Morphology of Axymyia Furcata

Entomology Publications Entomology

4-2012 Morphology of Axymyia furcata (Diptera: Axymyiidae), including scanning electron microscopy of all life stages Matthew .W Wihlm Iowa State University, [email protected]

Rebecca B. Sam Iowa State University

Gregory W. Courtney Iowa State University, [email protected]

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This Article is brought to you for free and open access by the Entomology at Iowa State University Digital Repository. It has been accepted for inclusion in Entomology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Morphology of Axymyia furcata (Diptera: Axymyiidae), including scanning electron microscopy of all life stages

Abstract The morphology of all life stages of the semi-aquatic nematocerous fly, Axymyia furcata McAtee (Diptera: Axymyiidae), is documented, with emphasis on use of scanning electron microscopy (SEM). Our data on some life stages (e.g., egg, first-instar larvae) represent the first SEM examination of these stages in axymyiids. Among the unexpected results is absence of an egg burster in first instars. Most characters provided little evidence of a phylogenetic relationship with any other clade of nematocerous Diptera, reflecting a need for comprehensive morphological studies of the immatures of many families.

Disciplines Entomology

Comments This article is from The Canadian Entomologist 144 (2012): 273–290, doi:10.4039/tce.2012.27. Posted with permission.

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/ent_pubs/40 273 Morphology of Axymyia furcata (Diptera: Axymyiidae), including scanning electron microscopy of all life stages

Matthew W. Wihlm,1 Rebecca B. Sam, Gregory W. Courtney2

Abstract—The morphology of all life stages of the semi-aquatic nematocerous fly, Axymyia furcata McAtee (Diptera: Axymyiidae), is documented, with emphasis on use of scanning electron microscopy (SEM). Our data on some life stages (e.g., egg, first-instar larvae) represent the first SEM examination of these stages in axymyiids. Among the unexpected results is absence of an egg burster in first instars. Most characters provided little evidence of a phylogenetic relationship with any other clade of nematocerous Diptera, reflecting a need for comprehensive morphological studies of the immatures of many families.

Re´sume´—Nous pre´sentons des informations sur la morphologie de tous les stades du cycle biologique de la mouche ne´matoce`re semi-aquatique Axymyia furcata McAtee (Diptera: Axymyiidae) principalement a` l’aide de microphotographies au microscope e´lectronique a` balayage (SEM). Nos donneés sur certains stades du cycle (par ex., l’oeuf, les larves de premier stade) repre´sentent le premier examen au SEM de ces stades chez les axymyiide´s. Parmi les dećouvertes inattendues, il y a l’absence de ruptor ovi (dent d’ećlosion) chez les larves de premier stade. La plupart des caracte`res fournissent peu d’indications de relations phylogeńe´tiques avec les autres clades de dipte`res ne´matoce`res, ce qui souligne la nećessite´ d’e´tudes morphologiques de´tailleés des stades immatures de plusieurs des familles.

Introduction Sinaxymyia Zhang, Juraxymyia Zhang, and Psocites Hong, are known from Kazakhstan and China Axymyiidae is a small family of semi-aquatic (Zhang 2010). lower (nematocerous) Diptera. The family contains Adult and larval A. furcata were first described, three described extant genera: Axymyia McAtee, respectively, by McAtee (1921) and Krogstad Mesaxymyia Mamaev, and Protaxymyia Mamaev (1959; see also Peterson 1960, fig. D9). Wihlm and Krivosheina (Krivosheina 2000). There are (2009) and Wihlm and Courtney (2011) have currently seven described species, of which all shown that immature axymyiids, once considered except Axymyia furcata McAtee are Palearctic. rare, can be abundant in suitable habitats. These Axymyia furcata is widespread in eastern North flies are dependent on woody debris in small America, ranging from the upper midwestern streams and seeps, where the larvae and pupae United States of America (Minnesota and Iowa) reside in galleries formed in wet, decomposing through the northeastern United States and wood. Adults emerge in early to mid spring, are southeastern Canada, and throughout Appalachia short lived, and appear to remain near their natal to South Carolina and Georgia (Wihlm and habitat. With oviposition also occurring on pro- Courtney 2011). An undescribed eighth species spective larval habitat, these flies are clearly has been recorded in Oregon (Wood 1981; Dudley dependent on a xylophilic lifestyle. and Anderson 1982; G.W. Courtney, unpublished Although distinct in all life stages, the phy- records). In addition, three extinct genera, logenetic position of Axymyiidae has remained

Received 21 March 2011. Accepted 23 May 2011. M.W. Wihlm,1 R.B. Sam, G.W. Courtney,2 Department of Entomology, Iowa State University, 3222 Science II Building, Ames, IA 50011-3222, United States of America 1Present address: Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, Johnston, Iowa 50131-0552, United States of America. 2Corresponding author (e-mail: [email protected]). doi:10.4039/tce.2012.27

Can. Entomol. 144: 273–290 (2012) ᭧ 2012 Entomological Society of Canada 274 Can. Entomol. Vol. 144, 2012 among the most problematic questions in lower will continue to be a major influence on students of Diptera. The family has in various classifications Diptera. At the core of the series is a solid under- been associated with Bibionidae, Anisopodidae, standing of Diptera morphology and homology or Pachyneuridae (see reviews by Mamaev and across taxa. Following this standard, our intent Krivosheina 1966; Wood and Borkent 1989). in this study is to present details on the structure Wood and Borkent (1989) identified no synapo- of all life stages of A. furcata as an exemplar of morphies to link Axymyiidae conclusively to any Axymyiidae, emphasizing use of scanning electron other major lineages, and therefore gave the family microscopy (SEM) to illustrate morphological an infraordinal rank. Subsequently, Oosterbroek characteristics. and Courtney (1995) suggested a sister-group relationship between Axymyiidae and Bibionomorpha Materials and methods sensu Wood and Borkent (1989). However, the character basis for this hypothesis remained weak. Eggs oviposited on wood by reared but Although a recent molecular phylogenetic analysis apparently unmated females of A. furcata were using four nuclear genes (Bertone et al. 2008) failed collected and preserved in 70% EtOH. Except to resolve the matter, a combined analysis using for first-instar larvae, which were obtained from multiple nuclear genes, complete mitochondrial theCanadianNationalCollectionofInsects, genomes, and 371 morphological characters Arachnids, and Nematodes, Ottawa, Ontario (Wiegmann et al. 2011) placed Axymyiidae within (CNC), most larvae and pupae used in this study a newly defined Bibionomorpha (Bibionomorpha were collected during 2005–2008. Field-collected sensu Wood and Borkent (1989) plus Anisopo- specimens were removed from their galleries and didae, Scatopsidae, and Canthyloscelidae). Despite immediately preserved in 70% or 95% ethanol. these results, the phylogenetic position of Axy- Most adult specimens were reared from larvae myiidae will likely remain a matter of further and pupae, following protocols described by interest and debate. Wihlm and Courtney (2011). Additional fresh This paper on axymyiid morphology is dedicated specimens were collected with Malaise traps. to the 30-year anniversary of the publication of Most reared and trapped adults were preserved in Volume 1 of the Manual of Nearctic Diptera 70% EtOH. Numerous dried adults were borrowed (McAlpine et al. 1981). Published while the junior from the following research collections: CNC; author (GWC) was an undergraduate assistant in an Cornell University Insect Collection, Ithaca, New aquatic entomology laboratory, this volume has York (CUIC); Museum of Comparative Zoology, been not only an invaluable resource on Diptera Cambridge, Massachusetts (MCZ); United States morphology and identification, but has provided National Museum of Natural History, Washington, information on many fascinating and obscure District of Columbia (USNM); University of groups of flies. For many young and aspiring Guelph Insect Collection, Guelph, Ontario (UGIC); students of Diptera, this publication was their University of Minnesota Insect Collection, St. Paul, first exposure to some families (e.g., Axymyiidae). Minnesota (UMIC); and University of Wisconsin The same can be said three decades since its pub- Insect Research Collection, Madison, Wisconsin lication. Volume 2 (McAlpine et al. 1987) had a (UWIC). Most of our specimens were deposited in comparable influence on students of Brachycera, the Iowa State Insect Collection at Iowa State and the final volume (McAlpine and Wood 1989) University, Ames, Iowa; additional material was established the framework and incentive for deposited in the CNC and USNM. many future investigations of Diptera phylo- Whole specimen observations of all life stages geny. Likewise, the series provided a template were made using Olympus SZX9, SZX12, and for many other regional Diptera ‘‘Manuals’’ (e.g., SZH dissecting microscopes. Light micrographs Papp and Darvas 1997, 1998, 2000a, 2000b; Brown were obtained using a SPOT RTs digital camera; et al. 2009, 2010) and the masterful illustrations by high-resolution images were created by combining Ralph Idema have populated not only books on a series of images using Helicon Focus 3.10s Diptera, but those on immature (e.g., Stehr 1991) software. and aquatic insects (e.g.,Merrittet al. 2008). In The structure of the terminalia and reproductive sum, the Manual of Nearctic Diptera has been and tract was studied by removing the distal half of the

᭧ 2012 Entomological Society of Canada Wihlm et al. 275 abdomen and clearing it in dilute ( , 10%) potas- Cranium sium hydroxide (KOH). The pharyngeal filter was First instar: Posterior three fourths of frontal prepared by clearing the larval head capsule in plate membranous. Egg burster greatly reduced/ diluteKOH,followedbytransfertoglycerinand absent. Dorsal sensilla forming two rows of three temporary slide mounts. Observations were made setae, progressively larger posteriorly to antero- using a Nikon E800 compound microscope. Light laterally, plus two short chaetiform setae near micrographs of slide mounted material were cap- base of clypeus; all sensilla simple (Fig. 3). tured using a Digital Sight DS-2Mv camera moun- Lateral surface of genae smooth, with five tufted ted on a Nikon Eclipse 50i compound microscope sensilla, dorsal three with three branches, ven- and NIS Elements 2.3 software. Drawings were tral, anteroventral sensilla each with four to made using Adobes Illustrators CS3. five branches (Fig. 4). Sclerotized bar between Material for SEM was sonicated in 70% EtOH clypeus and frontal plate less pronounced than for 10–15 s (repeated as necessary) to remove in later instars. Clypeus short, wide with four debris. Specimens were subsequently critical point prominent setiform sensilla (Fig. 3). dried and then sputter-coated with palladium-gold Fourth instar: Frontoclypeal apotome ovoid, alloy 60/40. Observations and digital images were elongate, narrowing posteriorly, not reaching made using a JEOL JSM-5800LV SEM. SEM was posterior cranial margin. Anterior margin with performed at the Bessey Microscopy and Nano- pair of short, branched sensilla (one on each imaging Facility (Iowa State University). side) on heavily sclerotized ridge separating Terminology for larval and adult morphology frontal plate from clypeus and spanning lateral is based primarily on Courtney et al. (2000) and margins at mandibular articulation. Genae meet Cumming and Wood (2009), respectively. at narrowed end of frontoclypeal apotome, forming posterior margin of cranium. Dorsal surface of Morphology of A. furcata gena smooth, with row of three sensilla adjacent to ecdysial suture; sensilla progressively larger Egg posteriorly to anterolaterally, posterior sensillum Approximately 1 mm in length, 0.5 mm in setiform, others two-branched (Fig. 9). Postero- width, oval in shape, burr-like in appearance lateral portion of gena less sclerotized where (Fig. 1). Ventral surface, which adheres to wood, overlapping portion of prothoracic cuticle attached. somewhat smooth in texture. All other surfaces Lateral surface of gena smooth, with five tufted covered in rows of raised tubercles, each bearing sensilla, arranged as in first instar but all with four an apical pore (Fig. 2). Surface microsculpture to five branches (Figs. 9, 12). Clypeus short, wide, appearing to enhance retention of thin water film with two pairs of tufted sensilla (Figs. 9, 11). on surface. Ventral cranium Larva Genae fused ventromedially, anterior margin General (Fig. 51) simple, not serrated. Postgenal bridge with scattered Eucephalic, head capsule partly invaginated papillae; single large tubercle on either side of into prothorax, sclerotization of cranium decreasing midline, three pairs of small tufted sensilla laterally gradually toward posterior margin, mouthparts (Fig. 12). Posterior margin of ventral cranium prognathous. Body cylindrical, white, divided into heavily sclerotized, forming postoccipital carina three thoracic and eight abdominal divisions. Eighth (Fig. 52). division (5 anal division) with elongated respiratory siphon, its length in mature larvae greater than Antenna combined length of head and body (not including Antenna reduced, on anterior margin of gena just the anal papillae). Pair of large anal papillae at base dorsolateral of mandible, comprising flat pad of of siphon, each elongate, branched. General cuticle sensilla within raised cuticular ring (Figs. 3, 9, 16). translucent, fat-body easily visible. Respiratory With eight blunt, peg-like sensilla, three large system amphineustic, with anterior pair of spiracles cushion-like sensilla, two of latter slightly located laterally on prothorax and posterior pair at smaller and bearing small bifurcated apical end of respiratory siphon. sensillum (Fig. 10).

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Figs. 1–8. Scanning electron micrographs of egg and first instar of Axymyia furcata. 1, Egg, dorsal view; 2, egg microsculpture; 3, first-instar head capsule, dorsal view; 4, first-instar head capsule, lateral view; 5, first-instar mouthparts, ventral view; 6, apex of first-instar right maxillary palpus; 7, first-instar habitus, ventral view; 8, first-instar respiratory siphon and anal papillae, ventral view. Abbreviations: ant 5 antenna, mnd 5 mandible, mxl 5 maxillary lacinia, mxp 5 maxillary palpus. Scale bars 5 100 mm(Figs.1,3,4,7,8),50mm(Figs.2,5),5mm (Fig. 6).

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Figs. 9–14. Scanning electron micrographs of fourth instar of Axymyia furcata.9,Headcapsule,dorsalview;10,left antenna; 11, fourth-instar labrum, clypeus, and mandibles, dorsal view; 12, head capsule, oblique-frontal view; 13, mouthparts, oblique-frontal view; 14, labrum and right mandible and maxilla, frontal view. Abbreviations: ant 5 antenna, cly 5 clypeus, fr ap 5 frontal apotome, lbm 5 labium, lbr 5 labrum, mnd 5 mandible, mx 5 maxilla. Scale bars 5 500 mm (Fig. 9), 200 mm (Figs. 11, 13), 100 mm (Fig. 14), 50 mm (Fig. 14), 20 mm(Fig.10).

Mouthparts Anteroventral surface (epipharynx) densely covered Labrum–epipharynx: Labrum somewhat con- with macrotrichia (Figs. 13, 14). Macrotrichia ical, projecting anteriorly from clypeus. Dorsal sur- mostly simple, setiform but those in posterior region face mostly ﬂat, with pair of tufted sensilla (Fig. 11). of epipharynx somewhat serrated.

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Figs. 15–20. Scanning electron micrographs of larval mouthparts of Axymyia furcata. 15, Labrum and right mandible, frontal view; 16, left antenna and mandibular articulation, dorsal view; 17, right mandible, lateral view; 18, right maxilla, ventral view; 19, apex of maxillary palpus; 20, base of labium, showing labial palpi. Abbreviations: ant 5 antenna, cly 5 clypeus, mnd 5 mandible, mxl 5 maxillary lacinia, mxp 5 maxillary palpus. Scale bars 5 100 mm (Figs. 15, 16), 20 mm (Fig. 20), 10 mm (Figs. 17, 18), 1 mm (Fig. 19).

Mandible: Large, heavily sclerotized, with prominent genal lobe, with deep groove between one large, sharp, apical tooth, two smaller, lobed genal margin and posterior margin of clypeus teeth on medial lateral surface (Fig. 11). First (Figs. 9, 16). Anterolateral surface with three instar with subapical teeth reduced but with tufted sensilla (Figs. 5, 11, 13, 15, 17). Adoral small, saw-like ventral lobe abutting maxillary surface with prominent prostheca (Fig. 17). palpus when mandible adducted (Fig. 5). Apical Maxilla: Reduced, comprising ﬂeshy lacinia, teeth overlap entirely when mandibles adducted elongate palpus (Figs. 5, 13, 14). Microtrichia on (Figs. 5, 12, 13). Posterodorsal surface with large medial surface of lacinia curved, serrate, those epicondyle functioning as fulcrum, articulating on near apex straight, smooth. Ventral surface of

᭧ 2012 Entomological Society of Canada Wihlm et al. 279 lacinia with prominent spiniform sensillum near base of respiratory siphon (Figs. 8, 26). Each (Fig. 18). Length of lacinia and palpus subequal papilla a central stem with three rows of approxi- in later instars (Fig. 18); palpus much longer mately six finger-like projections along length; than lacinia in first instar (Fig. 5). Palpus biarticled, projections simple in first instars, moniliform in with basal article (palpiger) greater in length and later instars. Abdominal segment VIII comprised circumference than distal article. Distal end of mostly of elongate, extensile, respiratory siphon. proximal article with single setiform sensillum Siphon circumference nearly constant throughout its arising near base of distal article (Fig. 18). Apex of length, with only gradual decrease from base to palpus concave, bearing two large, 10 smaller sen- apex. Cuticle of anterior portion of siphon similar to silla; arrangement of latter varying slightly between that of abdomen, cuticle of terminal third more first- and fourth instars (Figs. 6, 19). heavily sclerotized, darkly pigmented (Fig. 51). Labium: Small, mostly hidden by maxillae, Distal end of siphon separated from remaining forming conical setose lobe, distal portion cov- siphon by ring-like indentation (Fig. 23), beyond ered in curved serrate macrotrichia, similar in which siphon lacks compact rings of support appearance to apex of lacinia (Fig. 14). Base of tissue present in remaining siphon. Apex of siphon labium with small palpus, just ventral to opening in form of spiracular disc with five prominent of salivary duct (Fig. 20). Palpus short, button- lobes, a variety of setae, hooks of varying sizes like, with four apical sensilla. (Figs. 21, 22). Posterior spiracles elevated to level of Pharyngeal filter: With two, narrow, sclerotized lateral tubercles (Figs. 24, 25). plates extending from near anterior margin of eso- phagus to nearly length of frontoclypeal apotome Pupa (Fig. 52). Plates run subparallel, converging pos- General (Fig. 55) teriorly. Each medial surface of filter irregular, Cylindrical, blunt anteriorly, tapering poster- bearing approximately 10 parallel longitudinal ribs. iorly. Dorsal surface of head, prothorax, anterior Each rib running entire length of plate, bearing mesothorax heavily sclerotized, forming circular continuous row of microtrichia (Figs. 53, 54). disc covered with ornate ridges, numerous spines (Figs. 27, 31, 56). Prothoracic respiratory organs Body prominent, paired (Figs. 27, 28, 56). Diameter of first-instar head, thorax and abdomen subequal; thorax and abdomen of mature Head larva nearly twice diameter of head (Fig. 51). Pro- Dorsal surface of head heavily sclerotized, thoracic spiracles prominent, black, situated just covered with spines, tubercles (Figs. 27, 31–33). above lateral midline of invaginated head. Seven Lateral, ventral surface of head much less scler- abdominal segments present anterior to anal divi- otized, similar in sclerotization to metathorax. sion (segment VIII). Dorsal surface of segments Dorsolateral margin of head with large, semi- I–V each with transverse bands of spines, located circular, blade-like tubercles (Fig. 32). Antennal centrally on each tergite; bands on segments I and V sheath free, heavily sclerotized, extending along with two rows, with anterior row projecting ante- anterodorsal margin of eye. Cuticle medial to riorly, posterior row projecting posteriorly; bands on antennal base with single large sensillum. Postero- segments II–IV have three rows, with anterior two lateral margin of eye with prominent tubercle rows projecting anteriorly, posterior row projecting bearing two hair-like setae. Maxillary palpus posteriorly. Ventral surface of segments I–VI each sexually dimorphic; female palpus long, robust, with transverse bands of spines anteriorly; band on male palpus short, delicate. segment I comprising two rows, both projecting posteriorly; bands on segments II–IV each com- Thorax prising four rows, anterior two rows project ante- Dorsal portion of prothorax, anterodorsal riorly, posterior two rows project posteriorly; bands region of mesothorax heavily sclerotized, dark on segments V and VI each comprising two rows, brown to black in color, covered with irregular, with anterior row projecting anteriorly, posterior ornate microsculpture (Figs. 31, 56). Cuticle on row projecting posteriorly (Fig. 7). Anal papillae metathorax, posterior portion of mesothorax, large, paired, arising from abdominal segment VII wing- and leg sheaths lightly sclerotized, light

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Figs. 21–26. Scanning electron micrographs of larval abdominal features of Axymyia furcata. 21, Distal end of respiratory siphon, lateral view; 22, spines at distal end of respiratory siphon, lateral view; 23, apex of respiratory siphon, showing spiracular disc; 24, spiracular lobe; 25, spiracle; 26, anal papillae and base of respiratory siphon. Scale bars 5 200 mm (Fig. 23), 100 mm (Figs. 21, 26), 10 mm (Figs. 22, 24, 25).

brown to cream colored. Abdominal cuticle organ reveals plastron comprising cuticular pillars mostly membranous, translucent, pale brown to around inner margin (Fig. 30). deep gold in color. Ecdysial line extending along thoracic midline, Anterior respiratory organ large, produced bisecting heavily sclerotized ridge on anterior anteriorly from dorsolateral margin of prothorax, mesothorax (Fig. 31). Single, prominent setiform laterally compressed, curved anteriorly over sensillum posterior to ridge, on either side of lateral margin of head (Figs. 27, 56). Dorsal ecdysial line. surface with serpentine ridge (Fig. 28) lined with Wing- and leg sheaths tightly appressed to series of small, dome-like tubercles and small body. Base of each wing sheath with single pores (Fig. 29). Cross-section of respiratory setiform sensillum.

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Figs. 27–34. Scanning electron micrographs of pupae of Axymyia furcata. 27, Head and anterior part of thorax, dorsal view; 28, right thoracic respiratory organ, dorsal view; 29, microsculpture on thoracic respiratory organ, dorsal view; 30, cross-section of thoracic respiratory organ showing plastron; 31, microsculpture on dorsum of thorax, anterodorsal view; 32, microsculpture on left side of head, anterodorsal view; 33, microsculpture on left side of head; 34, abdominal segments V–VII, ventral view. Abbreviations: ant sh 5 antennal sheath, mes 5 mesonotum, pro 5 pronotum, ro 5 respiratory organ, sV–VII 5 sternites V–VII. Scale bars 5 0.5 mm (Fig. 27), 200 mm (Figs. 31, 34), 100 mm (Figs. 28, 32), 50 mm (Figs. 29, 30, 33).

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Figs. 35–42. Scanning electron micrographs of adults of Axymyia furcata. 35, Male head, oblique-frontal view; 36, female head, oblique-frontal view; 37, male eye at junction of upper and lower divisions; 38, female eye at junction of upper and lower divisions; 39, male ocelli, oblique-frontal view; 40, male mouthparts and tentorial pits, frontal view; 41, male antenna; 42, prescutal pits (female), anterior 5 top. Abbreviations: ant 5 antenna, atp 5 anterior tentorial pit, cly 5 clypeus, lbr 5 labrum, plp 5 palpus, pp 5 prescutal pit. Scale bars 5 0.5 mm (Fig. 35, 36), 200 mm (Figs. 40, 42), 100 mm (Figs. 37–39), 50 mm (Fig. 41).

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Figs. 43–50. Scanning electron micrographs of adults of Axymyia furcata. 43, Female tarsus, dorsal view; 44, female pretarsus, showing claws, empodium and pulvilli; 45, male terminalia, posterior view; 46, male terminalia, lateral view; 47, female terminalia, dorsolateral view; 48, female terminalia, lateral view; 49, female cerci, oblique-posterior view; 50, sensilla on female cercus. Abbreviations: aed 5 aedeagus, cer 5 cercus, emp 5 empodium, gst 5 gonostylus, hyl 5 hypandrial lobe, par 5 paramere, pul 5 pulvillus, sVIII 5 sternite VIII, t2–t5 5 tarsomeres 2–5. Scale bars 5 500 mm (Figs. 45–47), 200 mm (Figs. 43, 48, 49), 50 mm (Figs. 44, 50).

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Figs. 51–58. Light micrographs of larvae, pupae, and adults of Axymyia furcata. 51, fourth instar (inset 5 apex of respiratory siphon, in situ); 52, larval head capsule, showing pharyngeal filter; 53, larval pharyngeal filter; 54, details of larval pharyngeal filter; 55, pupa; 56, pupal head and thorax, in situ; 57, adult female; 58, adult male.

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Figs. 59–61. Thorax and wing of adult Axymyia furcata. 59, Thorax, dorsal view; 60, thorax, lateral view; 61, wing. Abbreviations: abd I 5 abdominal segment I, anepm 5 anepimeron, anepst 5 anepisternum, aprn 5 antepronotum; a spr 5 anterior spiracle, bas 5 basalare, cerv scl 5 cervical sclerite, cx 1–cx 3 5 coxa 1–3; epm 5 epimeron, hlt 5 haltere, kepm 5 katepimeron, kepst 5 katepisternum, ltg 5 laterotergite, mtg 5 mediotergite, mr 5 meron, mtanepst 5 metanepisternum, mtepm 5 metepimeron, mtkepst 5 metakatepisternum, presct pit 5 prescutal pit, presct sut 5 prescutal suture, pprn lb 5 postpronotal lobe, presct 5 prescutum, p spr 5 posterior spiracle, sctl 5 scutellum, trn sut 5 transverse suture.

Abdomen curving posteriorly. Anal division with promi- Cuticle translucent, membranous. Abdominal nent, ﬂeshy, dorsal lobe, presumably a remnant segments II–VII each with pair of dark brown to of larval anal siphon. Apex of abdomen sexually black spiracles near anterolateral margin. Small dimorphic; male with distinct sheaths of hypan- group of spines and single setiform sensillum drial lobes, female with distinct sheaths of large posteroventral to each spiracle, along lateral pad-like cerci. midline. Tergites III, V, VI each with single posterior row of delicate, posteriorly curved Adult spines, tergite IV with thick band of coarse, General (Figs. 57, 58) anteriorly curved spines. Sternites III and IV Mid-sized, heavy bodied, similar in appearance to each with single row of delicate spines, sternites Bibionidae, with prominent eyes and bulging thorax. V–VII each with row of delicate spines and band Color ranging from light mahogany brown to black. of coarse spines (Fig. 34). All ventral spines Cuticle covered in dense minute microtrichia.

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Sexual dimorphism apparent in structure of eyes Postpronotal lobe separated from posterior mar- and terminalia. gin of prescutum and anterior margin of scutum and delineated by inverted Y-shaped suture; Head stem of suture (5 prescutal suture) terminates at Cuticle: Large smooth tubercle at very pos- large dark spot or scar (prescutal pit) on anterior teroventral margin of eye. Posterodorsal margin scutum (Fig. 42). Prescutum and scutum swollen, lined with long hair-like setae. Similar setae giving hunchback appearance. Transverse suture between antennal bases. Anteriorly with two faint, slightly invaginated, arising laterally from large deep tentorial pits, each between antenna wing base, extending toward but terminating and maxillary palpus (Figs. 35, 36). before midline of scutum (Fig. 59). Scutellum Eyes: Large, bulging, without ommotrichia. narrowed transversely, strongly domed. Scutum Male eyes holoptic, contiguous dorsally (Fig. 35). and scutellum covered with long, fine, widely Female eyes smaller, dichoptic (Fig. 36). In both spaced setulae. Mediotergite nearly twice the sexes divided into upper and lower hemispheres by length of scutellum, lacking setulae. shallow groove and scattered row of small, delicate Episternum large, with distinct division between setae (Fig. 37). In male, upper hemisphere much anepisternum and katepisternum. Anepisternal cleft larger than lower hemisphere; arrangement oppo- dividing anepisternum into large anterior portion, site in females. In male, ommatidial facets of upper much narrower posterior portion. Basalare promi- hemisphere larger, lighter in color than facets of nent, dorsal to narrow portion of anepisternum. lower hemisphere (Fig. 37). In females, facets of Anepimeron broad, narrowing ventrally; katepi- upper and lower hemispheres uniform in both size meron subquadrate, clearly separated from meron. and color (Fig. 38). Meron large, subcircular, about half the size of Ocelli: Large, located on prominent, dome- katepisternum. Metepisternum and metepimeron like tubercle on posterodorsal surface of head reduced, represented by elongate bands below (Figs. 35, 36). Tubercle with narrower base, haltere (Fig. 60). Prescutum and lateral margin of more prominently raised in male than female, scutum (anterior to transverse suture) set with extending above dorsal margin of eyes (Fig. 39). numerous elongate, thin hairs; scutum dorsally with Antenna: Moniliform, comprising pedicel, sparse short hairs. Scutellum laterally with patches scape, and 14 flagellomeres. Pedicel and scape of short hairs, all oriented medially. Katepisternum similar in structure to flagellomeres, paler in and anterior part of anepisternum with few short color. Female antenna (Fig. 36) longer than, hairs. Nearly all thoracic sclerites set with dense nearly twice as robust as that of male (Fig. 35). microtrichia. All flagellomeres setaceous, each with additional crown of long sensilla (Fig. 41). Legs Mouthparts: Clypeus prominent, sclerotized, Legs somewhat short, subequal in overall triangular in shape. Labrum and labella greatly length, without spurs, with microtrichia and reduced, primarily membranous (Fig. 40). Apex scattered setiform sensilla (Fig. 43). Length of of labellum with small, sclerotized, circular pro- mid- and hind coxae approximately half-length cess covered in dark setae. Maxillary palpus short, of forecoxa. Pretarsus with prominent claws, delicate in males (Figs. 35, 40), longer, robust in empodium and pulvillus. Empodium spatulate, females (Fig. 36). Most palpal segments with about twice the size of pulvillus; ventral surface several prominent setiform sensilla. Maxillary of empodium and pulvillus densely set with palpus with five segments in most individuals, but long, spatulate macrotrichia (Fig. 44). with four to six segments in some individuals. Wings and halteres Thorax Wings large, broad, about equal in length to Cervical sclerite prominent but partially hidden the entire body; pale brown to slate gray, with by head; triangular in shape, with sharply acute distinct, but light, pterostigma. Membrane covered angle pointed inward toward posteromedial margin in fine microtrichia. Radial vein (R) with four of head. Antepronotum reduced medially but branches; R2 short, nearly perpendicular to R3;R3, increasing in width anteroventral of forecoxa. R4 1 5 long and subparallel. Medial vein (M) with

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Figs. 62–65. Terminalia of adult Axymyia furcata. 62, Male, dorsal view; 63, male, lateral view; 64, female, ventral view; 65, female, lateral view. Abbreviations: aed 5 aedeagus, cer 5 cercus, co 5 common oviduct, gen fk 5 genital fork, gnx ap 5 gonocoxal apodeme, gst 5 gonostylus, hyl 5 hypandrial lobe, par 5 paramere, spmth 5 spermatheca.

two branches, stem quite faint, branches more Abdomen prominent. Radial-medial cross vein (r-m) oblique. Abdomen large, especially in females (Fig. 57). Posterior cubital branch (CuP) present, but faint, Tergites and sternites mostly dark anteriorly, pale not reaching wing margin. Posterior branch of anal brown to cream-colored along posterior margin. vein (A1) weakly present, not reaching wing mar- Pleural region cream-colored to white, membranous. gin (Fig. 61). Alula absent. Halter prominent, with Anterolateral margins of segments I–VII each long stem; covered with ﬁne microtrichia; middle with spiracles in membranous pleural region. section of stem with cluster of long, stout setae. Segments I–VII with sparse short hairs. Tergite

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VIII small, partially hidden by tergite VII, with rotation, fused postgenae, simple postmentum) membranous posterior cleft. are either more generalized or widespread across many larval nematocerans. Still other characters Male terminalia are clearly autapomorphic and presumably rela- Sternite VIII small. Sternite IX large, fused ted at least partly to their xylophilic lifestyle. For with gonocoxites to form prominent hypandrium. example, the complex structure of respiratory Hypandrial lobes large, paired, hiding small siphon apex appears to function as a grapple, setaceous gonostyli in lateral view (Figs. 45, 46). securing the siphon to the gallery opening Cercus small, obscure, differentiated from proctiger (Fig. 51 (inset)). Other features (e.g., elongate primarily by texture. Aedeagus tube-like (Fig. 46, siphon with elastic qualities, structure of anal 62, 63). Parameres broadly flattened laterally, fused papillae) may be adaptations to periodical medially, flared at apex (Fig. 45). See Wood (1981, inundation by water and life in saturated wood. 1991) for further detailed illustrations. Details of A study of pupal structure and homology internal structure are presented by Borkent and across all nematocerous flies is desperately Sinclair (2012) in this volume. needed. Detailed studies of pupal structure in other groups (e.g., Arens 1995, 1997; Borkent 2010) have demonstrated that pupae can provide Female terminalia a rich source of phylogenetic information. Sternite VIII large, curved strongly upward Although we expect many features of axymyiid laterally nearly reaching dorsal surface. Sternite VIII with deep posterior furrow, covered in micro- pupae will be unique, again due to xylophyly, we hope that other characters may provide a useful trichia and numerous setiform sensilla (Figs. 47, 48). phylogenetic signal. Cercus large, pad-like, two-segmented; proximal Adult axymyiids have been the subject of segment with abundant setiform sensilla dorsally; several phylogenetic studies, usually as part of distal segment with numerous cuticular projections, a broader investigation of many nematoceran each with elongate macrotrichia basally and large, families (e.g., Wood and Borkent 1989; Ooster- rod-like sensillum apically (Figs. 49, 50). Genital broek and Courtney 1995; Michelsen 1996). fork Y-shaped in ventral view (Fig. 64), sinuate in lateral view (Fig. 65). Common oviduct unscler- Other studies have focused on particular character systems (e.g., Borkent and Sinclair 2012). For otized, indistinct posteriorly. Accessory glands not the most part, those studies have demonstrated apparent. Reproductive tract simple Spermathecae that axymyiids are fairly generalized in structure spherical, sclerotized, two in number; spermathecal (e.g., terminalia), or possess unique features ducts sclerotized distally, inflated and membranous (i.e., autapomorphies) that provide no evidence of basally (Figs. 64, 65). phylogenetic relationship. Discussion Acknowledgements Axymyiidae is an enigmatic family of semi- aquatic nematocerous flies whose phylogenetic The museums listed in materials and methods placement has remained problematic. Although generously loaned preserved specimens of Axymyia we have not attempted to resolve the phylo- furcata for this morphological study. We are also genetic relationships of this unusual family, we grateful to Art Borkent and Brad Sinclair for careful hope that some of the morphological data pre- review of an earlier draft of this paper. This project sented here may be useful to future investigations was supported in part by National Science Foun- of evolutionary relationships. Our data on first dation grants (EF-0334948 and DEB-0933218) to instars represents the first SEM study of this life Gregory W. Courtney and by funds from the Dean stage in Axymyiidae and uncovers some unex- of Agriculture and Life Sciences International pected results (e.g.,lackofeggburster).Other Research Grant and Biosystematics Travel Fund to larval characters suggest affinities with certain Matthew W. Wihlm. This journal paper of the Iowa bibionomorph and psychodomorph families (e.g., Agriculture and Home Economics Experiment larval antenna reduced); however, many other Station, Project no. 6693, was supported by Hatch features (e.g., conical labrum, horizontal mandibular Act and State of Iowa funds.

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