parasimulium to examine all the traits reported here. There is significant variation in the pattern of sensilla on the heads of Simuliidae. For example, Austrosimulium tillyardianum Dumbleton has only 1 anterolateral cephalic sclerite seta, all three clypeal sensilla as campaniform sensilla, and ocular sensilla 1 and 3 appear to be absent. Crozetia seguyi Beaucournu-Saguez & Vernon has both dorsal apotomals 1 and 2 as well developed setae situated on the lateral margin of the dorsal apotome, well posterior to the base of the antenna. There are also many deviations from the pattern of sensilla on the thorax and abdomen as described above. For example, Crozetia seguyi has numerous stout setae scattered all over the dorsum of the thorax. It seems clear that the distribution of thoracic sensilla will be of taxonomic significance with this family. The anterior portion of the mesosternum is fused with the forecoxae / trochanters in the pupae examined. This feature warrants further study in Simuliidae and other Culicomorpha. It may be present in at least some Chironomidae as well.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 29 A surprising degree of asymmetry was found in the cephalic setae in a few different species, with some setae missing on one side or the other, as well as varying in position. Nevertheless, sensilla could be identified in relation to those in Prosimulium formosum Shewell in most specimens of other taxa. Material examined: 1 pupa of Parasimulium crosskeyi Peterson (part of thorax; abdominal segments 4–9), Wahkeena Creek, Benson St. Pk., Oregon, USA; 5 pupae, 7 pupal exuviae of Prosimulium formosum, Dog Lake outlet, Yakima Co., Washington, USA; 2 pupae of Paraustrosimulium anthracinum (Bigot) e/ Huillunco y Chonchi, Rio Melataba, Chile; 2 pupae of Austrosimulium ungulatum Tonnoir, WD, SH6, near Fox Glacier, Ribbonwood Creek, S43.48880 E169.97733, South Island, New Zealand, 2 pupal exuviae, NN, Mt Arthur, Flora Hut, Flora Stream, South Island, New Zealand; 2 pupae of Austrosimulium tillyardianum MC, Banks Peninsula, Wainui, Wainui Stream, S43.81013 E172.89372, South Island, New Zealand, 2 pupal exuviae, Marlborough Snds, Kenepuru Head Strm, S41.16566 E174.12362, South Island, New Zealand; 3 pupal exuviae of Austrosimulium bicorne Dumbleton, MC. Arthurs Pass, Temple Basin, New Zealand; 4 pupal exuviae of Austrosimulium n. sp. (sister species of A. bicorne, to be described by D.A. Craig), OL, SH94 Homer Tunnel, left strm, upper Hollyford Rv, S44.76580 E167.98969, South Island, New Zealand; dorsal apotomes and respiratory organs of 5 pupal exuviae of Crozetia seguyi, Riv. Petit Manchotiere, S46° 23' 53" E 51° 49' 14", Ile de la Possession, Crozet Islands; 4 pupae, 3 pupal exuviae Gymnopais Stone sp., Iqaluit (as Frobisher Bay), Nunavut, Canada.

Dixidae (Figs. 1F, 9A–B, 13A, 15C, 17E, 20A, 21H, 23C, 26A)

Description: (sensilla listed in Table 2). Body (Fig. 1F) with cephalothorax much larger dorsoventrally than abdomen, apical portion of abdomen curved under cephalothorax in life; mouthparts and thoracic appendages loosely fused to each other and/or remainder of cephalothorax, easily separating in exuviae. Head (Fig. 13A): without tubercles; dorsal apotome nearly as long as wide; anterolateral cephalic sclerite well developed, broadly meeting medially, articulated with membrane to margin of antenna and anterior margin of mesonotum; antenna elongate, apex extending posteriorly along anterior margin of wing to near wing apex; mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum broadly rounded; mandible present, maxilla absent; palpus directed anteromedially, apices of palpi abutting or overlapping medially; labella divided medially; exuviae (Figs. 9A–B) with head attached with membrane to thorax dorsally (with anterolateral cephalic sclerites), head capsule folded longitudinally with each antennal base folded along margin of dorsal apotome, ocular halves folded along margin of clypeus/labrum, margin of dorsal apotome and clypeus folded transversely with both parts bent ventrally; sensilla: setae arising from flat cuticle; dorsal apotomals absent; anterolateral cephalic sclerite with 1 seta; clypeals absent; oculars—1–2 setae (2 in some Dixa Meigen (as Paradixa Tonnoir, Belkin 1968); antennals— absent. Thorax (Fig. 15C): without tubercles other than longitudinal short (Dixa) or broad (Dixella) flange arising anterior to base of respiratory organ and extending either to part of the antenna laterally (Dixa) or over anterolateral cephalic sclerite to its anterolateral margin (Dixella); prothorax without ventrolateral extension abutting lateral margin of palpus; respiratory organ (Fig. 21H) trumpet-shaped, surface without annulations, with spicules, tracheal tube extending partway into respiratory organ, well separated from external surface of respiratory organ, with filter apparatus at base of interior funnel-shaped portion at apex of respiratory organ, without posteromedial apodeme at base of respiratory organ; legs (Fig. 20A), foreleg extending to and curved dorsally along apical margin of wing; apex of foreleg ventroposterior to apex of midleg which is also curved dorsally around apex of wing margin; midleg extending to apex of wing; apical portion of hind leg not or barely visible in lateral view along posterior margin of wing, apex abutting apices of fore- and midleg; metathorax (Fig. 17E) a broad band undivided medially, extending posterolaterally posterior to anterior margin of abdominal tergite 2, apex poorly defined, approximating hind leg; sensilla: setae, non-bifurcated, arising from flat cuticle or from flange anterior to base of respiratory organ; anteromedial—1 seta (absent in Nothodixa Edwards; Belkin 1968), anteromedial to base of respiratory organ; anteromedial—1 seta; anterolateral—1 seta on short (Dixa) or broad (Dixella) flange anterior to base of respiratory organ; dorsals—2–3 setae (3 in some Dixa (as Paradixa); Belkin 1968); prealar—absent; supraalar—1 seta or campaniform sensillum; metathoracics—3 setae. Abdomen: somewhat squared in cross-section, with ventrum more or less flat; without tubercles; segments without open spiracles but segments 2–7 each with lateral spiracular scar; tergite 1 (Fig. 17E) without posterolateral extension; segments without pleural membrane; terminal processes (Fig. 26A) fused basally, each dorsoventrally compressed, directed posteriorly, tapering to strong point;

30 · Zootaxa 3396 © 2012 Magnolia Press BORKENT sensilla: setae, non-bifurcated, arising from flat cuticle; tergite 1 with 6–8 setae; segment 4 (Fig. 23C) with D-1-IV a minute seta, D-2-IV, D-5-IV, D-6-IV, D-8-IV, D-9-IV each a seta, D-7-IV a campaniform sensillum, L-1-IV, L-2- IV, L-3-IV, L-4-IV each a seta, V-1-IV, V-5-IV, V-5-IV each a seta. Segment 9 with 2 setae, D-1-IX at midlength, V-1-IX near apex of terminal process. Discussion: The homologies of some abdominal sensilla within the Culicoidea are interpreted differently here than in numerous papers by Belkin and his associates. This is discussed further in the glossary section of this paper. Descriptions of Dixidae pupae consulted for this work were Belkin (1962, 1968), Belkin et al. (1970) and Brundin (1966: 429). Material examined: 3 pupae, 4 pupal exuviae of Dixella lobata Chaverri & Borkent, Tapanti National Park, Pariso, Pr. Cartago, Costa Rica; 1 pupa, 4 pupal exuviae of Dixella serrata (Garrett), 5.6 km NW Enderby, BC, Canada; 1 pupa of Dixa pullogruma Peters, Brown Canyon, Apache Co., Arizona, USA; 1 pupal exuviae of Dixidae, Rock Chapel, Dundas, Ontario, Canada; 1 pupal exuviae of Dixidae, Grundlech, Germany; 3 pupal exuviae of Dixidae sp., Huckleberry Flat, Sequoia National Park, California, USA; 3 pupal exuviae of Dixella cornuta (Johannsen), Woods Lake, 10 km S. Westwold, BC, Canada.

Corethrellidae (Figs. 1G, 9C–D, 13B, 15D, 17F, 20B, 21I–J, 24A, 26B–C)

Description: (sensilla listed in Table 2). Body (Fig. 1G) with cephalothorax much larger dorsoventrally than abdomen, apical portion of abdomen directed posteriorly in life; mouthparts and thoracic appendages fused to each other and/or remainder of cephalothorax. Head (Fig. 13B): without tubercles; dorsal apotome nearly as long as wide; anterolateral cephalic sclerite well developed, broadly meeting medially, articulated with membrane to margin of antenna and anterior margin of mesonotum; antenna elongate, apex extending posteriorly along anterior margin of wing to near wing apex; mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum narrowly rounded; mandible present, maxilla absent; palpus directed posteromedially, apices of palpi separated medially; labella not divided medially; exuviae (Figs. 9C–D) with head attached with membrane to thorax dorsally (with anterolateral cephalic sclerites), head capsule folded longitudinally with each antennal base folded along posterolateral margin of dorsal apotome, each ocular half folded toward lateral margin of palpus, and margin of dorsal apotome and clypeus folded transversely with both parts bent ventrally; without head sensilla. Thorax (Fig. 15D): with or without short tubercles; prothorax without ventrolateral extension abutting lateral margin of palpus; respiratory organ (Figs. 21I–J) trumpet-shaped or broad and flattened with separate pores arranged along outer margin (these possibly not connecting with basal tracheae), surface without annulations, with or without spicules, with or without (only peruviana species group) tracheal tube extending into respiratory organ, well separated from external surface of respiratory organ, with finely perforated filter apparatus at base of interior funnel-shaped portion at apex of respiratory organ, without posteromedial apodeme at base of respiratory organ; legs (Fig. 20B), foreleg extending to and curved dorsally along apical margin of wing; apex of foreleg posteroventral to apex of midleg; apex of midportion of midleg well anterior to apex of wing; apical portion of hind leg not or barely visible in lateral view along posterior margin of wing, apex abutting apex of foreleg; metathorax (Fig. 17F) divided into three parts, two lateral (separated medially by wide extension of mesothorax) and slender medial band, metathorax extending posterolaterally to about 0.75 length of tergite 1, separate from hind leg; sensilla: setae, bifurcated or non-bifurcated, arising from flat cuticle or from short tubercles; anteromedial—1 seta, anterior to base of respiratory organ; anterolaterals—2 setae; dorsals—2 setae; prealar—absent; supraalar—1 enlarged, spherical campaniform sensillum present or absent (see discussion); metathoracics—1 seta, 1 spherical sensillum present or absent. Abdomen: dorsoventrally compressed in cross-section; segments without open spiracles but some taxa with segments 2–7 with lateral spiracular scar; tergite 1 (Fig. 17F) without posterolateral extension; segments without pleural membrane; terminal processes (Figs. 26B–C) fused basally, each dorsoventrally compressed, directed posteriorly, tapering to strong point or pair of strong setae; sensilla: setae, non- bifurcated, arising from flat cuticle or short tubercles; tergite 1 with 4–5 setae; segment 4 (Fig. 24A) with D-1-IV, D-2-IV, D-6-IV, D-8-IV, D-9-IV each a seta, D-7-IV a campaniform sensillum, L-1-IV, L-2-IV, L-3-IV, L-4-IV, V- 1-IV, V-5-IV, V-6-IV each a seta. Segment 9 with strong D-1-IX at midlength, D-4-IX, V-1-IX present or absent at apex of terminal process.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 31 Discussion: Borkent (2008) discussed the peculiar flattened respiratory organ of the peruviana species group (Fig. 21J) and identified the margin as bearing pores (his spiracles). In further study here, trachael connections between these marginal pores and the basal tracheae could not be distinguished, bringing into question whether they actually function as pores or perhaps serve some other purpose. The large and spherical campaniform sensilla present on the mesonotum (supraalar 2; Fig. 15D) and metathorax (metathoracic 2; Fig. 17F) are present in all Corethrella other than those of the otherwise strongly modified peruviana species group, and a few other species (see Borkent 2008: 219—character 48). The shape and size of the sensilla are unique and likely another synapomorphy of the family. Borkent (2008) identified these as "spherical sensory pits". The position of the one on the mesonotum is similar to that of the SA-2-T of Chironomidae and Ceratopogonidae indicating the "sensory pits" are likely large and modified campaniform sensilla. Some other Culicomorpha have a campaniform sensillum on the metathorax as well: Chironomidae, Ceratopogonidae, Thaumaleidae and Simuliidae (Figs. 17A–D), but they are otherwise unknown in the Culicoidea. The homologies of abdominal sensilla within the Culicoidea are interpreted differently here than in several papers by Belkin and his associates (Belkin 1962; Belkin & McDonald 1955; Belkin et al. 1970). This is discussed further in the glossary section of this paper. Borkent (2008) indicated the shape of the pupal abdomen was sometimes cylindrical. This is erroneous, as the abdomen is always at least somewhat compressed dorsoventrally. The arrangement of the two abdominal setae V-1-IV, with one being anterior to the other (Fig. 24A), is unique in Corethrellidae. In other Culicomorpha, they are lateral to one another (either side of the medial line). It is uncertain, however, how widely distributed this feature is within the family and further study is needed before designating this as a synapomorphy. The known pupae of Corethrellidae were described briefly by Borkent (2008) although details were not noted (especially chaetotaxy) for some features given in the above description. This stage requires more careful study within the family. Material examined: The specimens of Corethrellidae examined are listed in Borkent (2008). In addition, pupae and pupal exuviae of C. appendiculata Grabham from the colony maintained by L.P. Lounibos in Florida and two pupal exuviae of an unidentified species of the C. peruviana group were more intensely studied .

Chaoboridae (Figs. 1H, 6C, 9E–F, 10E, 13C, 16A, 17G, 20C, 21K, 24B, 26D)

Description: (sensilla listed in Table 2). Body (Fig. 1H) with cephalothorax much larger dorsoventrally than abdomen, in life apical portion of abdomen curved under cephalothorax in early lineages, posteriorly or posteroventrally in derived lineages; mouthparts and thoracic appendages loosely fused to each other and/or remainder of cephalothorax. Head (Fig. 13C): without tubercles; dorsal apotome variable, nearly as long as wide (Eucorethra, Chaoborus) or T-shaped (Mochlonyx), in whole pupae of Eucorethra, Mochlonyx, tucked under anterior margin of thorax and not visible externally; anterolateral cephalic sclerite small to well developed, narrowly separated medially, articulated with membrane to margin of antenna, only anterior margin visible in whole pupae of Eucorethra, Mochlonyx; antenna elongate, apex extending posteriorly along anterior margin of wing to about half wing length; mouthparts short, restricted to area anterior to forecoxae / trochanters; apex of labrum narrowly rounded; mandible present as small lobe at preapex (Mochlonyx, Chaoborus) or beyond apex of labrum (Eucorethra), maxilla absent; palpus directed posteromedially, apices of palpi separated medially; labella not divided medially, restricted laterally, leaving a sclerite between palpus and labellum; exuviae (Figs. 6C, 9E–F) with head attached with membrane to thorax (with anterolateral cephalic sclerites) and forecoxae / trochanters; head capsule folded longitudinally with each antennal base folded along posterolateral margin of dorsal apotome, each ocular half folded toward lateral margin of clypeus-labrum, and margin of dorsal apotome and clypeus folded transversely with both parts bent ventrally; sensilla: setae arising from flat cuticle; dorsal apotomals absent; anterolateral cephalic sclerite with 1 seta; clypeals absent; oculars—3 setae; antennals—absent. Thorax (Fig. 16A): with or without short tubercles; prothorax without ventrolateral extension abutting lateral margin of palpus; respiratory organ (Fig. 21K) trumpet-shaped or wide at midlength and tapered apically, surface without annulations, with or without spicules, tracheal tube extending into respiratory organ, well separated from (Eucorethra), partially appressed (Mochlonyx), or entirely appressed (Chaoborus) against external surface of

32 · Zootaxa 3396 © 2012 Magnolia Press BORKENT respiratory organ, with finely perforated filter apparatus at base of interior funnel-shaped portion at apex of respiratory organ (Eucorethra) or with small apical opening or slit (perhaps not opening externally?) (Chaoborus, Mochlonyx), without posteromedial apodeme at base of respiratory organ (but see discussion); legs (Fig. 20C), foreleg extending to and curved dorsally along apical margin of wing; apex of foreleg lateral to apex of midleg; apex of midportion of midleg well anterior to apex of wing; apical portion of hind leg not or barely visible in lateral view along posterior margin of wing, apex abutting subapices of fore- and midleg; metathorax (Fig. 17G) undivided medially, extending posterolaterally to anterolateral margin of tergite 2, just abutting hind leg under wing; sensilla: setae, plumose, arising from flat cuticle or from short tubercles; anteromedial—1 seta, anteromedial to base of respiratory organ; anterolaterals—2 setae; dorsals—3 setae; prealar—absent; supraalar—absent; metathoracics—2–3 setae. Abdomen: dorsoventrally compressed in cross-section; segments without open spiracles but segments 2–7 with lateral spiracular scar; tergite 1 (Fig. 17G) without posterolateral extension; segments without pleural membrane; membranous terminal paddles (Figs. 10E, 26D) each articulated basally on very reduced posterior margin of segment 9 (or segment 8?), with median rib (Chaoborus with additional thickened lateral margins); sensilla: setae, nearly all plumose, arising from flat cuticle; tergite 1 with 5, 6, or 8 setae; segment 4 (Fig. 24B) with D-1-IV, D-2-IV, D-3-IV, D-6-IV, D-8-IV, D-9-IV each a seta, D-7-IV a campaniform sensillum, L-1-IV, L-2-IV, L-3-IV, L-4-IV each a seta, V-1-IV a minute seta or a campaniform sensillum, V-5-IV, V-6-IV each a seta, V-3-IV absent (Eucorethra, Mochlonyx) or a seta (Chaoborus). Segment 9, other than genital lobe, reduced, D-4-IX, V-1-IX on midrib of terminal process. Discussion: The homologies of abdominal sensilla within the Culicoidea are interpreted differently here than by Belkin (1952, 1953, 1960). This is discussed further in the glossary section of this paper. In the three chaoborid genera examined, the labellum was restricted laterally, leaving a sclerite between the palpus and labium (Fig. 13C). This feature is unique and therefore possibly a synapomorphy of the family. However, the condition in Culicidae is uncertain because their highly modified mouthparts make them non- comparable. It is possible that the apomorphic condition is actually a synapomorphy of the two families, with further, unrecognizable modification in the Culicidae. In some Culicomorpha, there is an apodeme at the base of the respiratory organ. In Eucorethra underwoodi Underwood, there is a basal prong on the respiratory organ itself which appears to pivot against a flange. It is uncertain if this is homologous to the condition found in Culicidae, Simuliidae and Ceratopogonidae. Ogawa (2007) provides setal homologies between Eucorethra, Mochlonyx, Austromochlonyx Freeman and Chaoborus, and further details of their morphology. Other works describing pupae of Chaoboridae are listed by Borkent (1993). The identity of AL-1-T of Eucorethra (Fig. 16A) is based on assumed homologies with Culicidae, even though this sensillum is placed rather medially compared to that in Culicidae. Material examined: 2 pupae, 6 pupal exuviae of Eucorethra underwoodi, Takakka Falls, Yoho National Park, BC, Canada; 1 pupa of Eucorethra underwoodi, Woods Lake, 10 km S Westwold, BC, Canada; 5 pupae, 12 pupal exuviae of Mochlonyx velutinus (Ruthe), 5.6 km NW Enderby, BC, Canada; 1 pupa, 1 pupal exuviae of Mochlonyx cinctipes (Coquilett), Cheam Lake, BC, Canada; 14 pupal exuviae of Chaoborus trivittatus (Loew); 5 pupae of Chaoborus sp. (probably braziliensis (Theobald)) from Santa Elena ACG, Santa Rosa National Park, Costa Rica.

Culicidae (Figs. 1I, 10A–B, F, 13D, 16B, 17H, 20D, 21L, 24C, 26E)

Description: (sensilla listed in Table 2). Body (Fig. 1I) with cephalothorax much larger dorsoventrally than abdomen, in life apical portion of abdomen curved under cephalothorax; mouthparts and thoracic appendages loosely fused to each other and/or remainder of cephalothorax. Head (Figs. 13D, 20D): without tubercles; dorsal apotome variable, nearly as long as wide; anterolateral cephalic sclerite small, widely separated medially, articulated with membrane to margin of antenna; antenna elongate, apex extending posteriorly along anterior margin of wing to about half wing length; mouthparts extremely elongate, extending beyond apex of wing and curved anterodorsally; apex of labrum slender; mandible and maxilla elongate, extending beyond apex of wing and curved anterodorsally; palpus directed posteromedially (pharate adult palpus S-shaped with middle portion occupying swollen area near base of pupal lacinia, apices of palpi separated medially; labella short, narrow, apex ventral to abdominal sternite 1 or 2, bilobed, divided medially; exuviae (Figs. 10A–B) with head attached by

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 33 membrane to thorax (with anterolateral cephalic sclerites) and forecoxae / trochanters, head capsule folded longitudinally with each antennal base folded along posterolateral margin of dorsal apotome, basal portion of antenna and each ocular half folded toward lateral margin of clypeus-labrum, and margin of dorsal apotome and clypeus folded transversely with both parts bent ventrally; sensilla: setae arising from flat cuticle; dorsal apotomals absent; anterolateral cephalic sclerite with 1 seta; clypeals absent; oculars—3 setae; antennals—absent. Thorax (Fig. 16B): with or without short tubercles; prothorax without ventrolateral extension abutting lateral margin of palpus; respiratory organ (Fig. 21L) trumpet-shaped in most taxa, with a few autapomorphic modifications, surface without annulations, with spicules, tracheal tube extending into respiratory organ, well separated from or appressed against external surface of respiratory organ, with finely perforated filter apparatus at base of interior funnel- shaped portion at apex of respiratory organ (most taxa), with anteroventral apodeme at base of respiratory organ; legs (Fig. 20D), foreleg extending to and curved dorsally along apical margin of wing and then curving anteriorly; apex of foreleg anteroventral or ventromedial to apex of midleg; midleg extending to apex of wing and then curving anteriorly; apical portion of hind leg visible or hidden in lateral view along posterior margin of wing, apex abutting preapex of midleg; metathorax (Fig. 17H) undivided medially, extending posterolaterally to anterolateral margin of tergite 2, just abutting hind leg; sensilla: setae, most plumose or bifurcated, arising from flat cuticle or from short tubercles; anteromedial—1 seta, anteromedial to base of respiratory organ; anterolaterals—2 setae; dorsals—2 setae; prealar—absent; supraalar—absent; metathoracics—3 setae. Abdomen: dorsoventrally compressed in cross-section; segments without open spiracles but segments 2–7 with lateral spiracular scar; tergite 1 (Fig. 17H) without posterolateral extension; segments without pleural membrane; membranous terminal paddles (Figs. 10F, 26E) each articulated basally in membrane between abdominal segments 8 and 9, with median rib and thickened lateral margin; sensilla: setae, nearly all plumose, arising from flat cuticle or short tubercles; tergite 1 with 8 setae; segment 4 (Fig. 24C) with D-1-IV, D-2-IV, D-3-IV, D-6-IV, D-8-IV, D-9-IV each a seta, D-7-IV a campaniform sensillum, L-1-IV, L-2-IV, L-3-IV, L-4-IV each a seta, V-1-IV, V-5-IV, V-6-IV each a seta. Segment 9, other than genital lobe, reduced, D-4-IX, V-1-IX on midrib or membrane of terminal process. Discussion: The homologies of abdominal sensilla within the Culicoidea are interpreted differently here than by Belkin (1952, 1953, 1960). This is discussed further in the glossary section of this paper. The literature describing pupae of Culicidae is voluminous and some major works were surveyed to include some of the variation in this family (Belkin 1968; Belkin et al. 1970; Harbach & Knight 1980) as well as numbers of others too numerous to list. The palpus of the pharate adult is S-shaped with its middle portion occupying a swollen area at the base of the pupal lacinia (Fig. 13D). This feature is unique in the Diptera and likely another expression of the general modification of the mouthparts in all Culicidae (see characters 62–65). Harrison & Peyton (1984) described setae on the dorsal apotome of a specimen of Anopheles lesteri Baisas & Hu, the only Culicoidea with such sensilla, and this was likely an example of an individual atavism. Material examined: 16 pupae, 2 pupal exuviae of Anopheles gambiae Giles, colony at Simon Fraser University, originating from Ifakara, Tanzania; 20 pupae of Aedes fitchi (Felt & Young) from 31 km NE Argenta, BC, Canada.

PHYLOGENETIC ANALYSIS

In this analysis all synapomorphies were compiled which were considered to provide valid evidence of relationships between the families of Culicomorpha, incorporating those from all life stages. Autapomorphies for each family were also included not only to confirm the monophyly of each but also to include a number of newly discovered pupal features. The synapomorphies proposed below resulted in a single most parsimonius cladogram as shown in Figure 28A and are given as groundplan states for each family (Table 3). Bremer and bootstrap values are presented in Figure 28B. As a result of these new relationships the three families Ceratopogonidae + Thaumaleidae + Simuliidae are referred to as Simulioidea and Chironomidae is the sole family in the Chironomoidea. The pupae of Ptychopteridae were studied for outgroup comparisons for all pupal features and, for some character states, nearly all other families of nematocerous Diptera as listed in Materials and Methods and discussed further below were examined. Outgroup and ingroup character states are summarized as a groundplan (Table 3). Exceptions to groundplan character states in various families are given below in the discussion of character states.

34 · Zootaxa 3396 © 2012 Magnolia Press BORKENT The morphology section above indicates there are numerous similarities in the chaetotaxy within each of the Simulioidea and Culicoidea. Some of these have been included in the analysis, when I considered outgroup comparisons to be sufficiently broad, or at least a given state likely to be unique. Many others might be polarized by using the states in Ptychopteridae but I consider this too limited for outgroup comparisons. As well, there is the significant risk of 'splitting hairs'—of dividing a broader character into smaller and smaller expressions of that character. As such, future investigation of other nematocerous Diptera will likely show that a number of states within the Culicomorpha reported in the morphology section will be derived and, perhaps, further evidence of relationship. The following characters are grouped first according to egg (n= 1), larval (n= 21), pupal (n= 33) and adult (n= 37) stages and within these categories in descending order, head to thorax to abdomen, anterior to posterior, dorsal to ventral, male followed by female, and external to internal.

TABLE 3. Character state matrix used for phylogenetic analysis.

1111111111222222222233333333334444444 4 4 4 Taxa 1234567890123456789012345678901234567890123456 7 8 9 Groundplan 0 000000000000000000000000000000000000000000000 0 0 0 Chironomidae 0 000000000000100000000000000000000001000001000 0 0 0 Ceratopogonidae 0000000100100100000000001000100010000011011000 1 0 0 Thaumaleidae 1000000100000100000001001100100010000010011000 1 1 0 Simuliidae 1001001100010110000111011100100010010010111000 1 0 1 Dixidae 0000010111000000000000100001110001000000001000 0 0 0 Corethrellidae 0110110101001101111000100001101000100000001001 1 0 0 Chaoboridae 0 000110100001001112000100001100100000100001011 0 0 0 Culicidae 0000010101001001112000100011100100000100001111 0 0 0

TABLE 3. (continued)

55555555556666666666777777777788888888889 9 9 Taxa 01234567890123456789012345678901234567890 1 2 Groundplan 0 0 0 0 0 0 00000000000000000000000000000000000 0 0 Chironomidae 0 0 0 0 0 0 10000000000000000010000000000111000 0 0 Ceratopogonidae 1 0 0 0 1 0 10000000000000100000000000000121000 0 0 Thaumaleidae 00101000000000000000100101000000000121000 0 0 Simuliidae 01001001000000000001100001000001110121000 0 1 Dixidae 00000010000000000000011000010000000111000 0 0 Corethrellidae 00000010101000000010100000111110001101111 0 0 Chaoboridae 0 0 0 1 0 1 10000000000010000000110000000101111 1 0 Culicidae 00010010010111111110000000110000000111111 1 0

Eggs

1. Egg oval to elongate (plesiomorphic); egg with swollen dorsal transverse bulge (apomorphic). Borkent & Wood (1986) and Adler et al. (2004: 132; their character 23) stated that the derived condition was unique to Simuliidae. Actually, this is not the case. The eggs of Thaumalea testacea Ruthe and Androprosopa americana are also swollen dorsally (Mandaron 1963, Sinclair 1996). Outgroup comparisons are not entirely clear. All Culicoidea have elongate or oval eggs and in many Corethrellidae and Culicidae these having various pontoons and other modifications. The eggs of Ceratopogonidae are nearly all either elongate (plesiomorphic within this family) or oval. The single instance of a ceratopogonid egg looking similar to those of Simuliidae and Thaumaleidae is Leptoconops arnaudi Clastrier & Wirth described by Smith & Lowe (1948; as L. kerteszi Kieffer) but it may have been misdrawn as their written description differs and is similar to that of other Ceratopogonidae as "banana-shaped with one end slightly narrower than the other.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 35 They vary from straight to slightly curved and are circular in cross-section." The situation in Chironomidae is strikingly variable (Nolte 1993) with those of Telmatogetoninae (an unusual marine subfamily likely belonging well within the family; Cranston et al. 2012) being similar to the eggs of Thaumaleidae + Simuliidae. Otherwise eggs of Chironomidae vary from oval and oblong to elongate. The similarity of a few Chironomidae to those of Thaumaleidae + Simuliidae is considered to be due to homoplasy. The eggs of Ptychopteridae have not been described.

Larvae

2. Larva with anterolateral margin of head capsule complete (plesiomorphic); anterolateral margin of head capsule with groove in which the antenna lies when abducted (apomorphic). This feature was discussed by Borkent (2008: 210; as character 11). The antennae were referred to previously as adducted but the antennae are clearly at rest when in the groove.

3. Larva with posterolateral portion of the head capsule with, at most, only simple setae (plesiomorphic); posterolateral portion of the head capsule with row of strong spines (Fig. 81G) (apomorphic). This feature was discussed by Borkent (2008: 210; as character 12).

4. Larva without bacteria-covered multiporous sensillum near base of antenna (plesiomorphic); with bacteria-covered multiporous sensillum (apomorphic). Adler et al. (2004: 131; their character 18) noted that this feature was unique to Simuliidae. Although appearing to be unique, this feature has not been studied in a number of other families. It would be useful to know its homology to sensilla in other families.

5. Larval antenna not prehensile (plesiomorphic); antenna prehensile (apomorphic). Chaoboridae and Corethrellidae are unique in the Diptera in having prehensile antennae, used to capture and push food into the oral cavity. Wood & Borkent (1989; their character 65) suggested that the condition in the two families was not homologous because it was believed that the antennae of Corethrella were not used in feeding. Subsequent observations (Borkent 2008: 206) have shown that they are likely used in prey capture, but the mechanism is substantially different in Corethrellidae and Chaoboridae. In Chaoboridae the antennae are situated plesiotypically on the anterolateral margins of the head capsule, or in the highly modified Chaoborus, with their bases abutting medially. In all, the labral adductors simply move the apices of the antennae toward the oral cavity, helping to scoop up and push prey into the mouth. In Corethrellidae, the bases of the antennae nearly abut medially but, when abducted, their lengths rest on the anterolateral margin of the head capsule. When adducted, the antennal apices first move anteromedially and once closely approximated, swing downward to help push prey into the oral cavity. In short, they flip forward and then downward. This partially rotating movement is poorly understood morphologically and warrants further investigation. Regardless, the two mechanisms are quite different in Chaoboridae and Corethrellidae, lending support to the hypothesis that they are independently derived conditions in the two families. Oosterbroek & Courtney (1995; their character 16) suggested the prehensile antennae is a questionable synapomorphy of Corethrellidae + Chaoboridae + Culicidae, presumably with a secondary loss in Culicidae.

6. Larval antenna slender, tapering apically, usually rather short and multisegmented (plesiomorphic); antenna elongate, stout, and one segmented (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 64) and Oosterbroek & Courtney (1995; their character 14).

7. Larva with, at most, a relatively simple labral brush (plesiomorphic); with a complex labral brush (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 56) and Adler et al. (2004: 131; their character 16). Larvae of Simuliidae have a reduced number of rows of labral rays, the intertorma has an anteroventrally directed stem, and the ventral fascicles of the posterior frontolabral muscles are interdigitated (Craig 1974). All these are unique to Simuliidae.

36 · Zootaxa 3396 © 2012 Magnolia Press BORKENT 8. Larval premandible mainly an external sclerite, with a well developed external comb of setae and a small, invaginated apodeme for insertion of the labral retractor muscles (plesiomorphic); premandible mainly or entirely internal as a result of invagination, serving almost entirely as a point of insertion of the labral retractor muscle (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 58). Because of their phylogenetic conclusion that Chironomidae and Ceratopogonidae were sister groups, they viewed the plesiomorphic condition in Chironomidae as a secondary reversal. Here, the external premandible of some Chironomidae (many do not have an external premandible), is viewed as truly plesiomorphic, although it's use in a tong-like manner may be apomorphic (discussed further below). The presence of an external premandible in some Ceratopogonidae (but without a comb of setae) is here viewed as an independent reversal. The derived condition is susceptible to homoplasy in both the out and in groups. Further modifications of the premandible are discussed below under "Conflicting Synapomorphies".

9. Larval maxillary palpus reduced or barrel-shaped (plesiomorphic); maxillary palpus markedly elongate (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 60). The apomorphic condition is unique to Dixidae within the Diptera.

10. Larval labiohypopharynx not connected to paraclypeal phragma (plesiomorphic); anterolateral corner of labiohypopharynx connected via cibarial bar to paraclypeal phragma (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 61).

11. Larval pharyngeal apparatus absent to somewhat developed (plesiomorphic); pharyngeal apparatus markedly developed with two strongly diverging arms and rows of combs (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 63).

12. Larval salivary glands small or of moderate size, not folded (plesiomorphic); salivary glands large, folded (apomorphic). Adler et al. (2004: 131; their character 17) noted that this feature was unique to Simuliidae. Further outgroup comparisons would be useful as not all families are known in this regard (e.g. Corethrellidae, Dixidae, and a number of other families of nematocerous Diptera).

13. Larval thoracic segments distinct, not appreciably wider than first abdominal segment (plesiomorphic); all three abdominal segments fused to each other and enlarged (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 68).

14. Larva without prothoracic leg (plesiomorphic); prothoracic proleg present (apomorphic). Wood & Borkent (1989; their character 66) and Oosterbroek & Courtney (1995; their character 38) presented this as a synapomorphy of our earlier concept of the Chironomoidea as it was present in Chironomidae, early lineages of Ceratopogonidae, Thaumaleidae and Simuliidae. The discovery of the proleg in the first instar of Corethrellidae by Borkent & McKeever (1990) showed that the derived condition was more broadly distributed. As suggested by the latter, the derived condition is considered herein a synapomorphy of the Culicomorpha. The discovery that Chironomidae are the sister group to all remaining Culicomorpha further supports the interpretation of this as a synapomorphy of the infraorder. As such, the proleg has been lost in derived members of Ceratopogonidae, in Dixidae, second through fourth instars of Corethrellidae, and in all Chaoboridae and Culicidae. I have examined first instar larvae of unidentified Dixidae, Eucorethrea underwoodi and several species of Chaoborus and they do not have a proleg. In each of the taxa with a thoracic proleg, it bears at least one row of distinctive apical hooklets, further indicating the homology of the structure in those taxa that bear them. The presence or absence of a prothoracic proleg is related to the mode of larval movement. Culicomorpha with a proleg use it to propel themselves against substrate. The loss of the proleg in nearly all Culicoidea is likely related to their use of swimming to move about. It is unknown how first instar Corethrella larvae use their prolegs.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 37 The proleg has also been lost in derived members of Ceratopogonidae (all Ceratopogoninae other than first instar larvae of species of Culicoides) and larvae of these glide through wet substrate with a snake-like motion but also are the only Ceratopogonidae that use a rapid, lashing motion (differing from that used by Culicoidea) to swim from one local to another. In short, Culicomorpha with swimming larvae have lost the prothoracic proleg. Lukashevich et al. (2010) identified a Middle Triassic Culicomorpha larva (Anisinodus crinitus Lukashevich, Przhiboro, Marchal-Papier & Grauvogel-Stamm), unplaced to family, with prothoracic claws indicating this synapomorphy evolved more than 240 mya.

15. Larva with hooks of prothoracic and posterior prolegs in more or less circular arrangement or somewhat scattered (plesiomorphic); hooks arranged in longitudinal rows (apomorphic). Adler et al. (2004: 132; their character 22) noted that this feature was unique to Simuliidae.

16. Larval abdominal spiracles absent or flush with surface (plesiomorphic); abdominal spiracles elevated on a conical siphon (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 69) and Oosterbroek & Courtney (1995; their character 51).

17. Larval posterior abdominal spiracles, if present, surrounded by two pairs of flaps, namely one small pair flanking the spiracles and arising on abdominal segment 8 and a second, much larger pair posterior to them, arising on a separate and distinct segment 9 (plesiomorphic); pairs of flaps reduced, elevated with spiracle to apex of siphon (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 70) and Oosterbroek & Courtney (1995; their character 52).

18. Lobes surrounding abdominal spiracles (if present) immovable (plesiomorphic); lobes movable, folding together when the larva submerges (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 72).

19. Larval abdominal segment 10 without fan-like group of long setae ventrally (plesiomorphic); with some elongate ventral setae, with strengthened base (apomorphic'); with fan-like row of long setae on midventral surface, each seta having a transverse, T-shaped base (apomorphic"). This feature was discussed by Wood & Borkent (1989; their character 74) as a synapomorphy of Corethrellidae + Chaoboridae + Culicidae and further interpreted by Oosterbroek & Courtney (1995; their character 41). Here the feature is defined more specifically. Corethrellidae have a clump of four ventral setae which are arranged as two side by side pairs, all of which arise from one common strengthened base with three arms—two extending laterally and one extending anteriorly. In Chaoboridae and Culicidae, an extensive longitudinal fan is present, with each seta having a T-shaped base. The setae, although initially appearing to be a single row, are actually slightly staggered with setae alternately placed slightly lateral to the median line.

20. Larva with ventrally or posteriorly directed anus and without anal sclerite ventral to anus (plesiomorphic); with anus directed dorsally and with anal sclerite between anus and posterior proleg (apomorphic). Adler et al. (2004: 131; their characters 19, 21) suggest that the dorsal anus and the associated anal sclerite of the larvae of Simuliidae were synapomorphies for that family. Although they state that the most common condition in Diptera is the presence of a ventral anus, they indicated that Thaumaleidae have the anus in a similar dorsal position and that perhaps the dorsal anus is plesiomorphic within the Culicomorpha, throwing doubt on this as a synapomorphy of the Simuliidae. My observations suggest a different interpretation. Although some Diptera larvae have a ventral anus, most Culicomorpha have a terminal anus, surrounded by permanently external or retractible anal papillae (Oosterbroek & Courtney 1995, their character 47). When a posterior proleg is present, as some Ceratopogonidae, most Chironomidae and all Thaumaleidae, the anal papillae are in a relatively more dorsal position but not nearly as dorsal as in Simuliidae. Therefore, the completely dorsal anus is considered as unique to

38 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Simuliidae and a synapomorphy of the family. The anal sclerite between the anus and posterior proleg of Simuliidae is also unique within the Diptera. I consider it closely associated to the development of the dorsal anus (perhaps displacing the anus dorsally) and treat them together as a single synapomorphy.

21. Larva with either two or four anal papillae (plesiomorphic); with three anal papillae (apomorphic). Adler et al. (2004: 131; their character 20) noted that this feature was unique to Simuliidae. Other nematocerous Diptera either lack anal papillae (terrestrial groups) or have an even number (2, 4, 6, or 8) (Courtney 2000: 113).

22. Larval stage with four instars (plesiomorphic); with at least six instars (apomorphic). Adler et al. (2004: 131; their character 15) noted that most species of Simuliidae have 6–7 instars. The number of instars can vary intraspecifically, depending on characteristics of the larval habitat. They considered having a larger and unfixed number of instars to be a synapomorphy of Simuliidae. This feature, however, is also present in Thaumaleidae where 15–20 instars have been reported by Mandaron (1963). Bradley Sinclair (pers. comm.) measured 232 larval head capsules of Androprosopa americana collected at a seepage at Inglis Falls, near Owen Sound, Ontario (Canada). He found about nine groupings, strongly indicating more than four instars. Nearly all other nematocerous Diptera have four larval instars. Only Cecidomyiidae have three instars. Nannochoristidae have four instars and most Siphonaptera have three (rarely four).

Pupae

23. Pupal dorsal apotome with at least one seta (plesiomorphic); dorsal apotome without sensilla (apomorphic). The Culicoidea lack setae on the dorsal apotome (see also Reinert, 1999) and are the only nematocerous Diptera examined lacking them. They are present in Nannochorista philpotti. Harrison & Peyton (1984) described setae on the dorsal apotome of a specimen of Anopheles lesteri Baisas & Hu, like an aberrant specimen exhibiting an atavism.

24. Pupal exuviae with anterolateral cephalic sclerite articulated laterally and with 0–2 setae (Figs. 6C–E) (plesiomorphic); anterolateral cephalic sclerite fused laterally to remainder of head capsule and with 3 setae (Fig. 6F) (apomorphic). This sclerite is situated on the dorsum of the pharate adult head and abuts the lateral or dorsal margin of the dorsal apotome. In most Culicomorpha, upon eclosion of the adult, the dorsal apotome separates posterodorsally and laterally and, in some, hinges ventrally with the face of the pupa (Figs. 7C, D, 9A–F). The anterolateral cephalic sclerite hinges laterally in most Culicomorpha to allow more space laterally for the emerging adult head (Fig. 6A). As such, the head is released by the opening of three sclerites (the dorsal apotome and the two anterolateral cephalic sclerites) (see also "Adult Emergence" above). The fusion of the anterolateral cephalic sclerite laterally to the rest of the head capsule in Simuliidae is unique within the Culicomorpha. The sclerite is small, proportional to the broader dorsal apotome, and is fused against the medial margin of the antenna in female pupae (as a triangular sclerite) and to both the antenna and more lateral eye as a longitudinal band in male pupae (Fig. 6F). The broader dorsal apotome that splits posterodorsally and laterally and the fact that the entire head capsule plus most of the forecoxae / trochanters separates as a unit upon emergence probably is related to rapid emergence by simuliid adults underwater. In essence, an emerging adult simuliid pushes the pupal head capsule, with the help of the splitting dorsal apotome, off their head as a unit. In a derived lineage of Ceratopogonidae, the anterolateral cephalic sclerite is fused with the anterior margin of the thorax (Borkent, in prep.). The number of setae on the anterolateral cephalic sclerite may also be informative but were somewhat more difficult to interpret. Although Simuliidae were the only Culicomorpha examined with three setae, Sæther (1980) showed three setae in this area for Chironomidae (I was unable to confirm this). In Archaeochlus, there is one seta

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 39 and one campaniform sensillum. In some Ceratopogonidae, there are two setae and a campaniform sensillum but the earliest lineage (Leptoconopinae) has either two setae (Austroconops) or one seta and a campaniform sensillum (Leptoconops). Ptychopteridae have only one seta (perhaps an additional campaniform sensillum present).

25. Pupal exuviae with the face folded longitudinally (so that the bases of the antennae and the eyes are folded medially) and with at least the base of the mouthparts somewhat recessed dorsally (Figs. 6C, 7B,C–D, 91–F, 10A–B) (plesiomorphic); with antennae, face and mouthparts not distorted, with these having the same appearance as the whole pupa, other than ecdysial lines which have separated (Figs. 6B, D–F, 7E–F, 8A–E) (apomorphic). Within the Culicomorpha, the pupal exuviae of Simulioidea retain the shape of the various sclerites on the ventrum of the head (so the separate pieces can be easily understood as a somewhat separated jigsaw puzzle) (Figs. 6A, B, D–F). In Culicoidea, and nearly all Chironomidae, the face folds longitudinally, with each ocular half folded ventrally along the margin of the dorsal apotome and the junction between the dorsal apotome and the clypeus bent transversely, as discussed above in the section on "Adult Emergence" (Fig. 6C, 7C, D, 9A–F). As a result, it is often quite difficult to envision from the exuviae either what the whole pupa looked like or what the various parts of the head of the exuviae represent. The exuviae of Stictocladius (otherwise with an unusual leg arrangement) also retains the original appearance of the head but this is considered as homoplasy. In some groups with the plesiomorphic condition (e.g. Ptychopteridae and some Chironomidae), the folded face of the exuviae can be "popped" back into its original full pupal form with forceps and can likewise be "popped" back into the folded exuvial state (Figs. 7A, B). The plesiomorphic state is present in Ptychopteridae but the exuviae of other nematocerous Diptera have either unfolded or only a partially folded face. Nannochoristidae have a partially folded face, with the bases of the antennae closely approximated and a bend in the face/clypeal area. Therefore, as hypothesized here, the plesiomorphic condition in nematocerous Diptera is likely a partially folded face, with the evolution of a further folding of the face in Ptychopteridae + Culicomorpha and then the evolution to an entirely unfolded condition in the ancestor of Simulioidea.

26. Pupal antenna elongate, extending posteriorly beyond the wing (in some taxa with the apex of the antenna under the wing, others ventral to the wing) (Figs. 7A–F, 9A–F, 20C–D) (plesiomorphic); antenna short, at most extending just beyond the posterior margin of the head (Figs. 8A–E, 12A–B) (apomorphic). Simuliidae and Thaumaleidae are unique in the Culicomorpha in having such short antennae. The feature is also present in adults of these two families and likely related. However, some adult Chironomidae, such as Oreadomyia Kevan & Cutten-Ali-Khan, some Orthocladiinae and the females of a number of taxa also have quite short antennae and the pupae of at least some of these (others are unknown) have the apex of the antenna extended posteriorly past the posterior margin of the head. The antennae of the pupae of Ptychopteridae are elongate, as are those of most nematocerous Diptera. In some taxa, such as Scatopsidae and some Bibionidae (Brauns 1954), the antennae are also restricted to the head capsule but these are clearly instances of homoplasy.

27. Pupa with mouthparts short, not extending posteriorly beyond forecoxae / trochanters (Figs. 11A–B, 12A–D, 13A–C) (plesiomorphic); with mouthparts extremely elongate, extending posteriorly to apex of wings and then curving dorsally (following the rounded margin of the wing) (Figs. 13D, 20D) (apomorphic). Culicidae are unique within nematocerous Diptera in having elongate mouthparts in which their apices curve dorsally along the rounded apex of the wing. Obviously, this feature reflects the elongate mouthparts of the adults. Other nematocerous Diptera with elongate mouthparts (e.g. a few Tipulidae) have the pupal mouthparts directed posteriorly along the ventrum of body, indicating their independent evolution (and how the arrangement of pupal and adult features may independent).. Lygistorrhinidae, which have elongate adult mouthparts, are unknown as pupae.

28. Pupal head with clypeal/labral sensilla (Figs. 11A–B, 12A–D) (plesiomorphic); head without clypeal/ labral sensilla (Figs. 13A–D) (apomorphic). All Culicoidea lack clypeal sensilla. Ptychopteridae, Thaumaleidae, Simuliidae, most Ceratopogonidae (present in at least all early lineages), and some Chironomidae (e.g. Archaeochlus) have them. This evidence

40 · Zootaxa 3396 © 2012 Magnolia Press BORKENT indicates that the derived condition has occurred independently in a few Ceratopogonidae, at least some Chironomidae and all Culicoidea. Further outgroup comparisons were not made.

29. Pupal palpus directed laterally (Figs. 11B, 18C–D) (plesiomorphic); palpus directed posteriorly or medially (Figs. 12A–D, 13A–D) (apomorphic). The derived condition is nearly unique within the Diptera and it is considered of major importance in determining the monophyly of Simulioidea + Culicoidea. In large measure the posteriorly or medially directed palpus results in a major restructuring of the elements of the ventrum of the head. The plesiomorphic condition is present in Chironomidae and nearly all non-Culicomorpha, with the following possible exceptions. The Nymphomyiidae (Courtney 1994a) and Deuterophlebiidae (Kennedy 1981) have reduced mouthparts as pupae and adults and therefore cannot be appraised for this character. Perissommatidae (Colless 1962) have unusual pupae that remain in the last larval instar and are therefore poorly sclerotized. Their palpi are directed posteriorly. Although this is an instance of homoplasy, the condition in Perissommatidae looks quite different from that within Culicomorpha because the appendages, including the palpus, are only loosely appressed against the body. It is possible that Scatopsidae and Canthyloscelidae also have posteriorly directed palpi but no original material was examined and the palpi in the figures in Cook (1981) and Peterson & Cook (1981), respectively, are not entirely clear. Brauns (1954) illustrates a Scatopsidae with laterally directed palpi. Blephariceridae have their palpi initially directed laterally but their apices are bent anteriorly (Hogue 1981). Nannochoristidae and Siphonaptera have posteriorly directed palpi but at least some Lepidoptera have them directed laterally (Brauns 1954). Finally, although not a cladistic argument, it is interesting to note that Middle Triassic pupae, likely of the extinct family Grauvogeliidae, also have their palpi directed laterally (Lukashevich et al. 2010).

30. Pupal palpi separate medially and with each apex directed laterally, posteriorly or, at most, posteromedially (Figs. 11A–B, 12A–D, 13B–D) (plesiomorphic); palpi nearly abutting, abutting, or overlapping medially, with apices directed anteromedially (Fig. 13A) (apomorphic). The derived condition is unique to the Dixidae.

31. Pupa with ocular sensilla (Figs. 11A–B, 12A–D, 13A, C–D) (plesiomorphic); without ocular sensilla (Fig. 13B) (apomorphic). Corethrellidae are the only Culicomorpha without at least one ocular sensillum. In some Ceratopogonidae there is only a single campaniform sensillum but in most other taxa there is at least a seta and in most of those there are 2–3 setae (there may be additional campaniform sensilla). Ptychopteridae have four setae or three setae and a campaniform sensillum.

32. Pigment of adult eye first appearing in the pupa (plesiomorphic); pigment of adult eye developing precociously, becoming conspicuous as early as the second instar larva, and always well developed by the last instar (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 75). The precociously developing adult eye is located anterior to the pigment-containing cells characterizing the larval eye or stemma (apomorphic) in Chaoboridae and Culicidae.

33. Pupa with anterolateral margin of thorax restricted to dorsal portion of thorax (Figs. 11A–B) (plesiomorphic); anterolateral margin of thorax extending ventrolaterally along posterior margin of eye to lateral base of palpus as prothoracic extension (Figs. 2B–C, 7E–F, 8A–E) (apomorphic). Simulioidea are the only Diptera with the derived condition. This extension of the thorax in Simuliidae and Thaumaleidae and what is a separate sclerite in most Ceratopogonidae (because of the overlying antenna in this group), is here called the prothoracic extension. Dorsally, it covers the anterior margin of the mesonotum of the pharate adult, perhaps in part because the medial portion of the prothorax of the pharate adult develops further below the pupal cuticle. Laterally, the extension covers the postpronotum and antepronotum of the pharate adult.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 41 The situation is more complex in Ceratopogonidae than in Simuliidae and Thaumaleidae. In Leptoconopinae, the earliest lineage in the family, the extension sits as a separate rectangular sclerite, abutting the base of the palpus. In Forcipomyiinae + Dasyheleinae, the sclerite or extension is absent. In early lineages of the subfamily Ceratopogoninae (e.g. Culicoides), the extension is weaker in the area where the antenna lies over it, but the connection to the thorax is present. In remaining Ceratopogoninae, the sclerite is separate from the thorax and lies entirely between the medial margin of the antenna and the base of the palpus. There are a few further modifications in some lineages but these will be described elsewhere (Borkent, in prep.). When studying Ceratopogonidae, it was initially thought that this sclerite was a de novo feature that developed more strongly within the Ceratopogonidae (less developed in earlier lineages). However, examination of Simuliidae and Thaumaleidae made it clear that the feature was homologous in the three families. As such, the reduced presence of the extension in the earliest lineages of Ceratopogonidae as independent losses (reduced in Leptoconopinae; lost in Forcipomyiinae + Dasyheleinae, weak in Culicoidini). The entirely strong extension of the thorax in Simuliidae and Thaumaleidae warrants further comment. In early lineages of Ceratopogoninae, the portion of the extension covered by the antenna is weak and, as such, its origin from the thorax is somewhat obscure. Because the pupae of Simuliidae and Thaumaleidae have short antennae (see character 56), the extension is entirely exposed. Hence the fully developed extension (with no weak area) is likely related to the fact that the antenna does not reach and cover this portion of the thoracic extension in these two families.

34. Pupal thorax more or less flat or somewhat rounded anterior to respiratory organ (plesiomorphic); thorax with area anterior to the respiratory organ with longitudinal, protruding flange extending to posterior portion of head (Fig. 15C) (apomorphic). This feature, unique to the pupae of Dixidae, is best seen in whole pupae. The anterolateral seta is on the flange, indicating its origin from the thorax. The flange is very short in Dixa and barely overlaps the antenna laterally. It is well developed in Dixella and likely is a further derived condition in this genus. It is uncertain whether the feature is present in other genera and therefore the apomorphic condition needs further study within the family.

35. Metathorax of pupa either entire medially or narrowly separated (Figs. 17A–E, G–H) (plesiomorphic); metathorax divided into three parts (Fig. 17F) (apomorphic). In nearly all nematocerous Diptera, Ptychopteridae and nearly all Culicomorpha the metathorax forms a continuous transverse band posterior to the mesothorax. In Chironomidae and some Ceratopogonidae the metathorax is divided medially by the posterior, tapering medial extension of the mesothorax. In Corethrellidae, however, the posterior extension of the mesothorax is broad and its posterolateral margin indents abdominal tergite 1; as such it separates two lateral portions of the metathorax, which are each separate from a narrow medial band of metathorax lying posterior to the mesothoracic extension. The broad mesothoracic extension and the division of the metathorax into three parts are unique to Corethrellidae.

36. Pupal metathorax extending posterolaterally beside lateral margin of tergite 1 (Figs. 2A–B, 7F, 8D, 14A, 17C, E, F–H) (plesiomorphic); metathorax not extending posterolaterally beside lateral margin of tergite 1 (Figs. 8B, 17D, 27F) (apomorphic). The pupae of Simuliidae have a narrow metathorax with a distinct lateral margin anterior to that of abdominal tergite 1. In nearly all nematocerous Diptera and nearly all other Culicomorpha, the lateral margin of the metathorax extends posteriorly along the lateral margin of abdominal tergite 1 to near or posterior of the anterior margin of tergite 2 (and in Culicomorpha apically coming close to or abutting the posterior portion of the hind leg along the posterior margin of the wing). This extension of the metathorax contains at least part of the developing halter. In some, the halter develops only partially under the wing, in others it develops entirely dorsal to the wing margin. Instead, in Simuliidae the halter develops entirely under the wing, lateral to the exposed metathorax, and it is the lateral portion of abdominal tergite 1 which extends posterolaterally (coming close to the posterior portion of the hind leg along the posterior margin of the wing). This posterolateral extension of tergite 1 contains the developing elongate setae of adult tergite 1, previously identified as a synapomorphy of this family (Wood & Borkent 1982) and discussed elsewhere as character 83. The lack of a posterolateral extension of the metathorax

42 · Zootaxa 3396 © 2012 Magnolia Press BORKENT and the presence of such an extension of abdominal tergite 1 in Simuliidae may be developmentally related and thus not independent indicators of relationship. A similar, but differently expressed, derived condition occurs in Chironomidae (see character 37). Pupae of Deuterophlebiidae and Blephariceridae also lack a posterolateral extension of the metathorax but this is likely homoplasy resulting from their extremely modified pupae (and probably a synapomorphy of these two families).

37. Pupal metathorax extending posterolaterally beside lateral margin of tergite 1 (plesiomorphic); metathorax extending laterally or slightly posterolaterally, with apex under wing (apomorphic). Medially, the metathorax of Chironomidae is a narrow transverse band tucked under, in dorsal view, the large protruding posterior margin of the mesothorax (with the pharate adult mediotergite). The lateral portion of the metathorax is directed laterally or slightly posterolaterally (Oliver 1981, figs. 138–140). It contains the developing pharate adult halter and the base of this lobe-like extension is visible externally (best seen in dorsolateral view); the apex is tucked under the wing. This condition is unique in the Diptera. Simuliidae have a somewhat similar condition but in that family, the halter is entirely under the wing and there is no external lateral extension of the metathorax. I consider the condition in the two families only superficially similar, although a case might be made that the condition in Chironomidae is a precursor of that in Simuliidae. However, in at least some taxa (e.g. Podonomopsis Brundin) the small edge of the posterolateral extension of the metathorax is visible along the margin of the wing, indicating a somewhat intermediate condition between the fully posterolateral extension in most other Diptera (i.e. evidence that is independently acquired from that of Simuliidae) and that generally found in Chironomidae (i.e. fully under the wing). The condition of this area in Chironomidae differs in other ways. Not only does the apex of the halter develop under the wing but the anterolateral margin of tergite 1 is also loosely under the wing margin. As such, I consider the condition in Chironomidae to be synapomorphic. As discussed under character 36, pupae of Deuterophlebiidae and Blephariceridae also lack a posterolateral extension of the metathorax.

38. Pupal thorax and abdominal segments lacking plumose setae, or with, at most, some setae with a few bifurcations (Figs. 14A–D, 15A–D, 17A–F, 22A–C, 23A–C, 24A) (plesiomorphic); thorax and abdominal segments 1–8 with all, or nearly all, setae strongly plumose or bifid (Figs. 16A–B, 17G–H, 24B–C) (apomorphic). Chaoboridae and Culicidae are unique in the Culicomorpha in having all, or nearly all, setae strongly plumose or bifid. Some Corethrellidae (e.g. C. appendiculata, Fig. 17F) have a bifid or plumose seta on the metathorax, which may be a precursor to the fully developed condition in Chaoboridae and Culicidae. Ptychopteridae have simple setae or short bifurcated setae. There are a few Simuliidae (e.g. Simulium tescorum Stone and Boreham) with bifurcated thoracic setae and some Chironomidae (e.g. Pagastia Oliver) with some plumose or bifurcated setae on their abdomens but these are almost certainly secondarily derived and regardless, do not approach the condition in Chaoboridae and Culicidae. The derived state is also present in the larva of Chaoboridae and Culicidae and likely developmentally related (i.e. although they are different semaphoronts, it seems best to consider this as one condition).

39. Pupa with dorsal area of thorax with 2–4 sensilla (Figs. 14B, 15C–D, 16A–B) (plesiomorphic); with group of four setae and one campaniform sensillum (Figs. 3A, 14D, 15A–B) (apomorphic). Chironomidae have 2–5 dorsal setae and lack a campaniform sensillum in this area. However, early lineages within the family appear to have only two setae present (including only two for Archaeochlus). Culicoidea have 2–3 dorsal setae. In the Simulioidea, there is a group of five sensilla comprised of four setae and one campaniform sensillum. Simuliidae have an extra seta and campaniform sensilla (possibly homologous to those in Ptychopteridae). The dorsal sensilla D-1-T, D-2-T, D-4-T and D-5-T are setae in Simulioidea and D-3-T is a campaniform sensillum. Their arrangement is strikingly similar in Thaumaleidae and Simuliidae, whereas D-5-T is farther posteromedially in most Ceratopogonidae (Fig. 3A) (it is secondarily lost in some, Fig. 22B). I consider this arrangement of the five sensilla as well as one of them being a campaniform sensillum as derived for the Simulioidea. Further outgroup comparisons are limited at present. Ptychopteridae have a group of seven sensilla made up of five setae and two campaniform sensilla and this may contradict the conclusion here. However, in Ptychopteridae the sensilla are arranged linearly and are not like the pattern present in Simulioidea.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 43 The presence of 3–4 sensilla in some Ceratopogonidae (Fig. 14C) is restricted to the Forcipomyiinae and Dasyheleinae and a few other lineages (Fig. 14C), which are considered to be secondary losses (Borkent, in prep.).

40. Pupal respiratory organ with plastron or filter apparatus, without pores (Figs. 21C–D, F–L) (plesiomorphic); respiratory organ with pores (Figs. 3A–C, 21E) (apomorphic). Ceratopogonidae are probably the only Culicomorpha with open pores on the respiratory organ with each leading separately to the trachael tube. As such this is likely a synapomorphy of the family. However, there are problems of interpretation. The condition in the closely related Thaumaleidae is uncertain and using light microscopes I could not confidently interpret the state of the tight apical opening in this family. As such they too may have open pores—a SEM study is needed. If present, perhaps the open pores are a synapomorphy of the Simulioidea, with subsequent loss in Simuliidae. At least some other nematocerous Diptera (e.g. Sciaridae) and Ptychopteridae have pores along the length and apex of their respiratory organ (Fig. 21B), so this limited outgroup comparison suggests the condition may be plesiomorphic within the Culicomorpha. However, I am more inclined to consider the similar condition in most early lineages of Chironomidae and the Culicoidea (with their trumpet- like respiratory organs) (Figs. 21C, H–I, K–L) to be the plesiomorphic state in Culicomorpha but further study of details would be beneficial. Pupae in the Corethrella peruviana species group have a highly modified, disc-like respiratory organ (Fig. 21J) with what may be pores (Borkent 2008) (more study is needed) but this is clearly independently derived.

41. Pupal respiratory organ a single undivided structure (Figs. 21A–C, E–F, H–L) (plesiomorphic); respiratory organ divided into at least three branches (Fig. 21G) (apomorphic). This apomorphic state is nearly unique within the Culicomorpha and was discussed by Adler et al. (2004: 131; their character 11). Some Chironomidae have similarly divided respiratory organs (Fig. 21D) but the lineages characterized by these are all derived lineages well within the family's phylogeny (Ashe & O'Connor 2009), indicating that the divided respiratory organs are independently derived in the two families. The pupal respiratory organ of Deuterophlebiidae is divided into 3–4 branches, that of Blephariceriae into 4 plate-like lamellae, and that of the tipulid genus Antocha Osten Sacken into a few branches) and these (especially those of Deuterophlebiidae) superficially resemble those of Simuliidae. These are clearly instances of homoplasy. The pupal respiratory organ of all Simuliidae has a unique basal fenestra, an area of thin cuticle that bursts when the pupa first emerges, allowing water to enter the respiratory organ. The condition is undoubtedly derived and a synapomorphy (Adler et al. 2004: 130; their character 10), but perhaps not independent of the enclosed, divided condition described above.

42. Pupal exuviae with wings and legs loosely appressed to body and with the ventrum of the cephalothorax rounded (Figs. 1F, H–I, 9D, 10B) (plesiomorphic); with wings and legs firmly appressed to body and with the ventrum of the cephalothorax at least somewhat flattened (Figs. 7E–F, 8A–D) (apomorphic). Within the Culicomorpha, the pupal exuviae of Simulioidea are unique in having the legs, wings and legs all firmly appressed against the body, looking as if they are fused together ventrally and with the ventrum of the cephalothorax at least somewhat flattened. The Corethrellidae also have their wings and legs abutting and at least partially fused, but the ventrum of the cephalothorax is rounded. The partial fusion in Corethrellidae is regards as an instance of homoplasy. A related feature is reflected in the position of the apical portion of the foreleg. In Simulioidea, the foreleg is directed posteriorly and ends at the apex of the wing (Figs. 19A–C). In Culicoidea and nearly all Chironomidae the forelegs are more elongate and wrap around the edge of the wing apex and are directed posterodorsally (Corethrellidae), dorsally or anterodorsally (Figs. 1F, H–I, 9D, 10B). Brundin (1966: 427–432) discusses the variation in the legs of Chironomidae. A few rare genera have forelegs extending posteriorly, with nearly all of these extending beyond the apex of the wing (Figs. 18B–D). These are here interpreted as reversals to a condition outside of the Culicomorpha and are not similar to the condition in Ceratopogonidae, Simuliidae and Thaumaleidae. Outgroup comparisons are variable. In Ptychopteridae the appendages are fused to the body and all the legs are directed posteriorly (ending well posterior of the wing apex). As such, they exhibit the derived condition.

44 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Otherwise, only some Psychodidae share the derived condition among nematocerous Diptera. Other nematocerous Diptera, Nannochoristidae and Siphonaptera have loose or loosely appressed appendages. I consider the condition in Ptychopteridae and some Psychodidae to be instances of homoplasy.

43. Pupal hind leg straight and arranged longitudinally (Fig. 11A) (plesiomorphic); hind leg curved in a S- shape and tucked under wing (or with apical portion abutting posterior margin of wing) (Figs. 1F, J–I, 18B– C) (apomorphic). This feature was described by Wood & Borkent (1989; their character 76). There are some rare exceptions of a pupa with a straight hind leg in Chironomidae in the genera Harrisonina (Diamesinae), Stictocladius (Orthocladiinae) and Lopescladius (Orthocladiinae) (Brundin 1966) (Fig. 18D). The latter two are sister groups (Cranston & Sæther 2010, Cranston et al. 2012) and this lineage, as well as Harrisonina, are closely related to other Chironomidae with a S-shaped hind leg. Clearly, these instances of a straight hind leg are reversions to the plesiomorphic condition. The derived feature is unique in the Diptera. This feature is discussed further in the introduction to the family key and below under character 47.

44. Pupae with apical portion of foreleg medial or ventral to apical portion of midleg (plesiomorphic); with apical portion of foreleg dorsal to apical portion of midleg (apomorphic). The Culicidae are unique within the nematocerous Diptera in having the apical portions of the foreleg and midleg crossing over, just anteroventral to the apex of the wing, and then curving dorsally on the wing margin, so that the apex of the midleg ultimately lies dorsal to that of the foreleg.

45. Pupa with apex of hind leg abutting apex of fore- and/or midleg (plesiomorphic); apex of hind leg abutting fore- and/or midleg subapically (apomorphic). The apex of the hind leg, directed laterally from along the wing margin abuts the preapex of the closely appressed fore- and midlegs in Chaoboridae or just the preapex of the midleg in Culicidae. In nearly all other Culicomorpha the apex of the hind leg abuts the apex of the fore- and/or midleg. Because other nematocerous Diptera do not curve the hind leg under the wing, the condition cannot be appraised any further in other taxa. The interpretation of the hind leg abutting the subapical portion of the fore- and/or midleg as derived is dependent, therefore, on in-group comparisons. There are some rare exceptions approximating the derived condition in Chironomidae (Brundin 1966: 428– 432) but these are probably independently derived. The pupa of Buchonomyia thienemanni Fittkau has elongate fore- and midlegs and the apex of the hind leg abuts the subapical portion of the midleg (Murray & Ashe 1981) (Fig. 18B). This genus may form the sister group of all remaining Chironomidae (Cranston et al. 2010, Cranston et al. 2012) and calls into question the interpretation of the character states as proposed here.

46. Pupal abdomen somewhat squared or circular in cross-section (plesiomorphic); abdomen dorsoventrally compressed in cross-section (apomorphic). The abdominal segments of pupae of Corethrellidae, Chaoboridae and Culicidae are strongly compressed dorsoventrally. Those of Dixidae and Simulioidea are either somewhat squared or nearly circular in cross-section. At least some early lineages of Chironomidae (e.g. Archaeochlus) are either somewhat squared or circular in cross- section but at least some other Chironomidae have dorsoventrally compressed abdomens. Ptychopteridae and at least most other nematocerous Diptera have squared or circular abdomens. At least some Psychodidae have dorsoventrally compressed abdomens (hence some homoplasy is present). This suggests that the flattened condition of Corethrellidae, Chaoboridae and Culicidae is a synapomorphy of this group, with homoplasy in some Chironomidae. The shape of the abdomen in cross-section likely reflects the type of motion pupae are capable of. In most Chironomidae and Culicoidea, the flexion of the abdomen is used for swimming, with the primary dorsoventral motion being steady (some Chironomidae) or very brisk (some Chironomidae, nearly all Culicoidea (some Corethrella move slowly)). The pupae of Ceratopogonidae, Simuliidae (D. Currie, pers. comm.) and Thaumaleidae (B. Sinclair, J. Ogawa, pers. comm.) with their rounded or somewhat squared abdominal segments are capable of only relatively slow, methodical, and lateral or more or less circular movements of the apex of the abdomen. They also can extend and retract their abdomens to a slight degree, using their terminal processes and, in

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 45 some, their dorsal and/or lateral stout setae as anchors, to slowly move forward and backward. The condition in Dixidae may contradict this interpretation. They have somewhat squared abdominal segments and yet are capable of quick lashing movement, similar to that in Culicidae. However, larvae of Dixidae move to nearby emergent substrate to pupate (Nowell 1951), and the movement I have observed may be artificial because I have placed them in water after they have come to rest above the water line in lab containers. Regardless, the shape of the abdominal segments may be strongly influenced by the type of substrate the pupa occupies and its needs for locomotion.

47. Pupal abdomen curved ventrally or anteroventrally (Figs. 1B, 18F, H–I) (plesiomorphic); abdomen directed posteriorly (Figs. 1C–E) (apomorphic). All non-Culicomorpha Diptera have their abdomens directed posteriorly or, at most, curved somewhat ventrally. As such, outgroup comparisons would strongly indicate the posteriorly directed position within the Culicomorpha to be plesiomorphic. I do not, however, accept this conclusion for the following reason. The S-shaped hind leg of the pupae of Culicomorpha is a strong and unique synapomorphy of the infraorder (see character 43). This feature, along with the shortened and/or distally curved fore- and midlegs of the Culicomorpha allows the abdomen to be ventrally or anteroventrally curved at rest. It also allows the dorsoventral movement of the abdomen to produce a strong swimming behaviour (including escape responses) and, in the case of some Chironomidae, to also ensure water flow and hence oxygen through their pupal cases. The ventrally flexed abdomen is present in most Chironomidae (Fig. 1B), all Dixidae (Fig. 1F), in all early lineages of Chaoboridae (Fig. 1H) and all Culicidae (Fig. 1I). Corethrellidae do not have a ventrally flexed abdomen but at least some are capable of rapid dorsoventral flexions as an escape response (pers. obs.). I consider it highly likely that the S-shaped hind leg evolved to allow the swimming motion produced by the dorsoventral movement of the abdomen. As such, I view the ventrally flexed abdomen as plesiotypic to the Culicomorpha. The permanently posteriorly directed abdomen of the Simulioidea (Ceratopogonidae, Thaumaleidae and Simuliidae) would therefore be interpreted as a secondarily derived condition approximating that of the outgroup of the Culicomorpha. Members of the Simulioidea cannot swim actively through water but use the apex of their abdomens to push themselves about by pressing against substrate, similar to those of many non-Culicomorpha (hence this behaviour in Simulioidea is interpretation as a secondary reversal).

48. Pupal abdomen with closed spiracles (plesiomorphic); abdomen with open spiracles on at least segments 5–7 (Fig. 10D) (apomorphic). Thaumaleidae pupae are the only Culicomorpha with open abdominal spiracles. Nearly all other nematocerous Diptera are either terrestrial and may have open spiracles or are aquatic or semiaquatic and have closed spiracles. In Ptychopteridae, Ptychoptera has an open lateral spiracle between the first and second abdominal segment (apparently not reported previously). It is absent in Bittacomorpha. Within Thaumaleidae, at least some Androprosopa have open spiracles on segment 2–7. Trichothaumalea have open spiracles only on segments 3–7, 4–7 or 5–7.

49. Pupal abdominal segments without recurved strong spines (Figs. 22A–C, 22B, 24A–C) (plesiomorphic); pupa with at least abdominal tergites 3 and 4 with sensilla D–5, D–6, D–8 and D–9 and sternites 5–7 sensilla V-5 each a strong, recurved spine (Figs. 23A–B) (apomorphic). The Simuliidae are unique within the nematocerous Diptera in having sensilla D-5, D-6, D-8 and D-9 on abdominal tergites 3 and 4 and sternites 5–7 with sensillum V-5 as strong recurved spines (some lineages have them on additional sternites). There are some superficial similarities in other taxa. Pupae of Leptoconops have D- 5 and D-8, lateral sensillum L-3 and ventral sensilla V-5 and V-6 as strong recurved spines on abdominal segments 2–8. Pupae of some (e.g. Androprosopa americana) but not all Thaumaleidae have recurved or at least anteriorly directed stout setae D-5, L-1, and L-3. At best the presence of D-5 as a recurved spine might be considered a synapomorphy between the three families with subsequent and repeated losses within Thaumaleidae and Ceratopogonidae and the other, nonhomologous setae becoming recurved independently. This seems rather unlikely. The recurved setae in these different groups are almost certainly used to anchor the pupae in their respective but quite different habitats. The pupae of Simuliidae have a unique cocoon spun from larval silk that provides a holdfast for the recurved spines. Pupae of species of Leptoconops, a genus with numerous autapomorphies otherwise, are present in marine or freshwater beaches or alkaline wet soils and the recurved

46 · Zootaxa 3396 © 2012 Magnolia Press BORKENT spines likely are important in anchoring them in a sifting substrate which generally (always?) includes algal strands. Pupae of Thaumaleidae are present in seeps and trickles and the strong spines undoubtedly are important in anchoring the generally immobile pupae amongst detritus, flotsam and roots of small marginal plants in a habitat that would otherwise sweep them away. All of this strongly suggests that the condition in Simuliidae is a uniquely derived condition for the family.

50. Pupal abdominal tergite 4 with, at most, one campaniform sensillum (Figs. 22A, C, 23A, C, 24A-C) (plesiomorphic); abdominal tergite 4 with two campaniform sensilla (Figs. 5A, 22B) (apomorphic). Pupae of early lineages of Ceratopogonidae are unique in the Culicomorpha in having two campaniform sensilla D-4-IV and D-7-IV. Some Forcipomyiinae and higher lineages have only one campaniform sensillum, but these are almost certainly secondary losses. The condition in Ptychopteridae is uncertain because of the large number of sensilla on the abdomen. Further outgroup comparisons were not made.

51. Pupal abdominal segments without lateral membrane (plesiomorphic); abdominal segments 4–8 with lateral membrane (apomorphic). The Simuliidae are the only Culicomorpha with the derived condition. The feature was discussed more fully by Adler et al. (2004: 131; their character 13). Ptychopteridae also lack the lateral membrane.

52. Pupal abdominal segments 3–8 with lateral seta 1 slender and situated posteriorly along lateral margin of each segment (Figs. 22A–B, 23A, C, 24A–C) (plesiomorphic); abdominal segments 3–8 with lateral seta 1 stout and positioned on the anterolateral corner of each segment (Fig. 22C) (apomorphic). Thaumaleidae are unique in nematocerous Diptera in having the derived condition. In Thaumaleidae, other than some Austrothaumalea, there is also a thick anterolateral spine on segment 2.

53. Pupal segment 9 well developed, with conical, cylindrical or dorsoventrally flattened terminal process are which are continuous with base of segment 9 (Figs. 25A–G, 26A–C) (plesiomorphic); base of segment 9 very reduced, with two articulated apical paddles, each of which are dorsoventrally flattened, membranous, with a least one well developed median rib and mediobasally overlapping (Figs. 10E–F, 26D–E) (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 77). The derived condition is a highly distinctive and unique modification of Chaoboridae and Culicidae and one of the most convincing features indicating their monophyly. It is unique in the Diptera. As discussed in the glossary, these paddles appear to articulate with segment 8 (Chaoboridae) or in membrane between segment 8 and 9. Segment 9 is also uniquely and strongly foreshortened in both these families. Culicid workers consider the paddle to originate on segment 9 and this is probably correct. Members of the Chironomidae genus Lasiodiamesa have an articulated terminal process, clearly originating on an otherwise well developed segment 9 and lacking the membranous, ribbed condition of the paddle of Chaoboridae and Culicidae. It is clearly independently evolved in this genus.

54. Pupa with terminal process directed posteriorly (plesiomorphic); with terminal process directed dorsally or posterodorsally (Figs. 10C–D) (apomorphic). In Simuliidae and most Thaumaleidae the terminal process is directed dorsally, looking like an upwardly curved hook. The terminal process is absent in some Thaumaleidae (e.g. Trichothaumalea, here considered a secondary loss). In nearly all Ceratopogonidae the terminal process is directed posteriorly. However, in Austroconops, one of two genera in the Leptoconopinae (which is the earliest lineage of Ceratopogonidae), the terminal process is directed posterodorsally and this is here interpreted as the plesiomorphic condition within the Ceratopogonidae. The pupae of the other genus of Leptoconopinae, Leptoconops, have lobe-like terminal processes and are highly modified in this and many other features. I consider the posteriorly directed terminal process of most Ceratopogonidae to be a reversal to the plesiomorphic condition. All Culicoidea and Chironomidae have the terminal process directed posteriorly. Ptychopteridae have rounded terminal processes and further outgroup comparisons indicate the apex of other nematocerous Diptera to be highly variable. However, I have not observed the apomorphic state in these.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 47 Considering the homoplasy and limited distribution in Ceratopogonidae, this feature is likely only a weak indicator of the monophyly of the Simulioidea. The similarities between Thaumaleidae and Simuliidae, however, are convincing.

55. Pharate adult with abdominal setae separately pointing posteriorly (plesiomorphic); abdominal setae with each half in a grouped, brush-like arrangement, directed posterolaterally (apomorphic). This feature was noted by Borkent & Grimaldi (2004) for Chaoboridae and includes all pertinent fossil species. The feature is unique within the Diptera.

Adults

56. Adult pedicel not especially enlarged, about the same diameter as scape, and male flagellum not markedly plumose nor noticeably different from that of the female (plesiomorphic); pedicel enlarged, especially in males, usually globular and much wider than scape as a result of a greatly enlarged Johnston's organ, and male flagellum plumose, with much longer, denser setae than that of female (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 79) and Oosterbroek & Courtney (1995; their characters 64, 65). The well developed plume is almost certainly directly related to the enlarged pedicel containing the Johnston's organ, which acts as a receptor for vibrations coming from the flagellum. Oosterbroek & Courtney (1995) used the large pedicel to group the Culicoidea on their cladogram, although in their text they implied its correct interpretation as a synapomorphy of all the Culicomorpha (with exceptions in Thaumaleidae and Simuliidae). Furthermore, they indicated the plumose condition as having evolved twice—once in Corethrellidae + Chaoboridae + Culicidae and once in Ceratopogonidae + Chironomidae. Here, I support Wood & Borkent (1989) in considering the plume to have been lost in Dixidae and both the plume lost and pedicel reduced in Thaumaleidae + Simuliidae. I have scored Dixidae as derived even though the plume is absent, on the basis of the presence of the enlarged pedicel. The short, reduced flagellum of both Simuliidae and Thaumaleidae, without a plume, may be directly related to the short antenna of the pupa, discussed elsewhere as character 26. In general, a well developed antennal plume corresponds to an elongate antenna in the pupa to accommodate the long developing setae (in Dixidae the elongate pupal antenna corresponds primarily to the elongate adult antenna).

57. Adult pedicel large, much wider, especially in the male, than first flagellomere (plesiomorphic); pedicel cylindrical, not much wider than first flagellomere (apomorphic). This feature, a synapomorphy of Simuliidae, was discussed by Wood & Borkent (1982). Families outside of the Culicomorpha nearly all have a pedicel that is either equal in diameter or somewhat wider than the first flagellomere, suggesting that the derived condition here may actually be plesiomorphic. If it is, this feature would indicate that Simuliidae are the sister group of all other Culicomorpha, which seems very unlikely. Instead, Wood & Borkent (1982) used other families of Culicomorpha as the limited outgroup to polarize the feature (as is done here). Other Culicomorpha other than Thaumaleidae have large pedicels (see character 56).

58. Adult basal flagellomeres (generally 1–10) with elongate setae restricted to basal ring (plesiomorphic); basal flagellomeres with additional elongate setae on length of flagellomeres (apomorphic). This feature was discussed by Borkent (2008: 207; as character 2).

59. Adult with short proboscis (plesiomorphic); long proboscis (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 78) and Borkent & Grimaldi (2004; their character 4). The feature is likely correlated, at least in part, with the elongate pupal mouthparts described in character 27 and the two are likely not independent indicators of relationship.

60. Adult clypeus bare, with numerous setae of equal length, or with short, single seta (plesiomorphic); clypeus with an elongate, stout, single, medial seta (apomorphic). This feature was discussed by Borkent (2008: 207; as character 1).

48 · Zootaxa 3396 © 2012 Magnolia Press BORKENT 61. Adult clypeus with setae (plesiomorphic); clypeus without setae (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 9). A Burmese amber fossil, Burmaculex antiques Borkent & Grimaldi, which is the sister group of all extant Culicidae, has the plesiomorphic condition. The synapomorphy applies, therefore, only to extant lineages of Culicidae.

62. Adult female labrum relatively short, broad, and dorsal to remaining mouthparts (plesiomorphic); labrum stylate, partially enclosed by the labium (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 5).

63. Adult female lacinia either reduced or a flattened blade with retrorse hooks (plesiomorphic); lacinia a long, slender stylet bearing fine, flattened ridges (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 6).

64. Adult palpal segment 3 short or of moderate length (plesiomorphic); palpal segment 3 disproportionately elongate (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 7). A likely related feature is present as a unique feature in the pupa, where the midportion of the palpus is S-shaped in an enlarged area near the base of the lacinia (Fig. 13D).

65. Adult female palpus longer than other mouthparts (plesiomorphic); palpus equal in length or shorter than other mouthparts (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 8). A Burmese amber fossil, Burmaculex antiquus, which is the sister group of all extant Culicidae, has the plesiomorphic condition. The synapomorphy applies, therefore, only to extant lineages of Culicidae.

66. Adult palpus without scales (plesiomorphic); with scales (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 10). A Burmese amber fossil, Burmaculex antiquus, which is the sister group of all extant Culicidae, has the plesiomorphic condition. The synapomorphy applies, therefore, only to extant lineages of Culicidae. The palpus of all Culicidae other than B. antiquus have scales. These are absent in the fossil and all other Culicomorpha and therefore the scaled condition is considered derived. Otherwise, some nematocerous Diptera also have scales on their palpi (some Cecidomyiidae, some Psychodidae) but this is certainly independently derived.

67. Adult female palpus with five segments (plesiomorphic); with four or fewer segments, some with vestigial fifth (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 11).

68. Adult with anterior anepisternum undivided (plesiomorphic); anterior anepisternum divided by sinuous suture into dorsal and ventral portions (apomorphic). This feature was discussed by Borkent (2008: 212; as character 21). The derived condition is unique in the Culicomorpha and most nematocerous Diptera (including Ptychopteridae).

69. Adult wing narrow at base (plesiomorphic); wing greatly broadened at base (with large anal lobe) (apomorphic). This feature was discussed by Adler et al. (2004: 129; their character 3). Although they indicated it was unique within the Culicomorpha, in fact there are some Ceratopogonidae (e.g. Jenkinshelea Macfie) with equally broad anal lobes. However, it is clear that this is an instance of homoplasy and I agree with its interpretation as a synapomorphy of Simuliidae.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 49 70. Female wing with R1 elongate, more than 0.8 of wing length (plesiomorphic); wing vein R1 short, 0.52– 0.73 of wing length (apomorphic). This feature was discussed by Borkent (2008; as character 3). The interpretation of this character is somewhat uncertain because of homoplasy within and outside the Culicomorpha.

71. Adult wing with R1 moderately elongate and apically straight (plesiomorphic); R1 extending very near or to wing apex and curved apically (apomorphic). This feature is unique within the nematocerous Diptera, other than some derived lineages of Ceratopogonidae (e.g. Borkent et al. 2009, fig. 29.34), where it is clearly homoplastic. The feature might initially be thought to be related to the following feature of a strongly arched vein R2+3 but early fossils of Dixidae exhibit only character 72 (showing character 71 and 72 evolved sequentially).

72. Adult wing with R2+3 straight or slightly curved (plesiomorphic); R2+3 strongly arched (apomorphic). This feature was discussed by Wood & Borkent (1989; their character 82). The apomorphic condition is unique to Dixidae within the nematocerous Diptera. See character 71 for further discussion.

73. Adult wing with R4+5 either straight or with an even curve from base to apex (plesiomorphic); R4+5 dorsally curved at midlength (apomorphic).

Thaumaleidae are the only nematocerous Diptera with a dorsal bend in vein R4+5 and this condition is therefore derived.

74. Adult wing with both M1 and M2 present (plesiomorphic); M2 absent (apomorphic).

Chironomidae are nearly the only Culicomorpha without vein M2. A few Ceratopogonidae also have a reduced

M2 that is either very difficult to see or appears to have disappeared altogether. These, however, are in a few genera with tiny individuals (e.g. some Brachypogon Kieffer) and are almost certainly secondarily derived. Most other nematocerous Diptera have both veins but there are what appear to be independent losses of M2 in the following: some Ptychopteridae (Bittacomorpha, Bittacomorphella), Nymphomyiidae, a few derived Mycetophilidae (e.g. Adicroneura Vockeroth), some Cecidomyiidae (only derived lineages in Porricondylinae, Cecidomyiinae), and a few derived genera of Tipulidae.

75. Adult wing with fork of Cu situated about 0.4–0.6 the total length of the wing (plesiomorphic); with fork of Cu situated less than 0.25 the total length of the wing (apomorphic). Thaumaleidae have the fork of Cu in the basal 0.25 of the wing and in Simuliidae the fork is right at the base of the wing itself. The condition is unique within the Culicomorpha and uncommon in other nematocerous Diptera, although it also occurs in Blephariceridae, Deuterophlebiidae, Psychodidae, Canthyloscelidae, Scatopsidae, Rangomaramidae, Sciaridae, some Mycetophlidae (sensu lato), and some Cecidomyiidae. It appears to me that the derived condition has evolved independently in each of the Thaumaleidae + Simuliidae, Sciaroidea (with subsequent reversals), Blephariceridae + Deuterophlebiidae and Canthyloscelidae + Scatopsidae.

76. Posterior margin of adult wing with setae (plesiomorphic); with scales (apomorphic). This feature was discussed by Borkent & Grimaldi (2004; their character 1). The feature is unique within at least nematocerous Diptera.

77. Adult male with anterior claw of each leg without a basal prong (plesiomorphic); anterior claws with basal prong (apomorphic). This feature was discussed by Borkent (2008: 210; as character 15), in which it was pointed out that the prong is present in Dixidae, early lineages of Chaoboridae (Eucorethra underwoodi, Mochlonyx, Australomochlonyx) and Culicidae (Cretaceous fossil Paleoculicis minutus Poinar, Zavortink, Pike & Johnston (Poinar et al. 2000); most species of Anopheles, some other Culicidae). The derived condition otherwise appears unique within nematocerous Diptera.

50 · Zootaxa 3396 © 2012 Magnolia Press BORKENT 78. Adult midfemur of equal or lesser diameter than those of fore- and hind legs (plesiomorphic); midfemur thicker than those of fore- and hind legs (apomorphic). This feature was discussed by Borkent (2008: 208; as character 4).

79. Adult midtibia with spur (plesiomorphic); midtibia without spur (apomorphic). This feature was discussed by Borkent (2008: 208; as character 5).

80. Posterior face of hind tibia of adult with either undifferentiated or short, slender setae (plesiomorphic); hind tibia with posterior patch of thin, whip-like setae, some of which are apically expanded (apomorphic). This feature was discussed by Borkent (2008: 209; as character 6).

81. Adult with first tarsomere of hind leg simple and more or less cylindrical (plesiomorphic); first tarsomere of hind leg flattened laterally and keeled ventrally (apomorphic). This feature was discussed by Wood & Borkent (1982).

82. Male adult with each claw of each leg simple basally (plesiomorphic); each claw with short stout tooth on dorsal side of base (apomorphic). This feature was discussed by Wood & Borkent (1982).

83. Adult abdominal tergite 1 without posterior fringe of elongate setae (plesiomorphic); with fringe of elongate setae on each side (apomorphic). The derived condition is unique to Simuliidae and not present in any other Diptera. The feature is also expressed in the pupae of Simuliidae, which are unique in having a posterolateral extension of abdominal tergite 1 that envelops the developing elongate setae of the adult. This feature may not be independent of character state 36 in which the metathorax lacks the posterolateral extension present in nearly all other nematocerous Diptera (see discussion there).

84. Adult male gonostylus with or without a few small setae (plesiomorphic); most of length of gonostylus bare except for a single elongate subbasal seta (apomorphic). This feature was discussed by Borkent (2008: 209; as character 10).

85. Adult male aedeagus sclerotized (plesiomorphic); aedeagus membranous (apomorphic). This feature was discussed by Sinclair et al. (2007; their character 8).

86. Adult male genitalia without ventral plate (plesiomorphic); ventral plate present lobe-like (apomorphic'); ventral plate triangular or quadrate (apomorphic"). This feature was discussed by Wood & Borkent (1982) and Sinclair et al. (2007; their character 9). However, there may be misinterpretation as well as further resolution regarding this feature. As noted by Sinclair et al. (2007), there is a question as to whether Chironomidae actually have a ventral plate. Although Wood & Borkent (1982) identified it as such in Buchonomyia (a lineage forming the sister group of all other Chironomidae; Cranston et al. 2012), a ventral plate is not present or readily apparent in at least most other Chironomidae. If there is a homolog (perhaps called the pars ventralis; Sæther 1980), it is not as well developed as in other Culicomorpha (other than Corethrellidae and Chaoboridae where it is absent). As such, the presence of a well developed ventral plate may be a synapomorphy of the Simulioidea + Culicoidea, with subsequent loss in Corethrellidae + Chaoboridae. Further to this, the presence of a triangular or quadrate ventral plate as is present and distinctive in Ceratopogonidae, Thaumaleidae and Simuliidae, is unique within the Diptera and therefore a further derived condition. Simuliidae and the primitive ceratopogonid genus Austroconops (representing the earliest lineage of Ceratopogonidae; Borkent et al. 1987) have a very similar ventral plate, with a dorsal "median sclerite". Thaumaleidae appear to lack this median sclerite. Further to this, the ventral plate of Simuliidae and all Ceratopogonidae is basally articulated, whereas it is basally fused to the gonocoxites in Thaumaleidae. Further comparative study of the male genitalia of Culicomorpha and particularly Chironomidae is needed to provide a better understanding of this structure.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 51 87. Adult male with ejaculatory apodeme present (plesiomorphic); ejaculatory apodeme absent (apomorphic). This feature was discussed by Sinclair et al. (2007; their character 7). An ejaculatory apodeme is present in other nematocerous Diptera, except Nymphomyiidae where its absence is likely an independent loss.

88. Adult male accessory glands continuous with vasa deferentia (plesiomorphic); accessory glands separate from vasa deferentia (apomorphic). This feature was discussed by Sinclair et al. (2007; their character 2) and Borkent et al. (2008). Sinclair et al. (2007) indicated that a separate accessory gland was a derived feature for the Diptera and Mecoptera. As considered here, the derived condition approximates the plesiomorphic condition in taxa outside the Mecoptera + Diptera (as a reversal). Some other nematocerous Diptera also have a separate accessory gland, indicating such reversal is susceptible to homoplasy.

89. Adult male with vas deferens fused apically (plesiomorphic); vas deferens not fused apically (apomorphic). This feature was discussed by Sinclair et al. (2007; their character 4) and Borkent et al. (2008). The feature is susceptible to homoplasy in some non-Culicomorpha.

90. Adult male accessory gland divided into two or three chambers (plesiomorphic); accessory gland undivided (apomorphic). This feature was discussed by Sinclair et al. (2007; their character 5) and Borkent et al. (2008). The derived condition is a reversal to the earlier plesiomorphic condition found outside Bibionomorpha + Ptychopteromorpha + other Culicomorpha. Borkent et al. (2008) discussed how the undivided accessory gland is likely related to the obligatory rotation of the male terminalia between segments 7 and 8. This form of rotation is unique in the Diptera and further evidence of the monophyly of Corethrellidae + Chaoboridae + Culicidae.

91. Adult male with anterior portion of accessory gland not attached to vas deferens or forming a complex of fused accessory glands with the posterior portion of the vasa deferentia (plesiomorphic); with anterior portion of accessory gland attached to vas deferens or posterior portion of testis (apomorphic). The derived condition is unique within the Diptera (Borkent et al. 2008). It is possible that the derived condition is a remnant of the fused condition found in other Culicomorpha, with total loss in Corethrellidae a further derived condition (so that the derived condition in Chaoboridae and Culicidae is an intermediate condition).

92. Female adult with more than one spermatheca (plesiomorphic); with a single large median spermatheca and two lateral ducts (apomorphic). This feature was discussed by Wood & Borkent (1982).

Misinterpreted or Questionable Synapomorphies

Adler et al. (2004: 131; their character 12) presented a synapomorphy for Simuliidae which is actually more broadly distributed. The pupae of Simuliidae have an apodeme attached to the trachea just before it enters the respiratory organ (Hinton 1957) that is considered a respiratory apparatus to open and close the pupal spiracle. The evidence for control of respiration was not presented and is questionable. The apodeme occurs in at least some Thaumaleidae and some, but not all Ceratopogonidae. In the latter family, for some taxa, the apodeme is very thin and difficult to discern, but in others (e.g. Palpomyiini) it is stout. It is almost certain that in Ceratopogonidae at least, the apodeme and associated muscle allows for some movement of the respiratory organ (Murray 1987). The apodeme is present in Culicidae where it also functions to move the respiratory organ (Houlihan 1971) and is also present in at least Eucorethra (Chaoboridae). It was not observed in Chironomidae, Corethrellidae or Dixidae. Further research is needed before this often obscure feature can be determined as a synapomorphy for a given clade. Adler et al. (2004: 129; their character 2) stated that the line of discontinuity between large upper facets and small lower facets of male adult eyes was a synapomorphy of Simuliidae and the lack of the feature in

52 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Parasimulium was "undoubtedly" a reversal. I fail to see the need to invoke the plesiomorphic condition as a reversal and, as did Wood & Borkent (1982), consider the line of discontinuity as a synapomorphy of only the Simuliinae. Adler et al. (2004: 129; their character 4) proposed the presence of the reduced basal medial cell of the wing as a synapomorphy of Simuliidae. Although present in some Simuliidae, it is absent in Parasimulium, in which crossvein m-cu is absent. The crossvein is also absent in Ceratopogonidae and many Chironomidae (probably secondarily lost). As it stands, this feature needs further study. Adler et al. (2004: 130; their character 9) suggested that diurnal feeding on blood by adult simuliids is a synapomorphy of Simuliidae. Aside from the fact that Parasimulium do not feed, Borkent & Craig (2004) showed that diurnal feeding is plesiotypic in Ceratopogonidae and varies in Culicidae. Corethrellidae feed only at night and the remaining Culicomorpha do not feed on blood. This strongly suggests that diurnal feeding in Simuliidae is a more broadly distributed symplesiotypy. They further suggested that the considerable period of time required by female Simuliidae to initiate feeding after landing on a host is distinctive and may be a synapotypy of the family. This behaviour is difficult to appraise in outgroups. I have seen females of species of Culicoides take considerable time to feed, rather similar to that of some black flies. Further to this, even if the behaviour is considered a synapotypic feature, female Parasimulium do not feed and so this feature may be a synapotypy of Simuliinae alone. Hinton (1958) indicated that the pharate pupa of Simulium ornatum Meigen feeds and spins its own cocoon. Adler et al. (2004: 131; their character 14) presented this combination of feeding and silk spinning as a synapomorphy for Simuliidae even though it is unknown for the vast majority of black flies including, especially, Parasimulium (the sister group of all others in the family). These features are otherwise poorly understood or not studied in related families. There are no other studies of other Culicomorpha regarding pharate pupal feeding. Only Chironomidae and possibly a few Ceratopogonidae (only Dasyhelea but questionably so) are otherwise known to spin silk and, although there is a quiescent period between larval cocoon-building and pupation in at least some Chironomidae, it is uncertain whether the pharate pupal stage is involved. This suggests that the interpretation of these features requires further study. Borkent & Grimaldi (2004) suggested that the small aperture of the pupal respiratory organ of Chaoboridae was a synapomorphy of that family. The most basal lineage, Eucorethra underwoodi, however, has a wide- mouthed respiratory organ, similar to that of most Culicidae, most Corethrellidae, Dixidae and many Chironomidae (early lineages; Cranston et al. 2012). As such this derived feature groups only a restricted clade within Chaoboridae. In the character analysis above, it was indicated in character 47 that the posteriorly directed abdomen of Ceratopogonidae, Thaumaleidae and Simuliidae was likely a secondarily derived condition approximating the plesiomorphic outgroup state because ventral flexion of the abdomen is likely related to the S-shaped hind leg of all Culicomorpha. A similar argument may be valid for the position of the respiratory organs. In Ceratopogonidae, Thaumaleidae and Simuliidae the base of the respiratory organ is near the anterior margin of the thorax, as it is at least superficially in Ptychopteridae and some other outgroups. In Chironomidae and Culicoidea, the base of the respiratory organ is well posterior of the anterior margin of the thorax. These states may also be related to the slow movement of pupae of Ceratopogonidae, Thaumaleidae and Simuliidae and therefore another synapomorphy of the Simulioidea.

Wood & Borkent (1989; their characters 80, 81) discussed the lack of either M3 or discal cell and a radial sector with three or fewer branches as tentative synapomorphies of the Culicomorpha. Considering the amount of homoplasy in the nematocerous Diptera in this regard, they do not appear to provide good evidence for the monophyly of the Culicomorpha. Wood & Borkent (1989; their character 73) and Oosterbroek & Courtney (1995; their character 43) considered the permanently exserted larval anal papillae as a synapomorphy of Corethrellidae + Chaoboridae + Culicidae. However, the feature varies in the outgroup sufficiently to make it at best a poor indicator of relationship. Oosterbroek & Courtney (1995; their character 83) indicated that the presence of pulvilli was a synapomorphy of Chaoboridae + Culicidae. However, only some members of each family have pulvilli and they are present in only some Chironomidae. In some Culicidae, they are highly reduced and difficult to see. Further study is needed to determine the character state distributions. Oosterbroek & Courtney (1995; their character 40) indicated the presence of anal prolegs was a synapomorphy of Thaumaleidae + Simuliidae + Ceratopogonidae + Chironomidae. Sinclair et al. (2007: 737) discussed homologies in Culicoidea and other nematocerous Diptera that strongly indicate the feature is a synapomorphy at a significantly more inclusive level.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 53 Wood & Borkent (1989; their character 83) and Oosterbroek & Courtney (1995; their character 93) recognized the two-chambered spermatophore in some Culicomorpha as a synapomorphy but these conclusions were modified by Sinclair et al. (2007) (see character 92). Wood & Borkent (1989; their character 71) suggested that the procerci on abdominal segment 9 of larvae of Chironomidae are homologous to the posterior flaps associated with the abdominal spiracles in Culicoidea and some other non-culicomorph Diptera. They considered the modification of these into cylindrical processes to be homologous in Chironomidae and Thaumaleidae (where they occur on fused segment 8+9). Oosterbroek & Courtney (1995; their character 103) correctly pointed out that there are problems with the interpretation of the posterior abdominal segments in Culicomorpha and that some similar structures occur in some other non- culicomorph groups. However, homology of the procercus of Chironomidae with the siphon in Culicoidea seems likely. The apices of the pupal terminal processes of at least some Podonominae develop inside the procerci (Cranston et al. 1987) just as those of at least Chaoboridae and Culicidae do within their siphons. This similar development of homologous pupal structures strongly suggests homology in the corresponding larval features, but regardless, further research is needed to confirm this. The procercus of Chironomidae, if considered distinct, would be unique and a synapomorphy for this family (with a few losses within the family). Oosterbroek & Courtney (1995) noted four synapomorphies for Simuliidae + Ceratopogonidae + Chironomidae. Their characters 53 (as questionable) and 55 refer to arrangements of the larval tracheal system. As they point out, there is missing information for many families of nematocerous Diptera and it would be best to have further comparative information before confidently interpreting these features. Even so, synapomorphy 53 is absent in Ceratopogonidae, so at best this is evidence of Simuliidae and Chironomidae being sister groups. Their characters 71 (abbreviated costa) and 72 (reduced posterior veins and anterior veins concentrated along the costal margin) are probably related to one another (as they point out) and are features found in a number of other nematocerous Diptera. The features are likely susceptible to homoplasy, and at least the extent of the costa varies considerably with both Ceratopogonidae and Chironomidae. Wood & Borkent (1989; their character 55) and Oosterbroek & Courtney (1995: their character 5) suggested the presence of a larval labral brush complex was a synapomorphy of the Culicomorpha. In Simuliidae, Dixidae and Culicidae the labral brush is composed of numerous, tightly packed, long setae or a single row of long, sickle- shaped setae, is capable of rapid closure, and has an elongate, labral retractor muscle clearly divided into two portions, each originating farther back on the enlarged cephalic apotome. This complex feature is clearly homologous between the three families and this suggests that it was present in the ancestor of the Simulioidea + Culicoidea. However, because it must then have been lost independently in each of Ceratopogonidae, Thaumaleidae, Corethrellidae and Chaoboridae, it can at best be considered only as a weak indicator of relationship. A further modification of the labral brush in Simuliidae is discussed above under character 7 as a valid synapomorphy of that family. A related feature, the infolding of the larval torma, proposed by Wood & Borkent (1989; their character 57) and Oosterbroek & Courtney (1995: their character 13), shows exactly the same character state distribution as the labral brush complex and is therefore not utilized here for the same reasons as given for the labral brush complex in the preceding paragraph. Dallai et al. (2007) described the ultrastructure of sperm of some Chironomidae, Ceratopogonidae and Simuliidae. Those of the Chironomidae had a distinctive centriole adjunct composed of nine kidney-shaped structures, not known otherwise in the Diptera. As such, this was suggested as a synapomorphy of the family. However, the taxa studied were all Chironominae, Orthocladiinae or unidentified (two species) and it would be important to study sperm of some more basal lineages before this might be proposed as a synapomorphy of the family. So too, further comparisons to other families of Culicomorpha would provide better outgroup comparisons.

Conflicting Synapomorphies

Wood & Borkent (1989; their character 62) and Oosterbroek & Courtney (1995; their character 27) used the absence of a larval pharyngeal filter as a synapomorphy to group Simuliidae + Ceratopogonidae + Chironomidae. It is also absent in Corethrellidae and Chaoboridae (which swallow whole prey and don’t need a filter) and in a number of other nematocerous Diptera. I consider the loss to be a weak indicator of relationship. The phylogenetic relationships proposed here would indicate that the pharyngeal filter has been lost independently five times within the Culicomorpha.

54 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Wood & Borkent (1989; their character 67) and Oosterbroek & Courtney (1995; their character 49) used the loss of the prothoracic spiracle in larvae of Simuliidae + Ceratopogonidae + Chironomidae as synapomorphy but it is also absent in all Culicoidea and numbers of other nematocerous Diptera. As stated by Wood & Borkent (1989) it is at best a weak indicator of relationship. Wood & Borkent (1989; their character 59) and Oosterbroek & Courtney (1995; their character 11) used the tong-like premandibles of larvae of Chironomidae and some Ceratopogonidae as a synapomorphy grouping these two families. However, the homology of this state is now unlikely. Firstly, as previously recognized by Wood & Borkent (1989), the elongate and well-developed premandibles of Ceratopogonidae are present only in Forcipomyiinae and their function there was uncertain. The larvae in this subfamily are mostly semiterrestrial or terrestrial (as a derived state), with strikingly modified head capsules (and bodies). Lieven (1998) provided an elegant and detailed study of the mouthparts of Forcipomyiinae larvae and showed that the premandibles do not function as tongs, as do those of Chironomidae, but are intimately connected to the mandibles (which are not articulated) to push food into the oral cavity (the mandible is drawn into the preoral cavity by retraction of the premandible). As such, the proposed synapomorphy for the Chironomidae and Ceratopogonidae can no longer be considered valid. There is a possibility that the presence of tong-like premandibles is a synapomorphy of the Chironomidae but they are absent in the semifamily Tanypodoinae and the earlier lineages Buchonomyiinae and Aphroteniinae (Ashe & O'Connor 2009; Cranston et al. 2012) and further study is needed regarding its distribution within the family before further conclusions can be made.

PHYLOGENETIC CONCLUSIONS

The descriptions of the pupae of Culicomorpha herein provide the first attempt at homologizing the many features of this life stage. Although taxonomists and morphologists of each of the included families have previously developed their own terms, it is hoped that the current paper will provide a common vernacular that will not only permit better comparisons across families but also increase interest in interpreting the phylogenetic significance of such features. Understanding homologies is the basis for cladistic analysis. This work also needs to be extended to other families of Diptera so that we might have one set of terms (and understood homologies) across the order as we have for the larval and adult stages. Although numerous homologies for pupal structures have been proposed in this paper, more work needs to be done. The variation within each family needs to be better understood and interpreted (especially Chironomidae). In addition, the specific homologies of many sensilla are uncertain and there is a need to study both larval and pupal innervation of each of the families to determine which sensilla are homologous in the different families. So too, the details of the respiratory organs need more study. The single most parsimonius cladogram presents a unique arrangement for the families of Culicomorpha (Fig. 28A) and is generally well supported by Bremer and bootstrap analysis (Fig. 28B). The low Bremer and bootstrap values for the Simulioidea + Culicoidea reflects the presence of only two synapomorphies. The loss of the external premandible is susceptible to homoplasy. I consider the second synapomorphy, the posteriorly or medially directed pupal palpus, to be of major significance in determining the monophyly of this group (see discussion under character 29). The Chironomidae are newly recognized as the sister group of all remaining Culicomorpha and the Simulioidea, with the three families Ceratopogonidae, Thaumaleidae and Simuliidae, is a new concept reflecting their monophyly. The Culicoidea remain the same as previously recognized by most recent workers. Wiegmann et al. (2011) supported the old concept of a monophyletic Corethrellidae + Chaoboridae, primarily on molecular evidence in a combined analysis, but the morphological synapomorphies (if any) were not reported. The presence of only two synapomorphies for the Chironomidae, with its striking intrafamilial morphological divergence, indicates the need for further study of this family. As indicated elsewhere in this paper, the presence of a procercus (see "Misinterpreted or Questionable Synapomorphies") warrants further investigation as a possible synapomorphy for Chironomidae. The recognition of the newly defined Simulioidea, comprised of Ceratopogonidae, Thaumaleidae and Simuliidae, is well supported by six pupal and one adult synapomorphy. The presence of an extension from the anterolateral margin of the pupal thorax to the palpus is particularly strong evidence of the monophyly of this group. The discovery of synapomorphies in each life stage in the ancestor of Thaumaleidae + Simuliidae also is strong support for this as a monophyletic group, as proposed by Bertone et al. (2008).

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 55 There have been a number of previous publications interpreting the phylogenetic relationships between the families of Culicomorpha based on morphology (Wood & Borkent 1989; Oosterbroek & Courtney 1995; Borkent & McKeever 1990; Sæther 2000 (with two concluding cladograms); Beckenbach & Borkent 2003; Sinclair et al. 2007; Blagoderov et al. 2007; Borkent et al. 2008; Woodley et al. 2009) or a combination of molecular and morphological data (Wiegmann et al. 2011). As far as possible, I have either incorporated previously proposed synapomorphies into the analysis or discuss them further above (under 'Misinterpreted or Questionable Synapomorphies' and 'Conflicting Synapomorphies'). The analysis of the families of Culicomorpha by Sæther (2000) is difficult to appraise. Character states are listed and a matrix provided but no synapomorphies are presented in the figures of the cladograms. As such it is difficult to know which features support which nodes, without going back and rerunning the data set and hoping to get similar results. Nevertheless, I have read his list of characters and incorporated any which I thought were supported by outgroup comparisons. Sæther (2000) included the Nymphomyiidae as part of the Culicomorpha, citing Michelsen (1996) as providing evidence for this phylogenetic relationship. In fact, Michelsen (1996) did not include Nymphomyiidae in his study but positioned the family as questionably close to the Culicomorpha based (Michelsen, pers. comm.) on an abstract by Oosterbroek & Courtney (1994). Oosterbroek & Courtney (1995) retained the Nymphomyiidae as part of the Blephariceromorpha but noted that features of the larval postmentum and prementohypopharyngeal apparatus may indicate the family is most closely related to the Culicomorpha (but more study was suggested). At present there remains no convincing evidence to place Nymphomyiidae as sister group to or within the Culicomorpha. Ogawa (2007) provided a detailed study of the Chaoboridae and the homology of their structures with other Culicoidea. He proposed some additional synapomorphies for the monophyly of each of Corethrellidae and Chaoboridae that warrant further outgroup comparisons. Blagoderov et al. (2007) used an exemplar approach, including both extant and fossil taxa, to reinterpret the families of nematocerous Diptera and brachyceran stem groups. Included was a novel cladogram of the Culicomorpha based on 28 synapomorphies (six of these were homoplastic; two were reversals; two were multistate). For the Culicomorpha they did not incorporate any previously supported synapomorphies. I consider their analysis cursory and primarily based on weak and/or functionally related features (i.e. many of these treated as separate are actually only one character). Thirteen characters are wing vein characters but some are likely a function of each other (e.g. character 8—apex of vein CuP: (0) in distal half, (1) in basal half; character 20—base of discal cell: (0) proximal to CuP apex or at the same level, (1) distal; character 23—CuP: (0) complete, (1) reduced, not meeting wing margin; see also their characters 24 and 25 and characters 75 and 76). Other synapomorphies are invalid when more taxa are included. For example, their synapomorphy 40 for all Culicomorpha (parameres separate) is clearly not true for numbers of taxa (e.g. Corethrellidae, many Ceratopogonidae). Other features are not true for all or some of the included taxa. For example, their synapomorphy 10 (branching of R2+3+4+R5) grouping all Culicomorpha is not true since R4+5 is always a single fused vein in Culicomorpha. So too, their synapomorphy 76 (vein thickness with medial veins weakened) is not restricted to Austrochlus, a chironomid (given as Austrochlea), but is also present in Simuliidae and Ceratopogonidae (including their exemplars). Their synapomorphy 48, state 2 was not listed but should have been "MA (arculus) short and spur-like" (V. Blagoderov, pers. comm.); it is given as the only synapomorphy grouping Dixidae with Thaumalea (Thaumaleidae) + Leptoconops (Ceratopogonidae) + Austrochlus (Chironomidae). It is also used to group Corethrella (Corethrellidae) + Chaoborus (Chaoboridae) and excludes the fossil Burmaculex Borkent & Grimaldi (Culicidae). All other Culicidae have a short and spur-like MA and knowing that the base of the wing of Burmaculex is somewhat deteriorated and difficult to see in the 90-100 million year old fossil specimen likely explains its apparent absence. Almost certainly, a short and spur-like MA is present in all Culicomorpha. As such, none of their synapomorphies have been incorporated into the current study because they are considered to be poorly supported. Further cladograms, based solely on molecular information, have presented yet other proposals of the relationships between the families of Culicomorpha (Pawlowski et al. 1996; Miller et al. 1997; Bertone et al. 2008). Sæther (1992) listed seven apomorphic "trends" that he proposed supported the monophyly of the Chaoboridae. It is uncertain what outgroup comparisons he made and whether these are therefore synapomorphies.

56 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Cranston et al. (2012) presented a molecular analysis of the Chironomidae and outgroup relationships which showed Chironomidae as the sister group of Ceratopogonidae (Thaumaleidae + Simuliidae). The Culicoidea were not included but their conclusions regarding the relationships between the four included families are congruent with that presented here. Finally, Wiegmann et al. (2011) have presented a purported comprehensive interpretation of the phylogenetic relationships between the families of Diptera. Their interpretation of the families of the Culicomorpha concludes with yet another unique arrangement. Unfortunately, it is uncertain which character states support the various nodes, and consequently I cannot comment on their character state hypotheses (Mooi & Gill 2010). The Culicomorpha have traditionally been divided into two superfamilies, the Chironomoidea with Chironomidae, Ceratopogonidae, Simuliidae, and Thaumaleidae and the Culicoidea with Dixidae, Corethrellidae, Chaoboridae and Culicidae. The results of this paper show that this classification is no longer tenable. The recognition of the Culicoidea is retained, combining a number of larval, pupal and adult features. As such, I believe that superfamily classification within the Culicomorpha remains useful but with a need to revise the concept of Chironomoidea. Bertone et al. (2008) found strong support from molecular analysis for Simuliidae + Thaumaleidae as sister groups, a conclusion supported here, and they placed the two families in the new superfamily Simulioidea. My evidence indicates that Ceratopogonidae are the sister group of these two families and I have expanded the idea of Simulioidea to include all three, with Chironomoidea restricted to only the Chironomidae. The cladogram presented here incorporates character states from egg, larval, pupal and adult stages and this increases the likelihood that the pattern accurately reflects true phylogenetic relationships between these families. Nevertheless, it is clear that much needs yet to be done. There are whole character systems that have not been properly described, let alone interpreted. The phylogeny presented here can be used to address the diversification of the Culicomorpha into families. Figure 29 gives the dating for the earliest known fossils for each lineage. I have included only those fossils at the family level that have at least one synapomorphy of that family (more comment on specifics below). The cladistic relationships between the families proposed here and the confident identification of fossil Chaoboridae and Simuliidae at 176 mya (based on immature stages, see below) and Chironomidae at 200 mya (Krzemiński & Jarzembowski 1999) suggest an explosive radiation at the family level for both the Simulioidea and Culicoidea. The oldest Diptera are from 240 mya (Krzemiński et al. 1994; Krzemiński & Krzemińska 2003; Lukashevich et al. 2010) and include the extant infraorders Culicomorpha, Tipulomorpha, Anisopodomorpha, and Tabanomorpha. The fossil infraorders Grauvogeliomorpha and Nadipteromorpha from this deposit may be members of the extant infraorder Ptychopteromorpha (Blagoderov et al. 2002). Amazingly, by 216 mya the order had rapidly diversified into most of the various extant infraorders of nematocerous Diptera (Blagoderov et al. 2007), including a variety of Culicomorpha (one having elongate mouthparts), described but not named by Blagoderov et al. (2007). Unfortunately the wing venation and some other important details of these early Culicomorpha were not visible and they could not be identified to family (or recognized as a stem group). The Chironomidae existed by 200 mya. By 176 mya the Chaoboridae and Simuliidae were established and their derived positions on the cladogram indicates that their sister group relationships must have occurred earlier than that (Fig. 29). It appears, therefore, that cladogenesis of the families of the Culicoidea occurred within a relatively short period of time, between 240 and more than 176 million years ago, resulting in an impressive diversification of larval, pupal and adult morphology. Cranston et al. (2012) used a molecular clock (using the program BEAST) to suggest that Chironomidae originated in the Permian but pointed out that this dating was improbable considering that the oldest fossil Diptera are Triassic (at 240 mya). The oldest Simulioidea (Ceratopogonidae + Simuliidae + Thaumaleidae) is Simulimima grandis (Kalugina & Kovalev 1985), described as a unique uppermost Lower Jurassic (Ichetui Formation, at least 176 mya) pupa and originally tentatively placed in the Ptychopteridae (as the fossil family Eoptychopteridae). Crosskey (1991) redescribed the specimen and considered it a Simuliidae based on its bifurcated respiratory organ (see character 41), form and placement of details of abdominal sensilla (as spine-combs and especially the "hooks" present only on tergites 3, 4 and sternites 5–7, see character 49) and the presence of a dorsally directed hook-like terminal process (also present in Thaumaleidae and some Ceratopogonidae; see character 54). Evenhuis (1994) suggested

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 57 the fossil was similar to the pupa of the tipulid genus Antocha Osten Sacken but without giving specifics. Currie & Grimaldi (2000) also questioned the placement of the pupa as a simuliid on the basis of overall habitus but also did not further interpret specific features listed by Crosskey (1991). Crosskey (2002) provided a further summary of the fossil's salient points that indicate it to be a simuliid and pointed out further that the apparent large size of the fossil is not significantly different from some Ectemnia Enderlein (based only on the thorax; see below for further discussion). Lukashevich (2004), after restudy of the holotype, agreed with Crosskey (1991) in placing the specimen as a simuliid. Because of the age of the fossil and the reluctance of others to accept this fossil as a simuliid, the original photomicrographs used by Crosskey (1991) (Figs. 27A–C, E) as well as further photomicrographs sent by Elena Lukashevich were examined and compared to a pupal exuviae of Prosimulium formosum (Figs. 27D, F). These show (Figs. 27E–F) that the fossil definitely has the terminal portion of the hind leg (part of the tarsus) lying along the posterior margin of the wing, a feature of a recurved S-shaped hind leg and a unique synapomorphy of the Culicomorpha (see character 43). Further to this, the metathorax is short (without a posterolateral extension) and tergite 1 has an extension (see character 36) abutting the hind leg (Figs. 27E–F); these features are unique synapomorphies of Simuliidae. A prothoracic extension is also present (Figs. 27C–D), a feature of Ceratopogonidae + Thaumaleidae + Simuliidae (see character 33). The dorsal area of the head is poorly preserved but a short antenna, a synapomorphy of Simuliidae + Thaumaleidea (see character 26), is questionably present (flagellomeres seem to be present but the outline of the antenna is unclear) (Figs. 27C). This evidence convincingly supports Crosskey's (1991 2002) conclusion that this fossil is a simuliid. Although some simuliid workers have noted that the large size and overall habitus of the fossil do not fit their understanding of the family (Currie & Grimaldi 2000), specific data have been lacking. To make more objective comparisons, the wing length (Fig. 27F) and length of abdominal segments 3–9 (those posterior to the apex of the wing) of the fossil were measured (taken from Crosskey 1991: fig. 2 and compared to Fig. 27E) and a few Culicomorpha (these exemplars appear characteristic of other taxa in each family in this regard). Table 4 shows that extant Simuliidae have a relatively shortened abdomen, with a ratio of wing length/ length of abdominal segments 3–9 of about 0.7. The fossil Simulimima grandis has a proportionally longer abdomen, with the ratio being 0.33. This, however, is similar to that of other Culicomorpha, indicating that the relatively short pupal abdomens of extant Simuliidae is apomorphic. Considering the suite of apomorphic features shared by S. grandis with other Simuliidae, the proportionally longer abdomen of S. grandis is evidence that it forms the sister group of all other known extant and fossil Simuliidae. As such, it provides a source of outgroup features to better interpret extant lineages (e.g. features differing between Parasimuliinae and Simuliinae).

TABLE 4. Comparison of wing length and length of abdominal segments 3–9 of pupae of some families of Culicomor- pha. Abbreviation: abd. seg. = abdominal segments.

Taxon N Wing length (mm) Abd. seg. 3–9 length (mm) Wing length/ abd. seg. 3–9 length Prosimulium formosum 1 2.1 3.1 0.68 Simulimima grandis 1 2.7 8.0 0.33 Androprosopa striata 1 1.2 3.0 0.41 Ceratopogonidae 2 0.5–0.9 1.3–1.8 0.32 Dixella serrata 1 1.2 2.7 0.55 Archaeochlus 1 1.5 3.2 0.47 Crosskey (1991) reported dorsal recurved hooks as present on tergites 2–4 of S. grandis. Although extant Simuliidae have the dorsal hooks restricted to tergites 3–4, this could easily mean loss of hooks on tergite 2 by extant Simuliidae or a gain of tergite 2 hooks by the fossil. Kalugina & Kovalev (1985) showed the palpus of S. grandis as being directed laterally, a plesiomorphic condition found only in Chironomidae within the Culicomorpha (and in other nematocerous Diptera). However, this feature was not present in the photographs in Crosskey (1991), or those provided by Elena Lukashevich and it is clear it was misdrawn. Fossil black flies are rare, with Upper Cretaceous adults known from only five specimens, one in New Jersey amber (95 mya; Currie & Grimaldi 2000) and four as compression fossils from Upper Jurassic / Lower Cretaceous deposits in Siberia (145 mya; Kalugina 1991). Larval fossils are recorded from the 120 mya Lower Cretaceous Koonwarra deposits in Australia (Jell & Duncan 1986).

58 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Because Simuliidae occur in the Jurassic, its sister group Thaumaleidae must also. However, only a single male Thaumaleidae is known from Upper Jurassic / Lower Cretaceous deposits in Siberia (145 mya; Kovalev 1989), the only Mesozoic record of the family. Extant members of both Simuliidae and Thaumaleidae are restricted to running waters, likely also limiting their presence at both amber and compression fossil sites. Thaumaleidae are even more specialized, with immature stages restricted to water trickling over rock faces. The presence of a Jurassic simuliid also indicates that Ceratopogonidae must go back to at least this time and they are likely older yet. Why are no early compression fossils of this family known? Two aspects of the group may explain their absence. First, early members of this family are relatively small in size. The earliest records of Ceratopogonidae are from the oldest amber known to have insect inclusions (Lebanese amber). They are notably diverse in that amber, with 24 species in seven genera and with only one of these species having conspecific members in a single piece of amber (indicating the presence of many species, each with a relatively low population level) (Borkent 2000, 2001). The small size of these Lower Cretaceous species, with a wing length of no more than 1.1 mm (most significantly smaller than that) likely means that they are difficult to find or interpret as earlier compression fossils. However, Lukashevich (pers. comm.) has studied the small Chironomidae from the Upper Jurassic deposits at Shar Teg, Mongolia, without finding Ceratopogonidae. Second, all early extant lineages of Ceratopogonidae are in small water bodies (as are those of other families of Culicomorpha; Borkent & Craig 2004), while most Early Cretaceous and Jurassic deposits represent lake habitats. As such, the Simulioidea are likely poorly represented as Jurassic / Cretaceous compression fossils because of their small size and/or restriction to small aquatic habitats. Lukashevich (1996) described fossil Dixidae from the mid and late Jurassic and these all share synapomorphy

72 (elongate and apically curved R1). She also described Syndixa liasina Lukashevich from the Lower Jurassic, but this species has a shorter R1 and its placement in the Dixidae is questionable. Fossil Chaoboridae are diverse and well represented in Jurassic and Cretaceous fossils (Borkent 1993; Lukashevich 1996). Unfortunately, we have no synapomorphies for the adult stage and therefore cannot be certain whether some fossils represented by adults might not be an early lineage of Culicidae (see Borkent & Grimaldi 2004) or possibly an even earlier lineage. Larvae and pupae each have a synapomorphy (Fig. 28A), and these are present in such Lower Jurassic fossils as Hypsocorethra jurassica Kalugina and Praechaoborus tugnuicus Kalugina (Kalugina & Kovalev 1985). I consider the Lower Jurassic genus Rhaetomyia Rohdendorf, known only from adults, as questionably placed in Chaoboridae. The Culicomorpha identified by Blagoderov et al. (2007) from 216 mya Triassic deposits in Virginia, USA, warrant comment. These authors used eight features to identify their material to the infraorder, only two of which have been shown to be synapomorphies (enlarged antennal pedicel and elongate flagellar setae; Wood & Borkent 1989, their character 79). The enlarged pedicel appears to be present in at least some of their specimens. Although Blagoderov et al. (2007) state the antennal flagellomeres have long setae they do not look so in their figures. It is especially important to note that at least some of their specimens are males and their flagellar setae appear short (i.e. similar to many other nematocerous Diptera). This casts some doubt on the interpretation of these fossils as Culicomorpha considering it is male Culicomorpha that have particularly elongate setae. Another possibility is that these fossils form the sister group of all remaining Culicomorpha based on an enlarged pedicel) and that the remaining Culicomorpha are monophyletic based on elongate flagellar setae. If so, the enlarged pedicel and elongate flagellar setae are therefore no longer to be considered as one functionally related character. Unfortunately, none of these Triassic fossils present a clear view of their wing venation. Future material will certainly be valuable in further interpreting their phylogenetic position. Lukashevich et al. (2010) described a 240 million year old fossil larva of an unplaced Culicomorpha from Grés-a-Voltzia in France (site of oldest known Diptera). Other than the presence of a clawed prothoracic proleg, there are no other visible features that could place it to family.

BIONOMIC DIVERGENCE

The phylogenetic relationships indicated here can be used to interpret some bionomic features within the Culicomorpha (Fig. 30) and provides insight into their diversification. There is little doubt that the female adult biting habit is plesiotypic to the Culicomorpha. The fundamental structure and arrangements of the mouthparts are the same in each family (Borkent 1995 with errata, 1996), with an elongate labrum, stylet-like mandibles and laciniae, and a partially encompassing elongate labium. In species in which females bite, the apex of each mandible has a row of teeth and, in most biters, the lacinia has retrorse teeth. The process of biting functions

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 59 basically in the same way in each family, with some striking modifications in the highly modified Culicidae. These features are shared by some non-Culicomorpha familes, indicating the biting mouthparts are plesiomorphic at a more inclusive level within nematocerous Diptera. The biting condition has been repeatedly lost within the group (confusing some workers who use limited exemplars). The Thaumaleidae, Dixidae and Chaoboridae have no biting members but have sister groups that do. Even so, members of all three families have retained slender but reduced mandibles and maxillae. Within the Ceratopogonidae, there have been independent losses in each of several subgenera of Forcipomyia, within Atrichopogon Kieffer, the ancestor of Dasyhelea, within a number of subgenera and species groups of Culicoides, and repeatedly among the diverse predaceous Ceratopogoninae. There have been numerous independent losses in Simuliidae (Adler et al. 2004: 79), at least one in Corethrellidae (Borkent 2008), and at least twice in the Culicidae (Malaya Leicester, Toxorhynchites Theobald). There are two extant genera of Chironomidae with species in which the females have biting mouthparts (Archaeochlus, Austrochlus; Cranston et al. 2002), and although these are members of a relatively early lineage within the family, they are still five or six bifurcations from the very base of the Chironomidae (Cranston et al. 2010, 2012). Azar et al. (2008), however, showed the presence of biting mouthparts in fossil Tanypodinae (subfamily placement doubted by Cranston et al. 2012), as well as in the extinct subfamily Aenneinae which is the assumed sister group of all other Chironomidae. These observations provide further evidence that the biting condition is plesiotypic to this family but also with repeated subsequent losses. Although blood-feeding itself is clearly plesiotypic to the Culicomorpha, there remains a question as to whether the plesiotypic feeding strategy is for females to take blood from vertebrates or invertebrates. Within the Simulioidea, all biting Simuliidae feed on either birds or mammals (Adler et al. 2004). Early lineages of Ceratopogonidae feed on fish, anurans, turtles, lizards, birds and mammals (Borkent 1995, 1996, 2000). Within the Culicoidea, there are only two biting families. Corethrellidae feed on anurans and Culicidae on a wide array of vertebrates. The feeding behaviour of mandibulate Chironomidae has not been observed. However, the form of the mouthparts provides evidence regarding host type in this family. As shown by Borkent (1995 with errata 1996), nematocerous Diptera feeding on invertebrates have large, coarse teeth. Those that have finely toothed mandibles, retrorse teeth and small claws feed on a variety of vertebrates. Those with finely toothed mandibles and simple laciniae feed on anurans. There are only three families of nematocerous Diptera in which females of some species have mandibles with large, coarse teeth, namely Blephariceridae, the Chironomidae noted earlier and numerous Ceratopogonidae. Both Blephariceridae and those Ceratopogonidae with large coarse teeth feed on other insects. The large mandibular teeth are clearly used to cut into the tough cuticle of their insect hosts. This strongly suggests that extant mandibulate Chironomidae do the same. It would be fascinating and very informative to observe the feeding habits of any female adult Archaeochlus or Austrochlus to confirm this hypothesis. There are further clues to the feeding behaviour of mandibulate Chironomidae. In the extant genera Archaeochlus and Austrochlus, the laciniae lack teeth. In size and overall habitus, their mouthparts are nearly identical to those of female Heteromyiini, Sphaeromiini, Palpomyiini and Stenoxenini in the Ceratopogonidae, and which are known to prey on other insects of similar size. They probably do not need retrorse teeth on their laciniae to anchor their mouthparts to the host surface because the prey (in some, a conspecific male during copula) is held firmly with their legs. The fossil species of mandibulate chironomids likely belonging to early lineages of Chironomidae described by Azar et al. (2008), however, not only have large mandibular teeth (Peter Cranston, pers. comm.), but also toothed laciniae, suggesting they fed on invertebrates but in a mode requiring the anchoring of the mouthparts. This combination of mouthpart modification is otherwise known only in some extant Ceratopogonidae: a few Atrichopogon that feed on Meloidae and Oedemeridae blister beetles and some species of Culicoides (Trithecoides Wirth & Hubert) that feed on adult mosquitoes. This limited evidence suggests that the earliest lineages of Culicomorpha fed on the haemolymph of insects (retained in the extant chironomids Archaeochlus and Austrochlus) and that the ancestor of the Simulioidea and Culicoidea evolved the capacity to feed on vertebrates sometime before 176 mya. The Triassic (250–200 mya) already had a diverse vertebrate fauna of parareptiles (sauropsids) and paramammals (therapsids) that would provide likely candidates as hosts for bloodsucking Culicomorpha during that time and later. Alternatively, the apparent invertebrate feeding habit of Archaeochlus and Austrochlus may be derived, with the earliest ancestral Culicomorpha being a vertebrate blood-feeder.

60 · Zootaxa 3396 © 2012 Magnolia Press BORKENT Lukashevich & Mostovski (2003) discussed the evolution of haemophagy in the fossil record and indicated that feeding on the blood of vertebrates did not occur in Culicomorpha (and others) until the Early Cretaceous. The phylogenetic analysis here contradicts their conclusion. Day- or night-time feeding also appears to be reflected in cladistic relationships (Borkent & Craig 2004). Female adults of all Simuliidae and the early lineages of Ceratopogonidae are day-time feeders and this is therefore a plesiotypic condition within the Simulioidea. Within the Culicoidea, Corethrellidae and most Culicidae (all Anophelinae and many Culicinae) are nocturnal feeders, suggesting that this is the plesiotypic condition in this superfamily. The time of feeding of mandibulate Chironomidae will be informative for interpreting this feature within the Culicomorpha. Indeed, with the conclusion here that Chironomidae are the sister group to all remaining Culicomorpha, the incentive to study further the extant mandibulate Chironomidae and all other early lineages within this diverse family is all the higher. Understanding the plesiomorphic and plesiotypic features of the chironomids will help to interpret an array of other morphological and bionomical features of the remaining Culicomorpha (Borkent & Craig 2004). Borkent & Craig (2004) showed that the plesiotypic habitat for immature Culicomorpha is small lotic and lentic waters and that the plesiotypic feeding mode of their larvae is feeding on microorganisms. Both these conclusions are unaffected by the new phylogenetic arrangement of the families presented here. However, it has now become increasingly important to improve our understanding of the phylogenetic relationships within the Chironomidae and to better identify the earliest lineages within that family. These early lineages, close as they are to the origin of the Culicomorpha, will provide better insight into plesiotypic features of the infraorder (i.e. provide outgroup comparison for the rest of the Culicomorpha). For example, Cranston et al. (2012) indicate that Buchonomyiinae are the sister group to all remaining extant chironomids. Although records are meager, it appears that at least one species of Buchonomyia (the sole genus) is present in rivers and streams and the larvae may be ectoparasites on Trichoptera (Ashe & O'Connor, 2009). Otherwise, early lineages of Chironomidae appear to be restricted to small lotic and lentic aquatic habitats, as are at least early lineages of each of the other families of Culicomorpha. Numbers of species in each lineage within the phylogeny of the Culicomorpha is informative in interpreting the diversification of lineages within the infraorder when these numbers are examined the light of some bionomic features and habitat types. Figure 30 plots the number of extant species of each lineage of Culicomorpha, the occurrence of blood sucking females (underlined family name) and their predominant larval habitat on a phylogeny showing the relationships between the families. There are four families that include the bulk of the currently described 18,103 species of Culicomorpha, namely, Chironomidae, Ceratopogonidae, Simuliidae and Culicidae. The last three, along with Corethrellidae, are those with biting females. The Chironomidae have only two genera (with three species each) with females with biting mouthparts and are here considered to be a non-biting family. Their great diversity, with 5,850 species, is likely related to their presence as larvae in nearly every conceivable aquatic habitat on the planet. Indeed, their early invasion of such habitats likely resulted in the specialized aquatic niches present in their sister group (by competitive exclusion). Ceratopogonidae are predominately in wet mud and/or detritus where the larvae move snake-like through the substrate seeking food. Such interstitial microhabitats may be found in a wide array of aquatic and semiaquatic habitats. Early lineages are in smaller aquatic habitats and it is only some predaceous higher lineages that have invaded the benthos of larger lotic and lentic habitats. Only the derived larvae of Forcipomyiinae have become semiterrestrial or terrestrial, clearly as a secondary adaptation. Thaumaleidae are restricted to seeps trickling over vertical rock faces and Simuliidae have specialized larvae that use their highly modified anal proleg to anchor on substrates and filter microorganisms and detritus from flowing water, from tiny seeps to large rivers. Dixidae larvae feed on microorganisms and detritus as they occupy the meniscus on the periphery of lentic and lotic habitats. The small predaceous larvae of Corethrellidae are present only in small ground pools (some at the margins of marshes and lagoons in dense aquatic vegetation), phytotelmata, and rarely, the hyporheic. The larvae of Chaoboridae are relatively large predators and nearly all are planktonic. Only the earliest lineage, represented by a single species Eucorethra underwoodi, is non-planktonic but has the remarkably specialized habit of feeding at the water surface by detecting struggling adult insects at the surface and pulling them under to ingest. All early lineages of Chaoboridae are restricted to small woodland pools (often vernal). Only the highly modified larvae of species of Chaoborus, with 43 of the 51species in the family, are present in large ponds and lakes where they form an important component of the zooplankton community. Culicidae, as a group, are present in small ground pools and phytotelmata, with many restricted to temporary

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 61 ground pools where the larvae feed on microorganisms and detritus (a few derived lineages have become predaceous). In large measure, the Simulioidea and Culicoidea occur where Chironomidae do not, with each of their component families present in separate habitats. Chironomidae, as a group, are the lineage that invaded a wide array of aquatic habitats, with its sister group, the Simulioidea and Culicoidea, specializing in select habitats or using them in specialized manners. The sister group of the Chironomidae, with 12,253 species, includes more than double the number of species present in Chironomidae, with 5,850 species (Fig. 30) and it is striking that it is the three big biting fly families Ceratopogonidae, Simuliidae and Culicidae which make up 96% of this diversity. It seems likely, therefore, that feeding on vertebrates has enhanced speciation within each of these groups. Thaumaleidae, Dixidae and Chaoboridae, without any biting females, have very small numbers of species in comparison. Even though Thaumaleidae and Dixidae have been less vigorously studied taxonomically, the Chaoboridae are well known and not likely to increase much in number of species on the planet. It is highly unlikely that Thaumaleidae and Dixidae would each measure above a few hundred species each. The species diversity in Corethrellidae is low compared to other biting fly families. Female adults are specialists restricted to feeding on calling anurans, predominately in tropical and subtropical continental areas. The group has only recently become better known (Borkent 2008, Borkent and Grafe 2012) and the first concerted effort to interpret diversity showed that Costa Rica, previously with three recorded species, now has 38 species, with 25 of these being described as new. Borkent (2008) appraised the potential diversity in the group and suggested that there may be as many as 35–50 more species in Costa Rica (indeed in the last few years, another six new species are already at hand) and that the world likely has more than 500 (and perhaps, many more than that). However, it was pointed out that the crisis in anuran biodiversity may well have already impacted the diversity of Corethrella in many regions of the world. Regardless, it is likely that the specialized biting habit of females in this family has restricted its species diversity, when compared to that of other biting flies in the Culicomorpha. Of the three big families with biting females, are there any general features that may indicate why each has diversified to the extent they have? One obvious characteristic is that their hosts, largely endothermic vertebrates, have greatly diversified. Although speciation must also be related to other environmental and geographical events, a rich source of protein for biting flies everywhere on the planet likely enhanced their rate of speciation. It may be that there is a tremendous advantage for dispersing females to have a source of protein from which to develop their eggs. Most biting flies are not host specific and tend to feed on a spectrum of hosts, although generally within a broad taxonomic group (e.g. ungulates, birds). The Ceratopogonidae, Simuliidae and Culicidae differ as families in their strategy to blood sucking (as can be easily observed by camping outdoors in many areas of Canada!). The Simulioidea have more than twice the number of species than their sister group, the Culicoidea, and this difference is likely to increase with further taxonomic work. There are more mosquitoes to be described but nothing like the high number of especially Ceratopogonidae that require taxonomic attention. I estimate that there are more than 9,000 species in this family with a distinctive morphology in need of description and names. Although many species of Culicidae are likely to be species complexes (and hence the numbers of species in this family will increase), this is also true of Ceratopogonidae. As such, the Ceratopogonidae and Simuliidae, with their short biting mouthparts, are a remarkably successful lineage. As one common name suggests, most no-see-ums (Ceratopogonidae) are small. In reality, this name applies primarily to members of the genus Culicoides, which are often highly diminutive (and good at getting through most screening). There are presently 110 extant genera of Ceratopogonidae but only four genera, all early lineages, include females that feed on vertebrates. Others either do not feed or feed on other insects. Of the latter, one lineage, the Forcipomyiinae, are small and primarily feed on large insects (e.g. Odonata, Orthoptera). The other lineage, the majority of the Ceratopogoninae, feed mainly on nematocerous Diptera of similar size, and members of this group become, phyletically speaking, increasingly large so that some members of the Sphaeromiini, Palpomyiini and Stenoxenini have wing lengths of 5–7 mm. As noted, it is the earlier lineages of Ceratopogonidae that feed on vertebrate blood. Of the four genera with biting members, Leptoconops has 150 species, Austroconops has two, the subgenus Forcipomyia (Lasiohelea Kieffer) has 177 species, but it is Culicoides that has the preponderance of species with 1319 species (and many more unnamed). All these taxa are small and many are capable of working their way through hair and loose feathers to get at their host's skin. This distinctive strategy may have enhanced their diversification.

62 · Zootaxa 3396 © 2012 Magnolia Press BORKENT The Simuliidae are generally larger than the vertebrate-biting Ceratopogonidae and, with their short mouthparts, also need to get close to the skin. They can scrabble through sparse host vestiture and generally feed on the edge of dense hair or feathers. In addition, their bite is often painless (at least for humans), which often allows them to feed at will. The Culicidae, representing the bulk of the diversity of the Culicoidea, have females with distinctively elongate mouthparts, allowing them to penetrate through hair and feathers (or human clothing) to obtain blood from their hosts as well as penetrating selectively and more deeply to locate capillaries. This feature alone may have allowed them to diversify, as did the Ceratopogonidae and Simuliidae, with their vertebrate hosts. Borkent & Grimaldi (2004) described the oldest member of the family from 100 million year old Burmese amber. A number of features of the mouthparts and body were intermediate and indicate that this fossil represents an early stage of evolution of the group. More importantly here, the proboscis was relatively short. Although it is possible that more completely developed Culicidae were present at the time, if fully developed elongate mouthparts were not present in the Culicidae 100 mya, the group may have diversified more recently as a lineage, compared to the Simulioidea. In summary, the following evolutionary scenario is suggested by the patterns above. The Chironomidae is the sister group of other Culicomorpha and, with non-biting female adults, invaded and diversified in virtually all aquatic habitats as larvae. The sister group of Chironomidae, the Simulioidea + Culicoidea, had adult females that fed on vertebrates, a remarkably successful strategy for acquiring plentiful protein to develop eggs and, likely, enhanced dispersal. This lineage has over twice the number of species of Chironomidae, entirely due to the presence of the diverse biting fly families of Culicomorpha. The immatures of the Simulioidea + Culicoidea rapidly diversified into various, relatively limited, specialized aquatic habitats, either not or marginally occupied by Chironomidae. The Ceratopogonidae specialized in interstitial microhabitats, Thaumaleidae in grazing vertical rock faces, Simuliidae in filtering flowing water for microorganisms, Dixidae filtering surface and subsurface microorganisms while lodged in the meniscus, Corethrellidae as small predators in small ground pools and phytotelmata, Chaoboridae as predators in temporary ground pools or as planktonic larvae in large water bodies, and the Culicidae in largely temporary ground pools and in phytotelmata. It seems clear that the biting habit is associated with the strong diversification of lineages within the Simulioidea + Culicoidea. Furthermore, the non-biting chironomids have diversified by invading, as larvae, a vast array of aquatic habitats, likely forcing their sister group, the Simulioidea + Culicoidea, through competitive exclusion to have specialized aquatic habitats.

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PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 67 of the chaoborid genera. Aquatic Insects, 14, 1–21. Addendum: Aquatic Insects, 14, 193–194. Sæther, O.A. (2000) Phylogeny of the Culicomorpha (Diptera). Systematic Entomology, 25, 223–234. Sæther, O.A. & Cranston, P.S. (in press) New World Stictocladius Edwards (Diptera: Chironomidae). Tropical Entomology. Sinclair, B.J. (1992) A new species of Trichothaumalea (Diptera: Thaumaleidae) from eastern North America and a discussion of male genitalic homologies. The Canadian Entomologist, 124, 491–499. Sinclair, B.J. (1996) A review of the Thaumaleidae (Diptera: Culicomorpha) of eastern North America, including a redefinition of genus Androprosopa Mik. Entomologica Scandinavica, 27, 361–376. Sinclair, B.J. (2000) Immature stages of Australian Austrothaumalea Tonnoir and Niphta Theischinger (Diptera: Thaumaleidae). Australian Journal of Entomology, 39, 171–176. Sinclair, B.J. & Saigusa, T. (2002) A new species of the seepage midge genus Trichothaumalea Edwards from Japan (Diptera: Culicomorpha: Thaumaleidae). Insect Systematics and Evolution, 33, 175–184. Sinclair, B.J., Borkent, A. & Wood, D.M. (2007) The male genital tract and aedeagal components of the Diptera with a discussion of their phylogenetic significance. Zoological Journal of the Linnean Society, 150, 711–742. Smith, L.M. & Lowe, H. (1948) The Black Gnats of California. Hilgardia 18, 157–183. Sutou, M. (2006) Immature stages of three Japanese species of the genus Plecia Wiedemann (Diptera: Bibionidae). Zootaxa, 1223, 1–18. Stoffolano, J.G., Woodley, N.E., Borkent, A. & Yin, L.R.S. (1988) Ultrastructural studies of the abdominal plaques of some Diptera. Annals of the Entomological Society of America, 81, 503–510. Szadziewski, R. (1995) The oldest fossil Corethrellidae (Diptera) from Lower Cretaceous Lebanese amber. Acta Zoologica Cracoviensia, 38, 177–181. Szadziewski, R. (1996) Biting midges from Lower Cretaceous amber of Lebanon and Upper Cretaceous Siberian amber of Taimyr (Diptera, Ceratopogonidae). Studia Dipterologica, 3, 23–86. Trautwein, M., Wiegmann, B.M., & Yeates, D.K. (2010) A multigene phylogeny of the fly superfamily Asiloidea (Insecta): Taxon sampling and additional genes reveal the sister-group to all higher flies (Cyclorrhapha). Molecular phylogenetics and evolution, 56, 918–930. Wiederholm, T. (ed.). (1986) Chironomidae of the Holarctic region. Keys and diagnoses. Part 2. Pupae. Entomologica Scandinavica, Supplement, 28, 1–482. Wiegmann, B.M., Trautwein, M.D., Winkler, I.S., Barr, N.B., Kim, J.-W., Lambkin, C., Bertone, M.A., Cassel, B.K., Bayless, K.M., Heimberge, A.M., Wheeler, B.M., Peterson, K.J., Pape, T., Sinclair, B.J., Skevington, J.H., Blagoderov, V., Caravas, J., Kutty, S.N., Schmidt-Ott, U., Kampmeier, G.E., Thompson, F.C., Grimaldi, D.A., Beckenbach, A.T., Courtney, G.W., Friedrich, M., Meier, R., & Yeates, D.K. (2011) Episodic radiations in the fly tree of life. Proceedings of the National Academy of Sciences, 108, 5690–5695. Wihlm, M.W., Sam, R.B., & Courtney, G.W. (2012) Morphology of Axymyia furcata McAtee (Diptera: Axymyiidae), including scanning electron micrography of all life stages. The Canadian Entomologist, 144, 273–290. Wirth, W.W. & Stone, A. (1956) Chapter 14. Aquatic Diptera. pp. 372–482 In Aquatic Insects of California with Keys to North American Genera and California Species. R.L. Usinger ed., University of California Press, Berkeley, Los Angeles & London. Wood, D.M. (1981) 11. Axymyiidae. pp. 209–212 in Manual of Nearctic Diptera. Volume 1. Agriculture Canada Monograph 27. 674 pp. Wood, D.M. & Borkent, A. (1982) Description of the female of Parasimulium crosskeyi Peterson (Diptera: Simuliidae) and a discussion of the phylogenetic position of the genus. Memoirs of the Entomological Society of Washington, 10, 193-210. Wood, D.M. & Borkent, A. (1989) Phylogeny and classification of the Nematocera. Ch. 114, pp. 1333-1370 In Manual of Nearctic Diptera. Vol. 3. Agriculture Canada Mongraph 32. vi + pp. 1333-1581. Wood, H.G. (1952) The crane-flies of the south-west Cape (Diptera, Tipuloidea). Annals of the South African Museum, 39, 1– 327. Woodley, N.E., Borkent, A. & Wheeler, T.A. (2009) Phylogeny. pp. 79–94. In Brown, B.V., A. Borkent, J.M. Cumming, D.M. Wood, N.E. Woodley, & M.A. Zumbado (editors). Manual of Central American Diptera. Volume 1. NRC Research Press, Ottawa, Ontario, Canada. 714 pp. Yukawa, J. (1971) A revision of the Japanese Gall Midges (Diptera: Cecidomyiidae). Memoirs of the Faculty of Agriculture, Kagshima University, 8(1), 1–203. Zwick P. (1977) Australian Blephariceridae (Diptera). Australian Journal of Zoology, Supplementary Series, 46, 1–121.

68 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 1. A–I. Habitus of whole pupae of families of Culicomorpha. A. Archaeochlus bicirratus, preserved, in dorsal view. B. Chironomus anthracinus, live, in lateral view (by Klaus Peter Brodersen). C. Ceratopogon nr. abstrusus, preserved, in dorsal view. D. Androprosopa americana, preserved, in dorsal view. E. Austrosimulium longicorne, live, in dorsal view (by Doug Craig). F. Dixella cornuta, live, in lateral view. G. Corethrella appendiculata, preserved, in dorsal view. H. Eucorethra underwoodi, live, in lateral view. I. Culicid sp., live, in lateral view (by Walter Pfliegler).

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 69 FIGURE 2. A–C. Habitus of pupa of Culicoides sonorensis (Ceratopogonidae). A. Female, in dorsal view. B. Male, in lateral view. C. Female, in ventral view. A and C have the abdominal segments separated by expanded membrane, not shown in B. Shagreen not shown.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 71 FIGURE 4. A–B. Heads and anterior portion of thoraces of pupae, in anterior view. A. Archaeochlus bicirratus. B. Culicoides sonorensis (shagreen not shown).

72 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 5. A–B. Chaetotaxy of abdominal segment 4 of Culicoides. A. Culicoides haematopotus, in dorsal and ventral view. B. Culicoides arboricola, in lateral view (from Linley 1970).

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 73 FIGURE 6. A–F. Anterodorsal portion of pupae showing relative position and movement of sclerites before and after adult emergence. A. Culicoides nubeculosus pupa, in dorsal view (dorsolateral cephalic sclerite in gray). B. Culicoides denticulatus after adult emergence showing ecdysial fissures (stippled), in dorsal view (dorsolateral cephalic sclerite in gray); during adult emergence fissures are much wider and dorsal apotome is flipped completely forward. C. Eucorethra underwoodi pupal exuviae, in dorsal view. D. Culicoides denticulatus pupal exuviae showing ecdysial fissures, in anterodorsal view. E. Androprosopa striata pupal exuviae showing ecdysial fissures, in anterodorsal view (dorsolateral cephalic sclerites loosely attached, here partially overlapping). F. Prosimulium formosum female pupal exuviae showing ecdysial fissures, in anterodorsal view (most of respiratory organs removed).

74 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 7. A–F. Heads of pupal exuviae of families of Culicomorpha. A. Ptychoptera sp. (from Japan), in ventral view, unfolded position. B. Ptychoptera sp. (from Japan), in ventral view, medially folded position (natural position of pupal exuviae). C. Psectotanypus sp., in ventral view. D. Psectotanypus sp., in lateral view. E. Culicoides denticulatus, in ventral view. F. Culicoides denticulatus, in lateral view.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 75 FIGURE 8. A–E. Heads of pupal exuviae of families of Culicomorpha. A. Prosimulium formosum, ventral view. B. Prosimulium formosum, in lateral view. C. Androprosopa americana, in ventral view. D. Androprosopa americana, in lateral view. E. Androprosopa striata, in ventral view, showing detached head capsule.

76 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 9. A–F. Heads of pupal exuviae of families of Culicomorpha. A. Dixella serrata, ventral view. B. Dixella serrata, in lateral view. C. Corethrella appendiculata, in ventral view. D. Corethrella appendiculata, in lateral view. E. Eucorethra underwoodi, in ventral view. F. Eucorethra underwoodi, in lateral view.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 77 FIGURE 10. A–F. Heads and abdomens of pupae or pupal exuviae of families of Culicomorpha. A. Head of exuviae of Anopheles gambiae, ventral view. B. Head of exuviae of Anopheles gambiae, in lateral view. C. Abdomen of pupa of Prosimulium formosum, in lateral view. D. Abdomen of pupal exuviae of Androprosopa americana, in lateral view. E. Apical portion of abdomen of pupal exuviae of male Eucorethra underwoodi, in dorsal view. F. Apical portion of abdomen of pupal exuviae of female Culicseta morsitans, in dorsal view.

78 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 11. A–B. Pupal heads, in ventral view. A. Ptychoptera sp. from Iowa, USA, also showing ventrum of thorax and arrangement of basal portion of legs. B. Archaeochlus bicirratus.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 79 FIGURE 12. A–D. Pupal heads, in ventral view. A. Androprosopa striata. B. Prosimulium formosum. C. Austroconops mcmillani. D. Culicoides guttipennis, with mandible overlying maxilla.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 81 FIGURE 14. A–D. Mesonota, in ventral view. A. Ptychoptera sp. from Iowa, USA, also showing metathorax and tergite 1. B. Archaeochlus bicirratus. C. Austroconops mcmillani. D. Androprosopa striata.

82 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 15. A–D. Mesonota, in ventral view. A. Prosimulium formosum. B. Parasimulium crosskeyi (showing only right half of crushed specimen). C. Dixella serrata. D. Corethrella appendiculata.

84 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 17. A–H. Metanota and tergites 1, in dorsal view. A. Archaeochlus bicirratus. B. Austroconops mcmillani. C. Androprosopa striata. D. Prosimulium formosum. E. Dixella serrata. F. Corethrella appendiculata. G. Eucorethra underwoodi. H. Anopheles atropos (modified from Belkin et al. 1970: fig. 10).

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 85 FIGURE 18. A–D. Arrangement of antennae, wings and legs of pupae of Culicomorpha, in ventral view. A. Archaeochlus bicirratus (Podonominae). B. Buchonomyia thienemanni (Buchonomyiinae) (from Murray & Ashe 1981: fig. 2a). C. Paraheptagyia cinerascens (Diamesinae) (from Brundin 1966: fig. 623). D. Lopescladius sp. (Orthocladiinae) (from Brundin 1966: fig. 622, as Cordites).

86 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 19. A–C. Arrangement of antennae, wings and legs of pupal exuviae or pupae of Culicomorpha, in ventral view. A. Austroconops mcmillani (of pupal exuviae, mesosternum not shown). B. Androprosopa americana (of pupa). C. Prosimulium formosum (of pupa).

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 87 FIGURE 20. A–D. Arrangement of antennae, wings and legs of pupae of Culicomorpha, in ventral view. A. Dixella serrata. B. Corethrella appendiculata. C. Eucorethra underwoodi. D. Anopheles gambiae.

88 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 21. A–L. Left respiratory organs, in lateral view (position of C uncertain). Surface reticulation not drawn for most. A. Ptychoptera lenis (from Alexander 1981: fig. 22.7). B. Ptychoptera sp. nr. taxenouchii, from Japan. C. Coelotanypus sp. (redrawn from Fittkau & Murray 1986: fig. 5.9A). D. Xestochironomus subletti (from Borkent 1984: fig. 54A). E. Austroconops mcmillani (from Borkent & Craig 2004: fig. 5C). F. Androprosopa linsayorum. G. Prosimulium clandestinum (from Adler et al. 2004: fig. 4.29). H. Dixella serrata. I. Corethrella appendiculata. J. Corethrella pallida. K. Eucorethra underwoodi. L. Anopheles gambiae.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 89 FIGURE 22. A–C. Abdominal segment 4 of families of Culicomorpha. A. Archaeochlus bicirratus. B. Austroconops mcmillani. C. Androprosopa striata.

90 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 23. A–C. Abdominal segment 4 of families of Culicomorpha. A. Prosimulium formosum. B. Parasimulium crosskeyi (reconstructed from crushed specimen. C. Dixella scitula (based on Belkin et al. 1970: fig. 125).

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 91 FIGURE 24. A–C. Abdominal segment 4 of families of Culicomorpha. A. Corethrella appendiculata (based on Belkin et al. 1970: fig. 119). B. Eucorethra underwoodi. C. Anopheles grabhamii (modified from Belkin et al. 1970: fig. 13).

92 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 25. A–F. Abdominal segment 9 of families of Culicomorpha. A. Ptychoptera sp., female from Japan. B. Archaeochlus bicirratus, male. C. Lopescladius fittkaui, male, in dorsal view (from Coffman 1986: fig. 9.34F). D. Stictocladius victoriensis, male, in dorsal view (from Cranston & Sæther 2010: fig. 6i). E. Austroconops mcmillani, male. F. Androprosopa linsayorum, male. G. Prosimulium formosum, male.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 93 FIGURE 26. A–D. Abdominal segments 8 and 9 of families of Culicomorpha. A. Dixella scitula, female (modified from Belkin et al. 1970: fig. 125). B. Corethrella appendiculata, male (modified from Belkin et al. 1970: fig. 119). C. Corethrella appendiculata, female (modified from Belkin et al. 1970: fig. 119). D. Chaoborus braziliensis, male (as Sayomyia lanei) (modified from Belkin et al. 1970: fig. 117). E. Anopheline, female (modified from Harbach & Knight 1980: fig. 77a).

94 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 27. A–F. Comparison of thoracic features of pupae of the Lower Jurassic fossil Simulimima grandis and extant Prosimulium formosum (male). A, B. Opposing faces of Simulimima grandis showing magnified views in E and C, respectively. D, F. Thoracic features of Prosimulium formosum, wing length in Fig. F = 2.1 mm; same distance in Fig. E = 2.7 mm.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 95 FIGURE 28. A. Single most parsimonious cladogram of the families of Culicomorpha with numbers representing character states described in the text. Semaphoronts are indicated and abbreviated as follows: E—egg, L—larva, P—pupa, A—adult. B. Strict consensus tree of single most parsimonious trees found in the phylogenetic analysis. Bremer support values are shown above the branches and bootstrap values are shown below.

96 · Zootaxa 3396 © 2012 Magnolia Press BORKENT FIGURE 29. Phylogeny of the Culicomorpha showing the earliest fossils for each lineage. Fossil records are based on the following references: Chironomidae: Krzeminski & Jarzembowski (1999); Ceratopogonidae: Szadziewski (1996), Borkent (2000); Simuliidae: Crosskey (1991) and discussion in text here; Thaumaleidae: Kovalev (1989); Dixidae: Lukashevich (1996); Corethrellidae: Szadziewski (1995), Borkent (2008); Chaoboridae: Kalugina & Kovalev (1985), Borkent (1993); Culicidae: Borkent & Grimaldi (2004). The records of undetermined Culicomorpha are from Lukashevich et al. (2010) at 240 mya and Blagoderov et al. (2007) at 216 mya. Dating of ages based on scale by International Commission on Stratigraphy (www.stratigraphy.org, accessed July 26, 2011) and reports the minimum age for a given stage or epoch (without the confidence limits). As such each fossil is likely to be somewhat older than shown. Maximum dates of fossils based on those with at least one known synapomorphy for that family.

PUPAE OF CULICOMORPHA Zootaxa 3396 © 2012 Magnolia Press · 97 FIGURE 30. Phylogeny showing the number of extant species in each lineage of Culicomorpha, predominant larval habitat, and families with biting females (names underlined) (Chironomidae, with only two genera with biting females are considered a non-biting family). Authorities for numbers of species in each family as follows: Chironomidae—Patrick Ashe (pers. comm.); Ceratopogonidae—Borkent 2012; Thaumaleidae—Bradley Sinclair (pers. comm.); Simuliidae—Adler & Crosskey 2012; Dixidae—Pape & Thompson 2011; Corethrellidae—Borkent 2008, Borkent & Grafe 2012; Chaoboridae—author's catalog; Culicidae—Harbach 2012. Exceptions to larval habitats: some Ceratopogonidae are intertidal and some walk on semiterrestrial and terrestrial substrates (Forcipomyiinae); some Dixidae are in lotic habitats (at meniscus at edges); the earliest lineage of Chaoboridae, Eucorethra underwoodi, is a predator of live insects landing on pool surfaces.