Dtsch. Entomol. Z. 67 (1) 2020, 51-67 | DOI 10.3897/dez.67.49493 Gy tuseue ror BERLIN A comparative description of the mesosomal musculature in Sphecidae and Ampulicidae (Hymenoptera, Apoidea) using 3D techniques Maraike Willsch!, Frank Friedrich?, Daniel Baum®, Ivo Jurisch!, Michael Ohl! 1 Museum fiir Naturkunde Berlin, InvalidenstraBe 43, 10115 Berlin, Germany 2 Institut fiir Zoologie, Universitat Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany 3 Zuse Institute Berlin, Takustrabe 7, 14195 Berlin, Germany http://zoobank. org/94793352-7C43-496C-83D1-A 10A355BC801 Corresponding author: Maraike Willsch (maraike.willsch@mfn.berlin) Academic editor: D. Zimmermann @ Received 17 December 2019 Accepted | April 2020 Published 11 May 2020 Abstract Conflicting hypotheses about the relationships among the major lineages of aculeate Hymenoptera clearly show the necessity of detailed comparative morphological studies. Using micro-computed tomography and 3D reconstructions, the skeletal musculature of the meso- and metathorax and the first and second abdominal segment in Apoidea are described. Females of Sceliphron destil- latorium, Sphex (Fernaldina) lucae (both Sphecidae), and Ampulex compressa (Ampulicidae) were examined. The morphological terminology provided by the Hymenoptera Anatomy Ontology is used. Up to 42 muscles were found. The three species differ in certain numerical and structural aspects. Ampulicidae differs significantly from Sphecidae in the metathorax and the anterior abdo- men. The metapleural apodeme and paracoxal ridge are weakly developed in Ampulicidae, which affect some muscular structures. Furthermore, the muscles that insert on the coxae and trochanters are broader and longer in Ampulicidae. A conspicuous character- istic of Sphecidae is the absence of the metaphragma. Overall, we identified four hitherto unrecognized muscles. Our work suggests additional investigations on structures discussed in this paper. Key Words Aculeata, anatomy, microCT, phylogeny, propodeum, thorax Introduction Hymenoptera form one of the largest insect orders and comprise more than 150,000 extant species (Aguiar et al. 2013). The group of interest examined in this paper con- stitutes a subclade of Hymenoptera, the Aculeata (sting- ing wasps, bees, and ants; Sharkey et al. 2012). Derived from the modified ovipositor, the stinger is a synapomor- phy of aculeate Hymenoptera and a key innovation for their evolutionary success (Sharkey et al. 2012; Schmidt 2016). The nature of phylogenetic relationships within the monophyletic Aculeata is still contested (e.g., Konig- smann 1978; Lomholdt 1982; Rasnitsyn 1988; Alexander 1992; Brothers and Carpenter 1993; Ronquist et al. 1999; Peters et al. 2011, 2017; Sharkey et al. 2012; Johnson et al. 2013; Branstetter et al. 2017). Traditionally, Aculeata is divided into three lineages: Chrysidoidea, Vespoidea, and Apoidea (O’ Neill 2001; Branstetter et al. 2017). About 10,000 species of digger wasps (also named apoid wasps) as part of the species-rich superfamily Apoidea are currently known (Pulawski 2020). The most obvious synapomorphy of Apoidea is the rounded pro- notal lobe (Ohl and Engel 2007). Apoidea is divided into the monophyletic Anthophila (bees) and the paraphyletic apoid wasps. The latter comprises Ampulicidae, Crabro- nidae, Heterogynaidae, and Sphecidae (e.g., Branstetter et al. 2017). Recent phylogenomic and molecular anal- yses suggest Ampulicidae is the sister to the rest of the Apoidea (Debevec et al. 2012 [ribosomal 28S and pro- tein-coding nuclear genes]; Sann et al. 2018 [target DNA enrichment and transcriptomic sequence data]). How- ever, contradictory evidence on the phylogenetic rela- Copyright Maraike Willsch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. BZ tionships within the apoid wasps (e.g., Lohrmann et al. 2008; Ohl and Spahn 2010; Debevec et al. 2012; Sharkey et al. 2012; Branstetter et al. 2017) remains unresolved. Based upon different research methods, most results sug- gest, that Sphecidae and Ampulicidae are well-supported clades (Ohl and Spahn 2010 [morphological study]; Bran- stetter et al. 2017 [ultraconserved element phylogenom- ics]; Peters et al. 2017 [protein-coding genes]), whereas Crabronidae are likely to be paraphyletic (Lohrmann et al. 2008 [nuclear long-wavelength-opsin and mitochon- drial cytochrome-c-oxidase]|; Debevec et al. 2012; Bran- stetter et al. 2017; Peters et al. 2017). However, Sann et al. (2018) found Crabronidae to be polyphyletic. Another unresolved issue is the position of Heterogynaidae within Apoidea (Ohl and Bleidorn 2006). Debevec et al. (2012) obtained two different results: Heterogynaidae nested within Crabronidae (maximum likelihood tree) and as sister to a monophyletic group of Sphecidae sensu stric- to, Crabronidae and Anthophila (Bayesian tree). The first result was already proposed by Ohl and Bleidorn (2006 [long-wavelength opsin]). Branstetter et al. (2017) found Heterogynaidae to be sister to a grouping of paraphyletic Crabroninae and Sphecidae. Morphological characters are still one of the major sources of phylogenetic inference (e.g., Friedrich and Beutel 2010; Ohl and Spahn 2010; Vilhelmsen et al. 2010; Zimmermann and Vilhelmsen 2016; Liu et al. 2019). Nev- ertheless, internal mesosomal structures are insufficiently studied across Hymenoptera, as predicated by Vilhelmsen et al. (2010), who provided detailed information for many apocritan wasps and other Hymenoptera; especially the mesosomal musculature of Pison chilense (Crabronidae) and external mesosomal characters for Pison chilense, Stangeella cyaniventris (Sphecidae), and Ampulex com- pressa (Ampulicidae) are described. They demonstrated, that the mesosomal region reveals considerable informa- tion for phylogenetic research. Previously, indispensable work about the mesosomal musculature in Hymenoptera was presented by Maki (1938), Snodgrass (1942; in par- ticular, for Apis), Heraty (1989), and Matsuda (1970), followed by Prentice (1998). Recent substantial work was accomplished by Miko et al. (2007). They dissected the musculature of the head and mesosoma in a review of the parasitic wasp family Scelionidae. Furthermore, a reinterpretation of the delimitation of the metapostnotum in Chrysidoidea was presented by Kawada et al. (2015). Moreover, Porto et al. (2016) defined internal mesosomal characters of bees and evaluated the potential of these structures, concluding that they are of great value to phy- logenetic investigations. Garcia et al. (2017) described several body parts of three new species of the rare ant ge- nus Zasphinctus, resulting in a comparative character ma- trix for species-level taxonomy. Subsequently, Liu et al. (2019) provided insights on the mesosoma of an ant work- er of Myrmecia for comparisons with other Aculeata and to gain new information about evolution and body function. A state-of-the-art method for morphological analyses is the three-dimensional imaging, using micro-computed tomography (microCT). It is a highly powerful technique dez.pensoft.net Maraike Willsch et al.: The mesosomal musculature in Apoidea (Faulwetter et al. 2013 and references therein; Garcia et al. 2017; Liu et al. 2019), as it makes internal structures visi- ble without destroying the specimen. Moreover, the digital 3D models can be created repeatedly to work on different goals and the data can easily be shared worldwide. By using 3D imaging, we aim to expand the basic mor- phological knowledge for phylogenetic investigations within Aculeata. In this paper we present data of muscular structures in the mesosoma of Sceliphron destillatorium (Illiger, 1807), Sphex (Fernaldina) lucae de Saussure, 1867 (both Sphecidae), and Ampulex compressa (Fab- ricius, 1781) (Ampulicidae) (Fig. 1). These wasps are solitary and nest-provisioning predators with different lifestyles (e.g., Williams 1942; Bohart and Menke 1976; Fouad et al. 1994; Haspel and Libersat 2003; Libersat 2003; Ohl and Spahn 2010). Both families were selected for their large number of plesiomorphic characters within digger wasps (Ohl and Spahn 2010), which might help to reconstruct the ancestral apoid anatomy. Primarily, we illustrate and describe mesosomal conformations of the skeletal musculature, with focus on the mesothorax, metathorax, and the first abdominal segment (propode- um). We also describe muscles that originate in the meso- soma and insert in the second abdominal segment (meta- soma) because of strong interrelations of these muscles in this transition zone between both tagmata. The wasp waist allows for increased movability of the abdomen and, therefore, is an important anatomical cluster for various physical activities requiring precise movements of the ab- domen below the body. This includes, for instance, sting- ing prey or enemies for defence, laying eggs (Williams 1942; Bohart and Menke 1976), carrying prey between mid or hind legs and abdomen while in flight, dragging prey forwards or backwards (Bohart and Menke 1976), and increasing balance in flight (at least when the second abdominal segment is petiolate; Bohart and Menke 1976). Material and methods Specimens and body parts examined Sphex and Ampulex were taken from the collection of the Museum fiir Naturkunde Berlin (MfN) and Sceliphron was collected in the field (Table 1). To examine and compare the muscle sets, specimens of the same sex (females) were selected. We analysed the musculature of the mesothorax, metathorax, and the first and second abdominal segments. Preparation, microCT, and 3D reconstruction The extremities of the specimens were removed to min- imize the scan field for optimizing the resolution of the data sets. Furthermore, the tip of the gaster was removed to facilitate the infiltration of the iodine, which intensifies the visibility of the musculature in the scan. Following Metscher (2009) and Gignac et al. (2016), our specimens were contrasted in a 25% iodine solution in pure ethanol Dtsch. Entomol. Z. 67 (1) 2020, 51-67 53 Figure 1. Portraits of the three specimens examined, lateral view. A. Sceliphron destillatorium, body size 20 mm; B. Sphex (Fernal- dina) lucae, body size 18 mm; C. Ampulex compressa, body size 21 mm. Table 1. Basic information about the specimen collection, classification, preparation, and settings for microCT scanning. Specimens MEN collection number Location/label data Sceliphron destillatorium MfN_Hym_Sph_1004239 GREECE, Crete, Afrata [little road], 35°34'38.31"N, 23°4'2.3"E Sphex (Fernaldina) lucae MfN_Hym_Sph_1000635 USA, New Mexico, Hidalgo Co., Gray Ranch, 20.6 mi S Ammas Ampulex compressa MfN_Hym_Amp_ 1000029 GERMANY, Berlin, MfN breed, Oviposition 6 Aug. 2015, Eclosion 27 Sept. 2015 Date of collection 24 June 2015 28 Aug. 2003 28 Sept. 2015 Leg. M. Willsch S. Schiller, I. Richert L. Kirschey Det. M. Willsch 2015 M. Ohl 2004 L. Kirschey 2015 Family Sphecidae Sphecidae Ampulicidae Sex Female Female Female Body size (mm) 20 18 21 Storage 96% ethanol 96% ethanol 96% ethanol Sample preparation 25% Iodine staining, critical point 25% Iodine staining, critical point 25% Iodine staining, critical point drying drying drying Scanning medium Air Air Air Voltage (kV) 48 48 50 Current (pA) 250 250 Oe Number of images 1000 1000 1440 Rotation steps 0.36 0.36 0.25 Exposure time (ms) 1000 1000 1000 Resolution (um/pixel) 3.40 4.69 5.00 (100%) for three days and washed out with pure etha- nol for 30 seconds. The wasps were dried using a critical point dryer (Leica EM CPD300; Table 1). Afterwards, the three specimens were scanned at the Visualisation Laboratory of the MfN using a Phoenix nanotom X-ray|s tube (General Electric) at 48—50 kV and 250-275 uA. At 1 second per image 1000-1440 projections were gener- ated per scan. The different kV- and projection-settings depended on the respective specimen size, which was also responsible for the range of the effective voxel size between 3.4—5 um (Table 1). The cone beam reconstruc- tion was performed using the CT reconstruction software PHOENIX|X-RAY DATOS|X version 2.0 (GE Sensing & Inspection Technologies GmbH). 3D segmentation and post-processing The raw microCT image data were visualised and an- alysed by using a Wacom Cintiq 22HD interactive pen display and the software AMIRA ZIB EDITION 2020.02 and former versions (provided by the Zuse Institute Ber- lin). All muscles were segmented and labelled manual- ly by using appropriate segmentation tools in AMIRA. Segmented materials were transformed into high-resolu- tion surfaces using the Isosurface-Tool in AMIRA. The reconstruction was accomplished for one body side of the specimens, as no structural asymmetries were observed in this region. Therefore, the number of muscles given in the results refers to one-half of the body. For post-editing (e.g., picture artefacts, file size reduction, file converting, figure compilation) we exported TIF-files from AMIRA into ADOBE PHOTOSHOP CS6. Terminology Skeletal musculature was categorised based on insertion sites. The muscle terminology of the Hymenoptera Anato- my Ontology (HAO; http://portal.hymao.org/projects/32/ public/ontology/) (Miko et al. 2007; Vilhelmsen et al. 2010; Yoder et al. 2010; Seltmann et al. 2012) has been adopted here. In this connection, we provide a list of Uni- versal Resource Identifiers (URI) for each muscular and cuticular term (Suppl. material 1: Table S1). It was created by using the “analyze” tool on the HAO website. Newly dez.pensoft.net 54 detected muscles, not listed in the HAO so far or found in other literature, were also named in the HAO-scheme by the areas of origin and insertion with additional topo- graphical orientation, 1f required (Table 2). The abbre- viations used for the designation of muscles and sclerite structures are composed of the basic terms as follows: Region of origin and insertion: 3ax2___ third axillary sclerite of fore wing 3ax3 third axillary sclerite of hind wing ba basalare cx coxa fu furca ism intersegmental membrane occ occlusor pe pectus ph phragma pl pleuron S sternum S thoracal sternum sa subalare sp spiracle Tl first abdominal tergite/propodeum T2 second abdominal tergite tr trochanter Divided thorax: 1 located on the prothorax 2 located on the mesothorax 3 located on the metathorax Positions: a anterior d dorsal | lateral m medial p posterior Vv ventral Order; mostly stated for functional groups of muscles: aorl first bor2 second cor3_ third Descriptions, that involve the meso- and metafurca, are based on the terminology of Porto et al. (2016). The descriptions in the results were ordered by the point of insertion from mesosoma towards metasoma and by rele- vant functional groups, if possible (Table 2). In this com- parative work, Sceliphron destillatorium serves as refer- ence species (Fig. 2). In addition, a homologisation with the generalised nomenclature for the thoracic muscula- ture of Neoptera following Friedrich and Beutel (2008) is presented in Table 2. dez.pensoft.net Maraike Willsch et al.: The mesosomal musculature in Apoidea Data availability The large image data sets accomplished for this study are available online as a data publication in conjunction with this paper. Thus, our images and raw data are freely acces- sible via the MfN data repository (Willsch 2019; https:// doi.org/10.7479/dft0-yy6m). Moreover, images will be available on the HAO portal (http://portal.hymao.org). Results We found 42 muscle pairs within the analysed tagmata of the three species (Table 2). There are 37 muscles in Sceliphron (mesothorax 18, metathorax 14, first and sec- ond abdominal segments 5), 39 in Ampulex (mesothorax 19, metathorax 16, first and second abdominal segments 4), and 40 muscles in Sphex (mesothorax 20, metathorax 15, first and second abdominal segments 5). The follow- ing description of the skeletal musculature in Sceliphron serves as structural basis. Subsequently, comparative de- scriptions of differing muscles in Sphex and Ampulex are given. Each muscle absent in one or two of the compared species examined is mentioned below (see also Table 2): Sceliphron destillatorium (Mlliger, 1807) Mesothorax. Ventral mesofurco-profurcal muscle (fu2-fulv; Fig. 3A) arises ventromedially from the me- sofurcal bridge, then runs horizontal and inserts ventrally on the base of the profurca. First mesopleuro-mesono- tal muscle (pl2-t2a; Fig. 3B) arises from the mesopec- tus and inserts on the mesoscutum. The muscle expands vertically and is the second largest muscle in the meso- thorax. Mesopleuro-mesobasalar muscle (pl2-ba2; Fig. 3C) arises anteroventrally from the mesopleuron, fuses with ism1,2-ba2, and inserts on the mesobasalare anterior to the pleural wing articulation. Anterior tho- racic spiracle occlusor muscle (sploce; Fig. 3C) arises proximally of the intersegmental membrane anteromedi- al to ism1,2-ba2, runs obliquely, and inserts posteriorly on the anterior thoracic spiracle. Externally, the spiracle is covered by the pronotal lobe. Intersegmental mem- brane-mesobasalar muscle (ism1,2-ba2; Fig. 3C) aris- es from both the intersegmental membrane between the pronotum and mesopectus, and from the mesopleuron, and inserts on the mesobasalare after fusing with pl2-ba2. First mesopleuro-third axillary sclerite of fore wing muscle (pl2-3ax2a; Fig. 3D) arises anterodorsally from the mesopleuron and inserts on the third axillary scler- ite of the fore wing; it is short and fan-shaped. Second mesopleuro-third axillary sclerite of fore wing mus- cle (pl2-3ax2b; Fig. 3D) arises anterolaterally from the mesopleuron. This vertical, fan-shaped muscle is situated ventral to pl2-3ax2a and inserts on the third axillary scler- ite of the fore wing. Third mesopleuro-third axillary sclerite of fore wing muscle (pl2-3ax2c; Fig. 3D) arises Dtsch. Entomol. Z. 67 (1) 2020, 51-67 55 mesopleural suture Figure 2. Volume rendering of the mesosomal exoskeleton of Sceliphron destillatorium, anterior to the left. A. Dorsal surface view; B. Lateral surface view; C. Ventral surface view. Abbreviations: N11 — pronotal lobe, N3 — metanotum, ex1 — procoxa, cx2 — me- socoxa, ¢x3 — metacoxa, pl2 — mesopleuron, pl3 — metapleuron, tr2 — mesotrochanter, $1 — prosternum, S2 — mesosternum, $3 — metasternum, scl2 — mesoscutellum, T1 — propodeum, tg — tegula. Scale bars: 0.9 mm (A, B), 1 mm (C). fu3m-cx3 'U2-ex2 r2 ~ pi2-34x2b.. fu2m-tr2 wee pl2-t2b P pl2-3ax2c pl2-cx2 Figure 3. Sceliphron destillatorium, volume rendering, mesosomal musculature, A—C: medial view, anterior to the right, D: lateral view, anterior to the left. A. Muscles discernible from the centre; B. Muscles positioned sublateral; C. Muscles located sublateral and lateral; D. Laterally positioned muscles. Abbreviations: fu2-fulv — ventral mesofurco-profurcal; pl2-t2a — first mesopleuro-mesonotal; pl2-ba2 — mesopleuro-mesobasalar; sploce — anterior thoracic spiracle occlusor; ism1,2-ba2 — interseg- mental membrane-mesobasalar; pl2-3ax2a — first mesopleuro-third axillary sclerite of fore wing; pl2-3ax2b — second mesopleu- ro-third axillary sclerite of fore wing; pl2-3ax2c — third mesopleuro-third axillary sclerite of fore wing; pl2-t2b — second meso- pleuro-mesonotal; ex2-sa2 — mesocoxo-mesosubalar; fu2a-ph2 — anterior mesofurco-mesolaterophragmal; pl2a-fu2 — anterior mesopleuro-mesofurcal; pl2-cx2 — mesopleuro-mesocoxal; s2-cx2 — mesosterno-mesocoxal; fu2-cx2 — mesofurco-mesocoxal; fu2l-tr2 — lateral mesofurco-mesotrochanteral; fu2m-tr2 — median mesofurco-mesotrochanteral; ph1-ph2 — prophragmo-me- sophragmal; pl3a-ba3 — anterior metapleuro-metabasalar;, t2p-t3 — posterior mesonoto-metanotal; pl3la-t3 — anterolateral meta- pleuro-metanotal; pl3d-3ax3 — dorsal metapleuro-third axillary sclerite of hind wing; pl3-sa3 — metapleuro-metasubalar; cx3-sa3 — metacoxo-metasubalar; pl3m-cx3 — median metapleuro-metacoxal; fu3l-cx3 — lateral metafurco-metacoxal; fu3m-cx3 — median metafurco-metacoxal; pI3I-cx3 — lateral metapleuro-metacoxal; fu3-tr3 — metafurco-metatrochanteral; pl3-tr3 — metapleuro-me- tatrochanteral; ph2m-ph3 — median mesophragmo-metaphragmal; ph3-T2 — metaphragmo-second abdominal tergal; T1-S2 — propodeo-second abdominal sternal; fu3-S2 — metafurco-second abdominal sternal; s3-S2 — metasterno-second abdominal sternal. Scale bars: 0.8 mm (A-C), 0.9 mm (D). dez.pensoft.net 56 Maraike Willsch et al.: The mesosomal musculature in Apoidea Table 2. Terminology of the thoracic and abdominal musculature of all specimens examined. Origination and insertion are described on the basis of Sceliphron. If a muscle is absent in Sceliphron, the description refers to Sphex or Ampulex, respectively, if absent in Sphex. The list is sorted caudad (from thorax towards abdomen) by insertions of the muscles and by assumed functional groups. X = muscle present; - = muscle absent; ? = uncertain homology. A homologisation with the generalised nomenclature for neopteran thoracic muscles of Friedrich and Beutel (2008) is presented. Abbreviation Name of muscle Origin Insertion Sceliphron Sphex | Ampulex Neoptera destillatorium compressa| terminology Mesothorax fu2-fulv ventral mesofurco-profurcal | mesofurcal bridge profurca x Ivlm7 pl2-t2a first mesopleuro-mesonotal mesopectus mesoscutum X Ildvm1 pl2-ba2 mesopleuro-mesobasalar mesopleuron mesobasalare x IIspm1 splocc anterior thoracic spiracle intersegmental anterior thoracic x x x - occlusor membrane spiracle | ee ee ism1,2-ba2 intersegmental membrane- intersegmental mesobasalare x x x IIppm2 mesobasalar membrane, pl2-3ax2a first mesopleuro-third axillary mesopleuron | third axillary sclerite x x x IItpm7 sclerite of fore wing of fore wing LL | pl2-3ax2b second mesopleuro-third mesopleuron __ | third axillary sclerite x x x IItpm9 axillary sclerite of fore wing of fore wing Es pl2-3ax2c third mesopleuro-third axillary mesopleuron __ | third axillary sclerite x x x IItpm9 sclerite of fore wing of fore wing Pe pl2-t2b second mesopleuro-mesonotal mesopleuron _ | lateral axillar area of x x x IItpm5 cx2-sa2 mesocoxo-mesosubalar mesocoxa mesosubalare xX Iidvm6 fu2a-ph2 anterior mesofurco- mesofurcal arm | mesolaterophragma x x x Iidvm8 mesolaterophragmal cee] sp3o0cc posterior thoracic spiracle mesofurcal arm posterior thoracic - xX x - occlusor spiracle ae pl2a-fu2 anterior mesopleuro- mesopleuron, mesofurcal arm x x x IIspm2 mesofurcal mesepimeral ridge Le ie | pl2-cx2 mesopleuro-mesocoxal mesopleuron mesocoxa x x x IIpcm4 (anterolateral) [=a is | pl2-cx2b* second mesopleuro-mesocoxal | mesopleuron, mesocoxa - xX x? IIpcm4? mesopleural (dorsolateral) spiracle apodeme el |nall $2-cx2 mesosterno-mesocoxal mesodiscrimenal mesocoxa x x xX IIscm3 lamella, (anterolateral) fu2-cx2 mesofurco-mesocoxal mesodiscrimenal mesocoxa x x x IIscm2 lamella (anteromedial) lm RE fu2l-tr2 lateral mesofurco- mesopleuron, mesotrochanteral x x - IIscm6 mesotrochanteral mesofurcal arm apodeme (lateral) fu2m-tr2 median mesofurco- mesofurcal arm mesotrochanteral x x x? IIscm6 mesotrochanteral apodeme (lateral) | phl-ph2 prophragmo-mesophragmal prophragma mesophragma x IIdlm1 Number of mesothoracic muscles (max. 20): 18 20 19 Metathorax pl3a-ba3 anterior metapleuro- metapleuron, metabasalare x x x IIspm1 metabasalar paracoxal ridge [ & iRel t2p-t3 posterior mesonoto-metanotal | mesoscutellum |mesophragmal spine x x x? IIdlm3 pl3la-t3 anterolateral metapleuro- metapleural metanotal apodeme x x xe IItpms5 metanotal apodeme, metafurcal arm pl3lp-t3 posterolateral metapleuro- metapleuron metanotum x x x Itpm6 metanotal pl3v-3ax3 ventral metapleuro-third metapleuron, | third axillary sclerite x x x? Itpm9 axillary sclerite of hind wing | mesepimeral ridge of hind wing pl3d-3ax3 dorsal metapleuro-third mesepimeral ridge | third axillary sclerite x xX x IItpm7 axillary sclerite of hind wing of hind wing pl3-sa3 metapleuro-metasubalar metapleuron, metasubalare x x xe Itpm11 metapleural apodeme cx3-sa3 metacoxo-metasubalar metacoxa metasubalare x x x IlIdvm6 (sublateral) pce3l-fu3* lateral metapecto-metafurcal metapectus paracoxal ridge - - x - fu3-cx3* metafurco-metacoxal metafurcal arm | metacoxa (medial) ~ — xX IIIscm3? dez.pensoft.net Dtsch. Entomol. Z. 67 (1) 2020, 51-67 5/7 Abbreviation Name of muscle Origin Insertion Sceliphron Sphex | Ampulex Neoptera pl3m-cx3 median metapleuro-metacoxal metapectus, metacoxa x IIscm1 metadiscrimenal (ventrolateral) fu3l-cx3 lateral metafurco-metacoxal | paracoxal ridge, | metacoxa (lateral) x? IIscm2 metadiscrimenal fu3m-cx3 median metafurco-metacoxal metafurca, metacoxa (medial) xd Iscm2 metadiscrimenal §3-cx3* metasterno-metacoxal metadiscrimenal | metacoxa (medial) - IHscm1? pl3l-cx3 lateral metapleuro-metacoxal metapleuron, metacoxa x I pcm4 paracoxal ridge (dorsolateral) Bere fu3-tr3 metafurco-metatrochanteral metafurcal arm metatrochanteral x IIscm6 apodeme (central) pl3-tr3 metapleuro-metatrochanteral metapleuron, metatrochanteral x? II pcm6 metapleural apodeme (central) apodeme Number of metathoracic muscles (max. 17): (hel S| LoS) ion First and second abdominal segment ph2m-ph3 median mesophragmo- mesophragma median process xX = UIdlm1 metaphragmal ph3-T2 metaphragmo-second propodeum second abdominal x x x - abdominal tergal tergite T1-S2 propodeo-second abdominal propodeum second abdominal x x x - sternal sternite (lateral) fu3-S2 metafurco-second abdominal | metafurcal arm second abdominal x xX xd IIvlm2 sternal sternite (ventro- submedial) §3-S2 metasterno-second abdominal | metadiscrimenal | second abdominal x x x4 - sternal lamella, sternite (lateral) metasternum Number of first and second abdominal segment muscles (max. 5): 5 5 4 Total number of muscles (max. 42): 37 AO 39 * = newly identified; d = difference in structure or position, amplified in chapter Results laterally from the mesopleuron, positioned farther ven- tral and posterior to pl2-3ax2b, and inserts on the third axillary sclerite of the fore wing. It is the most extended and fan-shaped of the three fore wing muscles. Second mesopleuro-mesonotal muscle (pl2-t2b; Fig. 3C) arises, somewhat dorsal to pl2-3ax2c, from the mesopleuron, is fan-shaped and inserts on the ventral surface of the lateral axillar area of the mesonotum. Mesocoxo-mesosubalar muscle (cx2-sa2; Fig. 3D) arises from the mesocoxal apophysis, which corresponds with the cuticular pit and the paracoxal ridge. This muscle is slim and elongated and inserts on the mesosubalare. Anterior mesofur- co-mesolaterophragmal muscle (fu2a-ph2; Fig. 3B) arises from the anterodorsal surface of the mesofurcal arm and inserts on the mesolaterophragma. Posterior thoracic spiracle occlusor muscle (sp3occ) and the cor- responding spiracle (sp2) are absent. The mesopleural pit, which corresponds to the mesopleural apodeme, is vis- ible. Anterior mesopleuro-mesofurcal muscle (pl2a- fu2; Fig. 3D) arises from the mesopleuron and from the mesepimeral ridge and inserts on the mesofurcal arm. Mesopleuro-mesocoxal muscle (pl2-cx2; Fig. 3D) aris- es from the mesopleuron and inserts anterolaterally on the mesocoxa. Second mesopleuro-mesocoxal muscle (pI2- cx2b) is absent. Mesosterno-mesocoxal muscle (s2-cx2; Fig. 3B) arises mainly from the mesodiscrimenal lamella and partly from the mesopectus; it is located ventrally of pl2-cx2 and inserts anterolaterally on the mesocoxa. Mesofurco-mesocoxal muscle (fu2-cx2; Fig. 3A) aris- es from the mesodiscrimenal lamella as far as the tran- sition to the free basal portion of the mesofurcal arm; it inserts anteromedial on the mesocoxal margin. Lateral mesofurco-mesotrochanteral muscle (fu2Il-tr2; Fig. 3C) arises partly from the mesopleuron (posteriorly of pl2a-fu2) and partly from the anterior surface of the later- al mesofurcal arm (anteriorly of pl2a-fu2), fuses with the medially adjacent muscle fu2m-tr2, and inserts laterally on the mesotrochanteral apodeme. Median mesofur- co-mesotrochanteral muscle (fu2m-tr2; Fig. 3C) arises from the posterior surface of the mesofurcal arm and 1s positioned medially to fu2l-tr2. After fusing with fu2I-tr2, both muscles insert laterally on the mesotrochanteral apo- deme. Prophragmo-mesophragmal muscle (ph1-ph2; Fig. 3A) arises from the prophragma and inserts on the mesophragma. This horizontal, beam-shaped muscle is the largest in all species examined. Metathorax. Anterior metapleuro-metabasalar muscle (pl3a-ba3; Fig. 3C) arises from both the meta- dez.pensoft.net 58 pleuron and from the anterior surface of the paracoxal ridge and inserts on the metabasalare. This longitudinal, lateral muscle extends between the mesopleural and par- acoxal ridge. Posterior mesonoto-metanotal muscle (t2p-t3; Fig. 3C) arises from the mesoscutellum and in- serts laterally on a spine-shaped apodeme, which is lo- cated dorsally on the mesophragma at the transition of the meso- and metascutellum; it is fan-like. Anterolat- eral metapleuro-metanotal muscle (pl3la-t3; Fig. 3B) arises anterolaterally from the metapleural apodeme and metafurcal arm and inserts laterally on the metanotal apodeme. It is short and fan-like. Adjacent muscles are fu3-S2 and fu3-tr3, which arise posterior to the metafur- cal arm. Posterolateral metapleuro-metanotal muscle (pI3lp-t3; Fig. 3D) arises from the metapleuron and in- serts on the metanotum by fusing with pl3la-t3, which lies ventral to the small pl3lp-t3. Ventral metapleuro-third axillary sclerite of hind wing muscle (pl3v-3ax3, Fig. 3D) arises from the posterior surface of the mesepimeral ridge and the metapleuron. The muscle is located lateral to pl3d-3ax3 and fuses with it, then both insert on the third axillary sclerite of the hind wing. Dorsal metapleu- ro-third axillary sclerite of hind wing muscle (pl3d- 3ax3; Fig. 3C) arises dorso-submedial of pl3v-3ax3 from the posterior surface of the mesepimeral ridge, fuses with pl3v-3ax3 along half its length, and inserts on the third axillary sclerite of the hind wing; it is small and compact. Metapleuro-metasubalar muscle (pl3-sa3; Fig. 3C) arises from the metapleuron and partly from the meta- pleural apodeme and inserts on the metasubalare, ventral to the hind wing. Metacoxo-metasubalar muscle (cx3- sa3;, Fig. 3C) arises from the sublateral margin of the metacoxa and inserts on the metasubalare by fusing with pl3-sa3; it is long and slim. Lateral metapecto-metafur- cal muscle (pc3l-fu3) and metafurco-metacoxal muscle (fu3-cx3) are absent. Median metapleuro-metacoxal muscle (pl3m-cx3; Fig. 3B) arises ventromedially from the metapectus and from the metadiscrimenal lamella, in- serts ventrolaterally on the metacoxa. Lateral metafur- co-metacoxal muscle (fu3l-cx3; Fig. 3C) arises sublat- erally from the posterior surface of the paracoxal ridge and the metadiscrimenal lamella and inserts laterally on the metacoxa. Median metafurco-metacoxal mus- cle (fu3m-cx3; Fig. 3A, B) arises posteromedially from both the metafurca and metadiscrimenal lamella and in- serts medially on the metacoxa. Metasterno-metacoxal muscle (s3-cx3) is absent (see Sphex). Lateral meta- pleuro-metacoxal muscle (pl3l-cx3; Fig. 3D) arises laterally from the metapleuron and posteriorly from the paracoxal ridge and inserts on the dorsolateral margin of the metacoxa. The muscle is located anteriorly along the metapleural ridge. Metafurco-metatrochanteral mus- cle (fu3-tr3; Fig. 3C) arises posteriorly of the metafurcal arm, inserts centrally on the metatrochanteral apodeme by fusing with pl3-tr3. Metapleuro-metatrochanteral muscle (pl3-tr3; Fig. 3D) arises from the metapleuron and partly from the metapleural apodeme, then fuses with fu3-tr3, and inserts centrally on the metatrochanteral apo- dez.pensoft.net Maraike Willsch et al.: The mesosomal musculature in Apoidea deme. It runs parallel to, and between, pl3l-cx3 and cx3- sa3 and dorsolateral of fu3-tr3. First and second abdominal segment. Median mes- ophragmo-metaphragmal muscle (ph2m-ph3; Fig. 3A) arises posteromedially from the mesophragma and inserts anterior to the median process of the propodeum; it is short and square. Metaphragmo-second abdominal tergal muscle (ph3-T2; Fig. 3A—D) arises dorsolaterally from the propodeum, inserts dorsally on the second ab- dominal tergite; it is a large muscle. Propodeo-second abdominal sternal muscle (T1-S2; Fig. 3B, D) arises dorsolaterally from the propodeum, right above pl3l-cx3 and laterally of ph3-T2; it 1s large and inserts on the lateral margin of the second abdominal sternite. Metafurco-sec- ond abdominal sternal muscle (fu3-S2; Fig. 3B) arises posteriorly from the submedial metafurcal arm, located dorsally of fu3-tr3, and inserts ventro-submedially on the second abdominal sternite; it is elongate and slightly fan- shaped. Metasterno-second abdominal sternal muscle (s3-S2; Fig. 3A) arises from the metadiscrimenal lamella and metasternum, inserts on the lateral margin of the sec- ond abdominal sternite, and is fan-shaped and bent. Sphex (Fernaldina) lucae de Saussure, 1867 Mesothorax. Posterior thoracic spiracle occlusor muscle (sp3occ; Fig. 4A—D) arises medial on the mese- pimeral ridge, anterior to the mesofurcal arm, inserts on the posterior thoracic spiracle (sp2), which additionally is surrounded by pl2-t2b (anterodorsal), pl2a-fu2 (pos- terodorsal), and pl2-cx2b (ventral). The small tracheal occlusor muscle sp3occ is located submedial of pl2a-fu2. Second mesopleuro-mesocoxal muscle (pl2-cx2b; first description; Fig. 4A, C, D) arises from the mesopleuron and partly from the mesopleural spiracle apodeme, fuses with pl2-cx2 and inserts dorsolaterally on the mesocoxa; it lies anteroventral to the mesepimeral ridge. Lateral mesofurco-mesotrochanteral muscle (fu2l-tr2; Fig. 4A, D) arises from the anterior surface of the mesofur- cal arm, runs lateral to fu2m-tr2 and fuses with the same, then both insert laterally on the mesotrochanteral apo- deme; fu2I-tr2 is half the size of that in Sceliphron. Metathorax. Lateral metapecto-metafurcal muscle (pce3l-fu3) and metafurco-metacoxal muscle (fu3-cx3) are absent. Metasterno-metacoxal muscle (s3-cx3; first description; Fig. 4B) arises from the metadiscrimenal lamella and inserts medially on the metacoxa. Ampulex compressa (Fabricius, 1781) Mesothorax. Second mesopleuro-mesocoxal muscle (pl2-cx2b; Fig. 5C, D, E, G, H) is slimmer than in Sphex. It arises from the mesopleural spiracle apodeme, fuses with pl2-cx2 (Fig. 5A, C, D, G, H), and inserts dorso- laterally on the mesocoxa. Lateral mesofurco-mesotro- chanteral muscle (fu21-tr2) is absent. Median mesofur- Dtsch. Entomol. Z. 67 (1) 2020, 51-67 es. ~ ye — = pl2-cx2 pl3m-cx3 fu3l-cx3 A 59 — $3-cx3 pl3m-cx3 fu2m-tr2 Figure 4. The mesosomal musculature of Sphex (Fernaldina) lucae divergent to S. destillatorium;, volume rendering, transparent exoskeleton. A. Lateral view, anterior to the left; B. Medial view, anterior to the right; C. Anterior view on the posterior thoracic spiracle occlusor; D. Dorsomedial view, anterior top right. Abbreviations: sp3oce — poste- rior thoracic spiracle occlusor; sp2 — posterior spiracle; pl2-cx2 — mesopleuro-mesocoxal; pl2-cx2b — second meso- pleuro-mesocoxal; fu2l-tr2 — lateral mesofurco-mesotrochanteral; fu2m-tr2 — median mesofurco-mesotrochanteral; s3-cx3 — metasterno-metacoxal; pl3m-cx3 — median metapleuro metacoxal: fu3l-cx3 — lateral metafurco metacoxal;: fu3m-cx3 — median metafurco metacoxal; pI3I-cx3 — lateral metapleuro metacoxal:; mepr — mesepimeral ridge. Scale bars: 0.7 mm (A), 0.6 mm (B), 0.3 mm (C), 0.5 mm (D). co-mesotrochanteral muscle (fu2m-tr2; Fig. 5B, D, E) is larger than in Sphecidae, arises from the ventral surface of the mesofurcal arm, and inserts medially on the me- sotrochanteral apodeme. Metathorax. Anterior metapleuro-metabasalar muscle (pl3a-ba3; Fig. 5C, E) arises from the metapleu- ron, posterior to the mesepimeral ridge, and inserts on the metabasalare. This muscle is shorter than in Sceliphron, as it originates farther up. The paracoxal ridge is not very distinct. Posterior mesonoto-metanotal muscle (t2p-t3; Fig. SA—D, F) arises from the upper sclerite of the mesoscutellum and inserts on the lower surface of the mesoscutellum; rectangular. There is no filament con- necting it to another structure. Anterolateral metapleu- ro-metanotal muscle (pl3la-t3; Fig. 5B, E, F) mainly arises anterolaterally from the metafurcal arm (touching pl3-tr3 and partly fu3-tr3, which originate on the poste- rior surface of the metafurcal arm) and partly from the metapleuron and inserts on the metanotum. Posterolater- al metapleuro-metanotal muscle (pI3Ip-t3; Fig. 5C, D) arises from the metapleuron fuses with pl3la-t3, which is covered dorsally by pl3lp-t3, and inserts on the metano- tum. It is larger than in Sceliphron and Sphex and fan- shaped. Ventral metapleuro-third axillary sclerite of hind wing muscle (pl3v-3ax3; Fig. 5A, E) arises from the posterior surface of the mesepimeral ridge. This slim muscle is fused with pl3d-3ax3 and inserts on the third ax- illary sclerite of the hind wing. Metapleuro-metasubalar muscle (pl3-sa3; Fig. 5C, D) arises from the metapleuron at the posterior face of the mesepimeral ridge, and inserts on the metasubalare. Lateral metapecto-metafurcal muscle (pc3l-fu3; first description; Fig. 5C, E, G, H) the slender muscle arises anterior to the metacoxa laterally from the metapectus, and inserts on the posterior surface of the paracoxal ridge. Metafurco-metacoxal muscle (fu3-cx3; first description; Fig. SC, G, H) arises medially dez.pensoft.net 60 Maraike Willsch et al.: The mesosomal musculature in Apoidea {2p-t3 pi3la-t3 t2p-t3 yw, pi3v-3ax3-_ ae cy y eg? ism ,2-ba2- > 5s Ss Ye sp1occ LS SLA PILES Ta | Ree ee ——— pI3v-3ax3 sp30cc. — Ps Ye & Si ta, L + pit-eb 7 S| sp3occ 1 gee . = — eee fu3-cx3 0m I2-cx2 pc3l-fu3 a= as Figure 5. The mesosomal musculature of Ampulex compressa divergent to Sphecidae; volume rendering, transparent exo- skeleton. A—C anterior to the right; D-H anterior to the left. A. Medial view, all relevant muscles visible from the centre; B. Medial view on submedial muscles; C. Medial view, further lateral located muscles; D. All relevant muscles discern- ible from lateral view; E. Muscles located sublateral, lateral view; F. Muscles located further medial, lateral view; G. All newly identified muscles (plus pl2-cx2), lateral view; H. Dorsolateral view on all newly identified muscles (plus pl2-cx2). Abbreviations: splocc — anterior thoracic spiracle occlusor; ism1,2-ba2 — intersegmental membrane-mesobasalar; pl2-t2b — second mesopleuro-mesonotal; sp3oce — posterior thoracic spiracle occlusor; s2-cx2 — mesosterno-mesocoxal; pl2-cx2 — mesopleuro-mesocoxal; pl2-cx2b — second mesopleuro-mesocoxal; fu2-cx2 — mesofurco-mesocoxal; fu2m-tr2 — me- dian mesofurco-mesotrochanteral; pl3a-ba3 — anterior metapleuro-metabasalar; t2p-t3 — posterior mesonoto-metanotal; pl3la-t3 — anterolateral metapleuro-metanotal; pl3lp-t3 — posterolateral metapleuro-metanotal; pl3v-3ax3 — ventral meta- pleuro-third axillary sclerite of hind wing; pl3-sa3 — metapleuro-metasubalar; pe31-fu3 — lateral metapecto-metafurcal; fu3- cx3 — metafurco-metacoxal; fu3l-cx3 — lateral metafurco-metacoxal; fu3m-cx3 — median metafurco-metacoxal; pI3I-cx3 — lateral metapleuro-metacoxal; fu3-tr3 — metafurco-metatrochanteral; pl3-tr3 — metapleuro-metatrochanteral; ph3-T2 — metaphragmo-second abdominal tergal; T1-S2 — propodeo-second abdominal sternal; fu3-S2 — metafurco-second ab- dominal sternal; s3-S2 — metasterno-second abdominal sternal. Scale bars: 0.7 mm (A-C), 0.8 mm (D, E), 0.6 mm (F—H). dez.pensoft.net Dtsch. Entomol. Z. 67 (1) 2020, 51-67 61 Figure 6. Comparison of the paracoxal ridge (per) and metapleural apodeme (pl3a). A. Sceliphron destillatorium, antero- lateral view, head left; B. Sphex /ucae, anterolateral view, head left; C. Weakly developed structures in Ampulex compressa, lateral view, head left; D. Ampulex compressa, paracoxal ridge, lateral metafurcal arms (fu3a) fused with reduced metapleu- ral apodeme, anterior view. Further abbreviations: pI3Ip-t3 — posterolateral metapleuro-metanotal muscle, pI3-tr3 — meta- pleuro-metatrochanteral muscle, fu3-tr3 — metafurcor-metatrochanteral muscle. Scale bars: 0.4 mm (A-C), 0.3 mm (D). from the metafurcal arm, fuses with fu3l-cx3, and inserts medially on the metacoxa; it is slender and flattened. The median metafurco-metacoxal muscle (fu3m-cx3; Fig. 5A, F) arises posteromedially from the metafurca and from the metadiscrimenal lamella and inserts medially on the metacoxa. The lower metafurcal area runs further cra- nial and offers more posterior space filled by this muscle. Lateral metafurco-metacoxal muscle (fu3l-cx3; Fig. 5B, D) arises from the metapectus and inserts dorsolater- al on the metacoxa. Metasterno-metacoxal muscle (s3- cx3) is absent. Metapleuro-metatrochanteral muscle (pl3-tr3; Fig. 5D, E) arises posteriorly from the metafur- cal arm, which merges into a spiracle at that position. The muscle is positioned laterally of fu3-tr3, fuses with it and inserts on the metatrochanteral apodeme. Metapleu- ral apodeme and paracoxal ridge weakly developed (Fig. 6C, D); metapleural apodeme fused with lateral metafurcal arms (Fig. 6D). Second abdominal segment. Median mesophrag- mo-metaphragmal muscle (ph2m-ph3) is absent. The mesophragma in Ampulex is rectangular like the outer cuticle and lacks a posterior notch for the insertion of a muscle. Metaphragmo-second abdominal tergal mus- cle (ph3-T2; Fig. SA—C, F) arises from the metaphragma and propodeum, inserts dorsally on the second abdomi- nal tergite; broad, large muscle extended to the posterior region. Metafurco-second abdominal sternal muscle (fu3-S2; Fig. 5A) arises posteriorly from the metafurcal arm, positioned posteromedial to fu3-tr3, inserts anter- oventrally on the second abdominal sternite. In length and width distinctly more gracile than in Sceliphron. Metasterno-second abdominal sternal muscle (s3-S2; Fig. 5A) arises from the metadiscrimenal lamella and 1n- serts on the anterolateral margin of the second abdominal sternite. It is noticeably smaller and neither fan-like nor bent, as in Sceliphron. Discussion The cladistic analyses by Vilhelmsen et al. (2010) inferred Crabronidae (Pison) as being the closest relative of Sphe- cidae (Stangeella) and Ampulicidae (Ampulex) and all three taxa constitute a monophyletic Apoidea. However, many anatomic structures of Ampulicidae and Sphecidae we studied differ significantly from each other, whereas dez.pensoft.net 62 the two species within Sphecidae show many similarities. Especially, the metathoracic musculature varies remark- ably between the families. The muscles that insert on the notum, coxae, and trochanters show distinct structural di- vergences. Furthermore, the number and origin of muscles varies, due to the less distinct metapleural apodeme and paracoxal ridge in Ampulex (additional muscles inserting on the coxae in Ampulex: pl2-cx2b, fu3-cx3; in Sphex: pl2-cx2b, s3-cx3; absent muscle in Ampulex: fu2l-tr2; or- igin different in Ampulex: t2p-t3, pl3la-t3, fu3l-cx3, pl3- tr3; Fig. 3-6; Table 2). In addition, some of the meso- and metacoxal muscles, as well as a mesotrochanteral and a metanotal muscle of Ampulex tend to be larger compared to Sphecidae (pl2-cx2, fu2-cx2, fu2m-tr2, pl3lp-t3, fu3m-cx3, pl3l-cx3). The pl3l-cx3 is also larger in Sphex (Fig. 4A) compared to Sceliphron (Fig. 3D). Strong leva- tors and depressors attaching on the coxae might be need- ed for backwards dragging of large prey and speaks for an adaptation to this conspicuous hunting behaviour (Wil- liams 1942). On the contrary, pl2-cx2b in Ampulex (Fig. 5C, D, E, G, H) 1s narrower than in Sphex (Fig. 4A, C, D); fu2I-tr2 in Sphex is smaller than in Sceliphron (Figs 3C, 4A, D). However, muscles supposedly involved in the movement of the notum, coxae, and trochanters should be checked carefully in subsequent studies. Mesothorax. The mesopleural pit in Sceliphron pre- sumably developed by muscle and spiracle reduction. Ac- cording to Vilhelmsen et al. (2010), the occurrence of the mesopleural pit shows high variances within and amongst superfamilies. Spiracle reduction likely occurred inde- pendently in different groups. Snodgrass (1942), for in- stance, found the posterior thoracic spiracle in honeybee workers without a closing apparatus. Each of the other spiracles 1s equipped with an occlusor muscle (Snodgrass 1942). Vilhelmsen et al. (2010) documented the absence of the posterior thoracic spiracle in Stephanidae and Pteromalidae, while they evidenced its presence (with- out sp3occ) in the apoid family Crabronidae, as well as in Rhopalosomatidae (Vespoidea), and the non-aculeate families Cynipidae, Evaniidae, and Trigonalidae. Hence, not only Apoidea but also Spheciformes sensu lato bear a high variance of the development of this spiracle-mus- cle-complex. Duncan (1939) presented an illustration of the closing mechanism of the posterior thoracic spiracle in Vespula. The occlusor muscles we found in Sphex and Ampulex (Figs 4A—D, 5F—H) show wider attachment points than the fan-shaped muscle described in Duncan’s work. In the neopteran representatives, like Zorotypus, examined by Friedrich and Beutel (2008; Table 2), sp3occ was not revealed. Concluding, other related specimens should be examined to exclude all doubts about the ho- mologisation of the posterior thoracic spiracle and sp3o0cc and to gain further insights into the different formations. In all species examined, pl2-cx2 is located as described by the HAO, with origin on the mesopleuron and antero- lateral insertion on the mesocoxa (Figs 3D, 4A, D, 5A, C, D, G, H). However, it is larger and extending farther ante- riorly in Ampulex (Fig. 5A, C, D, G, H). Ampulex distinct- dez.pensoft.net Maraike Willsch et al.: The mesosomal musculature in Apoidea ly shows the additional and slender mesocoxal muscle pl2-cx2b, which we describe here for the first time (Fig. 5C-E, G, H). In Sphex it is broader and closely adjacent to pl2-cx2 (Fig. 4A, C, D). It is absent in Sceliphron. Con- sequently, the development of pl2-cx2b should be exam- ined in other species to clarify the phylogenetic relevance. The muscles fu2]-tr2 and fu2m-tr2 in Ampulex, which insert on the mesotrochanter, seem to have been coalesced completely, making a separation impossible (compare Fig. 7A, B). Because of the insertion and the rather me- dial position, we reasonably homologized the structure with fu2m-tr2 by excluding fu2l-tr2 for Ampulex. The unambiguous identification of both muscles in Sphecidae appears to indicate an autapomorphic feature of Ampuli- cidae. However, Vilhelmsen et al. (2010; see also refer- ences therein) stated that both muscles were found in Eva- niidae, Platygastroidea, most Proctotrupoidea, Plumarius, and Apoidea, which might include all genera they exam- ined (1.e., Ampulex, Apis, Bombus, Pison, Stangeella). However, the authors noted the absence of fu2I-tr2 in Or- thogonalys (Trigonaloidea) and of fu2m-tr2 in Ceraphro- noidea, Chalcidoidea, and Stephanoidea. Nevertheless, they explained that a secondary subdivision of fu2m-tr2 may have led to the development of fu2I-tr2. In summary, the contrariness referring to fu2I-tr2 needs to be clarified by additional studies on Ampulex, in particular. In addition, fu2l-tr2 fills the mesopleural area in Sceliphron (Fig. 3C), whereas this muscle is smaller in Sphex (Fig. 4A, D). In contrast, pl2-cx2b extends over the mesopleural region in Sphex and Ampulex (Figs 4A, C, D, 5C-E, G, H). In Ampulex, the origin of this muscle is the same spiracle apodeme as that from which sp3occ arises (Fig. SE—H); in Sphex it partly originates from the posterior thoracic spiracle and partly from the mesopleu- ron (Fig. 4A, C, D). However, we recommend a closer look at these different formations in other species before drawing phylogenetic conclusions. Metathorax. The different constructions of the metatho- racic muscles mainly depend on variations of the skeletal structures. The slight difference in the metapleural origin of pl3a-ba3 in Ampulex (Fig. 5C, E) is a consequence of the less distinct development of the paracoxal ridge (Fig. 6). As shown by Vilhelmsen et al. (2010), the paracoxal ridge 1s weakly developed in Ampulicidae and non-apo- critan Hymenoptera, whereas it is highly variable within apocritan groups. Orthogonalys (Trigonalidae), which serves as reference species in the paper of Vilhelmsen et al. (2010), has a weakly developed paracoxal ridge, except for the ventralmost part. As no other information about the structure in Pison (Crabronidae) is available, it should be identical. We confirm the differences noted by Vilhelmsen et al. (2010), as the paracoxal ridge is weakly developed in Ampulicidae and well-marked in Sphecidae (Fig. 6). Additionally, Vilhelmsen et al. (2010) described a distinct paracoxal ridge in Chrysidoidea, Evanioidea, and Stephanoidea. The muscle t2p-t3 inserts laterally on a spine, which is located dorsally on the mesophragma in Sphecidae (Fig. Dtsch. Entomol. Z. 67 (1) 2020, 51-67 3 Ae ( ty 63 Figure 7. Comparison of fu2m-tr2 — median mesofurco-mesotrochanteral muscle and fu2]-tr2 — lateral mesofurco-me- sotrochanteral muscle, anterolateral view. A. Sceliphron destillatorium, B. Ampulex compressa. Scale bars: 0.4 mm (A), 0.5 mm (B). Mt + ‘ eo LE . J seat * ‘ £ W a Sane Mi? ae eS fF | i 7 + % vs j / Ane V} Pe , v Med iy) he /. Figure 8. Comparison of t2p-t3 — posterior mesonoto-metanotal muscle, posteromedial view. A. Sceliphron destilla- torium; B. Ampulex compressa. Scale bars: 0.2 mm. 8A). Vilhelmsen et al. (2010) revealed in Apoidea and Vespoidea a typical lateral insertion on the metanotum, which is not yet observed in other groups; this might in- dicate that this feature is synapomorphic in both super- families. Although we found the mesoscutellum to be of similar shape in all analysed species, t2p-t3 in Ampulex is instead located entirely between the upper and lower mesoscutellar sclerite (Fig. 8B). So far, this modification seems to be unique. To verify this, further representatives of Ampulicidae should be examined. The metanotal muscle pl3la-t3 in Ampulex differs from that in Sphecidae because of the weakly developed metapleural apodeme, which leads to a rather more lat- eral than submedial position on the thorax (Fig. 5B, F). We found a fusion of the lateral metafurcal arms with the metapleural apodeme in Ampulex (Fig. 6D), as already observed by Vilhelmsen et al. (2010) in the same species, other apoid taxa (Stangeella, Apis, Bombus, Pison), and in Vespoidea. Vilhelmsen et al. (2010) stated that most apocritan Hymenoptera have a metapleural apodeme that is often fused with the lateral metafurcal arms. In non-ap- ocritan Hymenoptera, the metapleural apodeme shows high morphological diversity. In many cases, this may not be easy to recognize (Vilhelmsen et al. 2010). Studies on more species from both families are necessary to deter- mine if the structures found in the present study are fam- ily-specific. Sphecidae has a well-developed metapleural apodeme, similar to Cynipoidea (Vilhelmsen et al. 2010), which is an important characteristic. Our results corrob- orate the conclusion by Vilhelmsen et al. (2010), that the development of the metapleural apodeme is highly varia- ble within Apocrita and, moreover, even within Apoidea. Additionally, the weakly developed metapleural apo- deme in Ampulex influenced the origin of pl3-sa3, which only originates from the metapleuron and inserts on the metasubalare (Figs 5C, D, 9B). The origin of the metatro- chanteral muscle pI3-tr3 is also affected in Ampulex (Figs 5D, E, 6C, D). This muscle originates from a delicate sclerite, which provides a narrow surface of origin. This sclerite arose from the fusion of the metafurcal arm and metapleural apodeme and is equal to the medial margin of the metapleural apodeme and metafurcal arm. dez.pensoft.net 64 Maraike Willsch et al.: The mesosomal musculature in Apoidea Figure 9. Comparison of pl3-sa3 — metapleuro-metasubalar muscle, dorsolateral view, anterior to the left. A. Sceliphron destillatorium: B. Ampulex compressa. Further abbreviations: mpa — mesopleural apodeme; mtpa — metapleural apo- deme; plI3 — metapleuron; sa3 — metasubalare; sp2 — posterior thoracic spiracle. Scale bars: 0.2 mm (A), 0.4 mm (B). Figure 10. Illustration of the metaphragma (ph3) in the propodeum (T1) of Ampulex compressa. A. Medial view, ante- rior to the right; B. Anteromedial view on ph3-T2 — metaphragmo-second abdominal tergal muscle; C. Anteromedial view on ph3; D. Posterior view on the vertical part of propodeum. Scale bars: 0.6 mm (A), 0.3 mm (B, C), 0.9 mm (D). C. Varo aa 22, The homology of the metanotal muscle, which we ten- tatively assign to pl3Ip-t3, according to the HAO termi- nology, cannot be assured. In the HAO, it is described as fan-shaped and posterolaterally originating from the metapleuron. However, size, structure, and position of pl3Ip-t3 are different among the species examined (Figs dez.pensoft.net 3D, 5C, D). In Ampulex, pl3lp-t3 shows great similarity to the description of it by the HAO (wide, fan-shaped, and arises laterally from the metapleuron), whereas in Sphecidae, pl3Ip-t3 is very small and compact but still fan-shaped and located sublaterally. It appears to origi- nate from the metanotum and to insert on the metapleu- Dtsch. Entomol. Z. 67 (1) 2020, 51-67 ron. Additional examination of pl3Ip-t3 in other speci- mens is required to resolve the homology of this muscle. The muscle s3-ex3 1s clearly identifiable in Sphex (Fig. 4B). It is located ventrally to fu3m-cx3 and might serve to strengthen the metacoxal function from the lower cen- tre of the body. From fu3m-cx3, s3-cx3 might be subdi- vided. This possibly forms a genus-specific character of Sphex, but not of the family Sphecidae. First and second abdominal segment. The metaphrag- ma is conspicuously absent in Sphecidae among all stud- ied taxa. Nevertheless, ph2m-ph3 (Fig. 3A) and ph3-T2 (Fig. 3A—D) in Sphecidae are homologue muscles. The metaphragma is usually located between the metanotum and the first abdominal segment (Snodgrass 1942). The HAO describes the metaphragma as the site of origin of the mesophragmo-metaphragmal and metaphragmo-sec- ond abdominal tergal muscles. Although the third phrag- ma was found to be absent in honeybees by Snodgrass (1942). However, Vilhelmsen et al. (2010) stated that most Hymenoptera have at least a weak laterally devel- oped metaphragma. This has been observed in Myma- rommatoidea (Terebrantia) and Chrysidoidea (Aculea- ta). Vilhelmsen et al. (2010) described a metaphragma medially continuous adjacent to the lateral metapleural apodeme for other apocritan taxa (1.e., Vespoidea, Trigo- naloidea, Megalyroidea, Stephanoidea, Evanioidea, most Ichneumonoidea, and Apoidea: Stangeella (Sphe- cidae), Pison (Crabronidae), and Ampulex (Ampulici- dae)). Stangeella and Ampulex were analysed by dis- section but not figured. We cannot confirm this specific pattern for Ampulex (Fig. 1OA—D). The absence of the metaphragma we observed in Sceliphron and Sphex may be a potential autapomorphy or an independent reduc- tion. Consequently, further investigation of this phragma is highly recommended. Conclusions We recommend additional investigations of the structures and features presented in this paper. It would be of great value to analyse the tagmata and other characteristics in the family Heterogynaidae and additional species of Crabronidae, Ampulicidae, and Sphecidae. Due to the unresolved phylogenetic position of Heterogynaidae and the paraphyly of Crabronidae, the study of more species from these taxa might be desirable. Structural investi- gations of more species of Vespoidea and Chrysidoidea would be helpful for clarifying controversial assumptions about phylogenetic relationships within Aculeata. Struc- tures of phylogenetic significance were mainly found in the metathorax, 1.e., the metapleural apodeme, paracoxal ridge, metaphragma, and the origin and insertion of asso- ciated muscles. Future studies should also focus on: the muscles that insert into the legs, the posterior thoracic spiracle as well as the occlusor muscle in closely related species, and the four muscles described here for the first time in Sphecidae and Ampulicidae. 65 Acknowledgements We gratefully acknowledge the project funding (project OH81/13-1 for MO) by the Deutsche Forschungsgemein- schaft. Technical facilities and continuous support was provided by Science Programme I of the MfN (in partic- ular, Kristin Mahlow, Prof. Dr Johannes Miller, Dr Na- dia Frobisch), as well as by Falko Glockler, Dr Carsten Luter, Anke Sanger, and the IT department. Furthermore, the research group Ohl gave reliable support, especially Annika Beckmann assisted with literature research. MW would like to thank Dr Michael Roggenbuck-Wedemeyer and Dr Martin Fritsch for useful advice. Moreover, we offer many thanks to Dr Matthew Dennis (University of Manchester) for proofreading the manuscript. 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Arthropod Systematics & Phylogeny 74(2): 195— 218. https://curis.ku.dk/portal/files/171586094/05_asp_74 2 zim- mermann_195_218.pdf Supplementary material | Table SI Authors: Maraike Willsch, Frank Friedrich, Daniel Baum, Ivo Jurisch, Michael Ohl Data type: Excel file Explanation note: Provision of Universal Resource Iden- tifiers (URIs) referring to structures mentioned in the paper. Automatic creation by using the Hymenoptera Anatomy Ontology "analyzer" (http://api.hymao.org/ projects/32/public/ontology/analyze). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.67.49493 suppl 1 dez.pensoft.net