Comparative analysis of worker head anatomy of Formica and Brachyponera (Hymenoptera: Formicidae)

133 ISSN 1863-7221 (print) | eISSN 1864-8312 (online) | DOI: 10.26049/ASP78-1-2020-06 78 (1): 133 – 170 2020 © Senckenberg Gesellschaft für Naturforschung, 2020. Comparative analysis of worker head anatomy of Formica and Brachyponera (Hymenoptera: Formicidae) Adrian Richter 1, 2, *, Francisco Hita Garcia 2, Roberto A. Keller 2, 3, Johan Billen 4, Evan P. Economo 2, # & Rolf Georg Beutel 1, # 1 Institut für Zoologie and Evolutionsforschung, FSU Jena, 07743 Jena, Germany; Adrian Richter [[email protected]]; Rolf Georg Beutel [rolf[email protected]] — 2 Biodiversity and Biocomplexity Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, 904-0495, Japan; Francisco Hita Garcia [fhitagar[email protected]]; Evan P. Economo [evaneconomo@gmail. com] — 3 Museu Nacional de Historia Natural e da Ciência and Centre for Ecology, Evolution and Environmental Changes, Universidade de Lisboa, 1749-016 Lisbon, Portugal; Roberto A. Keller [roberto.kellerper[email protected]] — 4 Ecological Networks, Zoological Institute KU Leuven, 3000 Leuven, Belgium; Johan Billen [[email protected]] — * Corresponding author; # joint supervision Accepted on May 8, 2020. Published online at www.senckenberg.de/arthropod-systematics on May 26, 2020. Editor in charge: Klaus-Dieter Klass Abstract. An organism’s morphology plays a crucial role in its interactions with its environment. Therefore, comparative anatomical analysis is a critical basis to understanding the ecology, behavior, and evolution. While our knowledge of ant internal anatomy has considerably improved in recent years, it is still highly fragmentary, and many evolutionary questions remain unsolved. The current work is a contribution of a series of studies with the larger goal to increase our knowledge of ant head morphology and reconstruct the evolution of this tagma across the ant phylogeny. We investigated the head anatomy of “generalized” ant species from phylogenetically distant clades to establish a very solid basis for future works on the formicid head and its transformations. To achieve a multifaceted documentation, we used a broad array of techniques, including microphotography, scanning electron microscopy, µCT-scan based 3D-reconstructions, and histological sections. This enabled us to show many anatomical features in unprecedented clarity and detail. Our results outline considerable conservation of the main structural features across the ant tree of life, but they also reveal many details that could prove phylogenetically informative and/or functionally important. The cephalic digestive tract with its sclerotization, associated musculature, and glands is more diverse than previously reported. More work will be necessary to clarify the functional and systematic significance of the observed differences. The cephalic endoskeleton, especially the tentorium and torular apodeme, is identified as a second structural complex of high potential. This previously neglected character system is apparently functionally important and very likely phylogenetically informative. Our results improve the basis for reconstructing the groundplan of the formicid head and evolutionary transformations in the stem group and crown group. Future studies focusing on functional aspects and evolutionary changes of different elements of the head will help to create a complete picture of the evolution of this highly successful group of insects. Key words. Ants, head, anatomy, skeletomusculatur system, 3D-reconstruction, µCT-scan, homology. 1. Introduction Formicidae is one of the most dominant groups of insects in nearly all terrestrial ecosystems outside polar regions (e.g. Lach et al. 2010). Like other organisms, ants interact with their environment using morphological structures such as mouthparts or locomotor organs. Consequently, comprehensive knowledge of structure and function of the body parts of extant and extinct species is necessary to understand their success and the evolution on the phenotypic level. With the ant phylogeny resolving in everincreasing accuracy and resolution (e.g. Branstetter et al. 2017; Borowiec et al. 2019), we have a basis to understand how the ant phenotype arose and was successively remodeled and diversified over time. The current study presents a comparative analysis of head structures among two major subfamilies, extending a previous analysis of the cephalic morphology of workers from the subfamily Myrmicinae (richter et al. 2019). These, combined with forthcoming studies on other sub- Richter et al.: Head anatomy of Formicidae 134 families, will form a basis for a broad analysis of how the anatomy of the ant head has evolved across the phylogeny, facilitating or at least accompanying the stunning diversification of ant morphology, ecology, and behavior. As the head is the body region where sensory (acquisition, processing) and feeding functions are concentrated, it plays a central role in the way insects interact with their environment. Its morphology is apparently linked with the particular ecology and behavior displayed by the organism. The external morphology of ants has been well studied within the framework of phylogenetic and taxonomic studies (e.g. Baroni UrBani et al. 1992; BoLton 2003; KeLLer 2011). However, with the exception of specific structures such as the glandular system (e.g BiLLen 1993; BiLLen et al. 2013; BiLLen & aL-KhaLifa 2015; 2016), the richness of internal character systems remains to be evaluated in a phylogenetic context. Key aspects of internal ant morphology such as the head musculature, its endoskeleton, and the cephalic digestive tract have never been studied in detail and only documented for very few species (e.g. Janet 1923; LiLLico-oUachoUr et al. 2018), making it presently impossible to evaluate them in the context of the phylogeny of Formicidae, thus preventing the development of meaningful hypotheses on their evolution. The first complete description of the head of a formicid species with modern methods was done by richter et al. (2019) for Wasmannia affinis Santschi, 1929 (Myrmicinae), revealing many novel aspects of ant anatomy including a modified dorsal mandibular articulation (secondary mandibular joint with respect to the groundplan of ectognathous insects), and the presence of a previously undescribed prepharyngeal muscle originating on a torular apodeme. That study also raised questions about the evolution of structures that have only rarely been documented in the literature, such as the cephalic digestive tract and cranial endoskeleton (see for instance KUBota et al. 2019 and Yamada et al. 2020 on the tentorium). Our ultimate goal is thus to provide a detailed and systematic documentation of external and internal head structures across the whole family, incorporating most lineages and several representatives for larger clades to achieve a solid assessment of the character evolution based on current phylogenetic hypotheses. The present work represents a further step towards this goal, documenting the cephalic anatomy of Formica rufa Linnaeus, 1761 (type species of Formicidae, Formicinae), and Brachyponera luteipes (Mayr, 1862) (Ponerinae). Formica rufa feeds mainly on honeydew of plant sucking arthropods and additionally uses species of different arthropod groups as prey (e.g. domisch et al. 2009). In contrast, Brachyponera luteipes is primarily predacious, like most members of the subfamily Ponerinae (schmidt & shattUcK 2014) but relies on seeds as additional food source (ZhoU et al. 2007). We also included in our anatomical investigation the closely related Brachyponera chinensis (emerY 1895), an invasive species in North America from its native Asian range (GUénard & dUnn 2010), which accepts a diverse array of different food substrates, including sugar and lipids (mo 2013). Both species are rather “generalized” forms, thus serving as good representatives of the two major ant clades formicoids and poneroids (informal ending -oid does not imply superfamily rank in studies on ants), an appropriate starting point to investigate the diversity of anatomical structures in ants as a whole. We chose generalized species to get an initial idea of the anatomical diversity across the ant phylogenetic tree, and to assess which structures may contain phylogenetic signal or may be of interest from a functional perspective, and thus provide new insights in the evolution of ants. While comparing the observed structures and discussing potential phylogenetic and functional implications, another aim is to provide the groundwork for the clarification of persisting homology problems. Well-established homology hypotheses are crucial for understanding the evolution on the phenotypic level of any group of organsism. However, this aspect has been rather neglected in ant morphology and taxonomy in recent decades, with few exceptions, such as Keller’s morphology-based phylogeny of ants (KeLLer 2011), the detailed treatment of male genitalia by BoUdinot (2013), or richter et al. (2019) addressing the homology of sections of the cephalic digestive tract for the first time. Related to the problem of homology is the use of a consistent, ontologized terminology as the basis for comparative morphological work, as recently discussed by siLva & feitosa (2019). Even though progress has been made, many issues of homology and terminology persist. 2. Material and methods 2.1. Material Adult workers of Formica rufa, Brachyponera chinensis, and Brachyponera luteipes were used in this study. Formica rufa shows a slight worker size polymorphism that does not affect our qualitative anatomical results. We used workers with a head width (measured as maximum head width in full face view) ranging from ca. 1.2 mm to ca. 2.2 mm. Brachyponera luteipes workers are monomorphic with a representative head width of ca. 0.79 – 0.81 mm (n = 3). For CT scanning specimens from the collection of OIST were chosen. The specimens of F. rufa with the CASENT numbers 0790267 and 0709411 were used, and of B. luteipes the individuals with the CASENT numbers 0709409 and 0709409. The specimens used for histological sectioning were collected in Belgium (F. rufa) and Taiwan (B. chinensis and luteipes). For the SEM images, specimens of F. rufa collected in Belgium and B. luteipes collected in Japan (Okinawa) were used. 2.2. Scanning electron microscopy Workers of F. rufa were fixed in 70% ethanol. The head of several specimens was severed and after removal of 135 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 the antennae with forceps macerated in 10% KOH overnight. Afterwards, the maxillolabial complex was either already in an extended position, or extended by slight pressure onto the head. The mouthparts of some heads were dissected using Dumont forceps and minute needles attached to a hobby knife handle. All samples were transferred to 100% ethanol (70, 80, 90, 95, 100%) and dried at the critical point in liquid CO2 with an Emitech K 850 Critical Point Dryer (Sample Preparation Division, Quorum Technologies Ltd., Ashford, England). The samples were glued on the tip of minute needles and sputter coated with gold using an Emitech K 500 (Sample Preparation Division, Quorum Technologies Ltd., Ashford, England). Using a rotable specimen holder (PohL 2010), SEM micrographs were taken with a Philips ESEM XL30 (Philips, Amsterdam, Netherlands) equipped with Scandium FIVE software (Olympus, Münster, Germany). Workers of B. luteipes with an extended maxillolabial complex were killed and fixed in 70% ethanol. The head of one specimen was slightly compressed with forceps to fully inflate the glossa (see Fig. 8). The elastic cuticle of the head was not damaged in this process. The head of several specimens was severed with Dumont No. 5 forceps and the antennae removed. Some of the heads were immersed in 10% KOH solution and macerated for six hours at room temperature. They were then washed in water twice for five minutes and subsequently transferred to 70% ethanol. The mouthparts were removed using Dumont forceps and minute needles attached to a hobby knife handle. All samples of B. luteipes (removed mouthparts, entire heads, heads without mouthparts, and heads without antennae) were dehydrated in a graded ethanol series (70, 80, 90, 95, 100%) and subsequently transferred to t-butyl (2 × for 10 minutes). In t-butyl, the samples were put into a Hitachi ES-2030 freeze dryer (Hitachi, Chiyoda, Japan) and vacuum dried overnight. The samples were then glued onto SEM stubs with double-sided adhesive tape, employing a dog hair glued to a glass pipette to manipulate and clean them. Samples were sputter coated with gold (Model: VE3030CVD of the OIST imaging section). SEM images were taken with a JEOL JSM-7900F (JEOL Ltd, Tokyo, Japan), using the manufacturer software. Two heads of B. luteipes without antennae and a few dissected mouthparts of this species were glued on minute needles and documented as described above for Formica. 2.3. Photomicrography Before heads of F. rufa and B. luteipes were sputter coated with gold as described above, photos were taken with a Canon EOS 7 D Mark II equipped with a Canon MP-E65 macro lense, in combination with an adjustable extension bellows. The samples were illuminated by two flashes through a transparent plastic cylinder for soft light. Zerene Stacker (Zerene Systems LLC, Richland, USA) was used to combine image stacks with a different focus. 2.4. Histological section series The heads were removed from the body with a transverse cut behind the compound eyes, thus creating a large posterior opening for the penetration of chemicals used during tissue processing. The heads were fixed in 2% cold glutaraldehyde in a buffer of 50 mM Na-cacodylate and 150 mM saccharose. Postfixation took place in 2% osmium tetroxide in the same buffer, and was followed by dehydration in a graded acetone series. Tissues were embedded in Araldite® and sectioned with a Leica EM UC6 ultramicrotome (Wetzlar, Germany). Transverse, longitudinal, and frontal serial semithin sections were made for both species twith a thickness of 1 µm. A 0.1% solution of methylene blue and thionin was used for staining. Sections were viewed under an Olympus BX-51 microscope (Tokyo, Japan), equipped with an Olympus Camedia C-3040 Zoom digital camera (Tokyo, Japan). Pictures were taken at 10 µm intervals employing a 10 × objective and used for anatomical comparisons and descriptions. Some additional images of anatomical details were taken with a 40 × objective. Selected section images were mounted as image plates as described below. 2.5. Micro-computed tomography scanning µCT-scanning was performed using a Zeiss Xradia 510 Versa 3D X-ray microscope operated with the Zeiss Scout-and-Scan Control System software (version 11.1.6411.17883) at the Okinawa Institute of Science and Technology Graduate University, Japan. Specimens fixed in 100% ethanol were immersed in a 2 M iodine solution for four days, except for one specimen of F. rufa (CASENT0709411) which was accidentally kept in the solution for several months. All specimens were washed in 100% ethanol for one hour before mounting them within a sealed pipette tip of appropriate size. 1601 projection images were taken for a full 360° rotation. Scan settings were selected in order to yield optimum scan quality. Specimens of B. luteipes were scanned at 40 kv and 3 W and voxel sizes of 1,1557 µm3 (CASENT0709409) and 1,2244 µm3 (CASENT0709419) were achieved. Specimens of F. rufa were scanned with the same energy values, and voxel sizes of 2.5527 µm3 (CASENT0709411) and 2,8337 µm3 (CASENT0790267) were achieved. 3D reconstructions of the resulting scan projection data were done with the Zeiss Scout-and-Scan Control System Reconstructor (version 11.1.6411.17883) and saved in DICOM file format. 2.6. 3D modelling and volume calculation Scans of CASENT0790267 (F. rufa) and CASENT 0709409 (B. luteipes) were segmented. As the F. rufa individual showed an abnormal muscle in the region of the mandibular musculature (see Results section), the mandibles and associated structures were also segmented for the Richter et al.: Head anatomy of Formicidae 136 scan of CASENT0709411 (F. rufa). Segmentation was performed in Amira 6.5 (Visage Imaging GmbH, Berlin, Germany). Large structures (cuticle, mandibles, brain, large muscles, pharyngeal gland) were pre-segmented by manually segmenting every 30th slice, and subsequently semiautomatically segmented using the online application biomedisa (LöseL & heUveLine 2016) and the watershed function of Amira. The resulting segmentations were compared to assess the anatomical accuracy. The ones with less spilling of segmented materials into wrong structures were selected for post-processing (biomedisa for CASENT0709409 and CASENT0709411, watershed for CASENT0790267) and then cleaned up and completed by adding the remaining structures with the interpolation function of amira. Finally, segmented materials were exported with the plugin script “multiExport” (enGeLKes et al. 2018) in Amira 6.2 as Tiff image stacks. The image series were then imported in VG-Studio 3.2.5 (Volume Graphics GmbH, Heidelberg, Germany) to create volume renders (Phong) of individual structures. To calculate the volumina of different structures, a label analysis was run in Amira. To obtain the volume of the head capsule, it was labelled as one material. As the antennae were not completely present on the scans, only the scape was labelled. The volumes of individual materials from the label analysis were divided by the head volume in Microsoft Excel (2016) to assess the percentage of the volume compared to the entire head. It is important to note that only one specimen per species was investigated in this way. Only selected values are reported within the manuscript, but the whole table of volume measurements is available in the supplementary material (File 2). 2.7. Data availability The µCT scans used in this study are available at the online repository Zenodo as DICOM files under the DOI 10.5281/zenodo.3786977. Additionally, videos of 3Dvolume renders of all the main anatomical structures are available there. 2.8. Image processing Image plates were arranged in Adobe Photoshop® CS6 (Adobe System Incorporated, San Jose, USA). All images were subjected to limited levels adjustment and smart sharpen (30%). Labels for the image plates were created in Adobe Illustrator® CS6 (Adobe Systems Incorporated, San Jose, USA). 2.9. Terminology The terminology largely follows richter et al. (2019), but several adjustments were made. We recognize the increasing demand for morphological data, which are computer-parsable and suitable for automated computation (see e.g. voGt 2019). Even though it would not be possible to present our descriptions as “knowledge graph” without losing a lot of detailed information, we follow the suggestion of this author to link the used terms to the Hymenoptera Anatomy Ontology (HAO, Yoder et al. 2010) wherever possible. This revealed many gaps in HAO concerning structures in ant morphology, without an established general designation. It is also apparent that some of the current definitions in HAO are not perfectly applicable to ants, for example if structures used in the definition are missing or the positional relationships are different. However, in many cases older alternative definitions, also deposited in HAO, reflect the meaning of the terms better when applied to Formicidae, making an assignment of HAO terms possible. For the musculature we do not use HAO terms, but instead follow the nomenclatures of v. KéLer (1963) and wiPfLer et al. (2011), which are commonly used in entomology, and were also previously applied to ants (richter et al. 2019) and other groups of Hymenoptera (e.g. Zimmermann & viLheLmsen 2016). As separate regions of the head capsule, e.g. “frons”, “vertex” etc., are not separated by any sutures or other unambiguous markings, we refer to them instead as “frontal region”, “vertexal region” etc. As indicated in richter et al. (2019), M. 41a (0hy1a) (described for the first time by the authors) is reinterpreted as M. 47 / 0hy2 (v. KéLer 1963; wiPfLer et al. 2011). The terminology of the processes/arms of the sitophore plate has been rather inconsistent in previous publications and no label exists for them in the HAO yet. Therefore, they will consistently be designated as “oral arms” following Zimmermann & viLheLmsen (2016). “Torular apodeme” is introduced as a term to describe the “phragma of the antennal acetabulum” of richter et al. (2019). The term torulus is used here as encompassing the whole sclerite forming the antennal insertion, including the acetabulum, external torular rims/ lobes as well as the internal torular apodeme, following KeLLer (2011). We introduce here the term “atala” (plural, “atalae”) (áh-tah-lah, from the Arabic for crowbar) for the prominent process on the lateral side of the mandibular base between the dorsal and ventral articulations of the mandible. This process receives the tendon of the abductor muscle, and the distance of its tip from the axis of mandibular rotation mechanically facilitates the opening of the mandibles, comparable with the use of a crowbar, providing an elongated lever arm for improved force transduction. A mandibular atala is universally present across all ants. Previous names for this structure include abductor apodeme (KeLLer 2011), abductor swelling (richter et al. 2019), and lateral articular process of the mandible (siLva & feitosa 2019). We prefer our new term because this structure is obviously of high functional (biomechanics of the abductor) and phylogenetic importance (with considerable variation in aculeate Hymenoptera, Keller unpubl. data), which justifies a specific term, instead of an ambiguous (abductor apodeme) or composite one. 137 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 3. Results A full description is provided for each of the documented cephalic elements of Formica rufa, whereas the treatment of head structures of Brachyponera luteipes focuses on the differences. When structures are not mentioned explicitly, it is implied that they are very similar to what was described for Formica. A table summarizing structural congruities and differences between the two genera is presented in Supplementary File 3. The structural differences between the examined Brachyponera luteipes and B. chinensis are negligible. 3.1. Head capsule Formica rufa The prognathous head (HAO_0000397) (moderately declined in working posture, i.e. subprognathous in general insect terminology; e.g. BeUteL et al. 2014) of workers is rectangular with rounded corners, appearing almost cubic in dorsal and ventral view (Figs. 1A,C, 2A,C) (heads of smaller specimens are slightly less wide anteriorly and more rounded) (Figs. 1A, 2A); it reaches its maximum width immediately behind the eyes (ce, Figs. 1B, 2B) (HAO_0000217); in lateral view it appears oval with a slightly flattened ventral side. The cervical articulation between the prothorax (HAO_0000874) and the margin of the posteroventrally located occipital foramen (HAO_0000347) is narrow; the area immediately surrounding it is countersunk into a concavity of the occipital region (HAO_0000658) (Figs. 1C, 2C); the strongly sclerotized postocciput (HAO_0000790) forms a collarlike enclosure of the foramen, which appears hourglassshaped due to the presence of mesally directed postoccipital condyles (HAO_0000654). The fissure-shaped posterior tentorial pits (HAO_0000768) are visible at the lateral edge of the postocciput (ptp, Fig. 2C). The postgenal bridge (HAO_0000777) is extensive and each side of it is slightly convex (pgb, Figs. 1C, 2C); the internal postgenal ridge (HAO_0001104) is externally marked by a thin sulcus (pgr, Figs. 1C, 2C). The large clypeus (HAO_0000212) appears roughly diamond-shaped in dorsal view; its main middle portion is slightly raised above the surrounding areas, together with the anterior frontal region between the antennae (cl, Figs. 1A,B, 2A,B); its anterior margin is strongly inflected (cli, Fig. 5B) and the inflected portion is thickened. The epistomal sulcus (es, Figs. 1A, 2A) (HAO_0000306) runs from the dorsal mandibular articulation (dma, Figs. 1B, 2B) diagonally to the anterior tentorial pits (HAO_0000126) (atp, Figs. 1A, 2A), from where it forms an arch in front of the antennal articulations; from the posterior, almost straight portion at the level of the scapal base, a pair of diagonal lines run posteromesad, forming a triangular supraclypeal area (HAO_0001821) (sca, Figs. 1A, 2A) with different cuticular sculpture; from the top corner of the triangle a thin frontal line is marked by a smoother surface sculpture compared to the surrounding cuticle (fl, Figs. 1A, 2A). The frontal carinae (HAO_0001533) are short and do not form distinct “frontal lobes” (frc, Figs. 1A,B, 2A,B); the toruli, which are thus uncovered dorsally, face dorsolaterad due to the raised frontal region between them and have a relatively simple circular rim (HAO_0000103) (to, Figs. 1A,B, 2A,B); a small lobe emerges from the median arch but does not cover the bulbus of the scape (tol, Fig. 3F). The compound eyes are well-developed (565 ommatidia in the smallest investigated worker), oval and located dorsolaterally on the posterior portion of the head. Additionally, three small ocelli (HAO_0000661) (oc, Figs. 1A, 2A) are located in shallow depressions on the vertexal region (HAO_0001077). The ventrally visible hypostoma (HAO_0000411) is the hypostomal carina (HAO_0000413) (hysc, Figs. 1C, 2C, 11A); it has a median indentation and a lateral inward twist (Fig. 11A); together with the main part of the hypostoma it forms the deep hypostomal cavity (HAO_0001316) (hyc, Fig. 11A), which contains the base of the maxillolabial complex; the hypostoma forms large triangular processes projecting into the oral foramen (hysp, Fig. 11A); the concave anterior surface of the processes (*, Fig. 11A) receives the lateral margin of the stipes (HAO_0000958), whereas their lateral margin encloses the mandibles (HAO_0000506) on the mesal side; a much smaller additional rounded projection (white arrowhead, Fig. 11A) is present mesad the socket (pleurostomal fossa HAO_0000732) (vma, Fig. 11A) that receives the ventral mandibular condyle (HAO_0000508); this rounded projection fits into a depression of the closed mandible (*, Fig. 4B). The cuticle is largely smooth, with a fine reticular microstructure and a short pubescence. Brachyponera luteipes The head is rectangular with rounded corners and distinctly longer than broad in dorsal view; the maximum width is reached in the middle region; in profile it appears also roughly rectangular but wedge-shaped anteriorly (Figs. 1D – F, 2D – F). The occipital region appears concave in ventral view; the occipital foramen is located further posterior and is narrower than in Formica; the concave area around it is more extended; a low carina delimits it along its dorsal edge (occipital carina HAO_0000653) (oca, Figs. 1F, 2F); the carina is almost completely flattened laterad the occipital region but continues into short distinct diagonal portions ventrally (white arrowhead, Figs. 1F, 2F). The postgenal bridge is raised in the middle region rather than depressed (pgb, Figs. 1F, 2F); the postgenal ridge is not externally marked by a different cuticular structure or a sulcus. The clypeus is posteriorly narrowed by the anteromesally shifted antennal insertions (cl, Figs. 1D,E, 2D,E); the area laterad the anterior tentorial pits is similarly shaped as in Formica, whereas rounded concavities for the antennal insertions are present mesad of them; the posterior median clypeal portion extends between the antennal insertions as a long, thin strip (mcl, Figs. 1A, 2A); it bears a mesal carina directly between the antennal sockets; the mesal clypeal Richter et al.: Head anatomy of Formicidae 138 area including the region between the antennal sockets (which consists of the anterior frontal region in Formica) is raised much more steeply than in Formica, which results in an almost vertical orientation of the anterior clypeal portion (Figs. 1E, 2E); distinct clypeal grooves are present directly laterad the anterior tentorial pits (black arrowhead, Figs. 1D, 2D). The clypeal inflection (cli, Fig. 5D) is much shorter; its posterior edge bears a flat ridge on the dorsal and ventral side. The epistomal sulcus is represented by an external furrow that is deeper and narrower in the part mesad the anterior tentorial pit (es, Figs. 1D,E, 2D,E). A supraclypeal area is presented as a small, elongated oval depression directly posterad the clypeus (sca, Figs. 1D, 2D). The frontal carinae are more approximated due to the mesal shift of the antennal insertions, but they are also short (frc, Figs. 1D,E, 2D,E); as in Formica, the frontal carinae do not form “frontal lobes”; they are slightly curved and anteriorly end above the very large torular lobes (tol Figs. 1D,E, 2D,E, 3E), which almost completely cover the torular acetabula (ac, Fig. 3E) in dorsal view. The lateral arch of the torolus (trl, Fig. 3E) is short and barrel-shaped, which results in a more horizontal orientation of the antennal base. The compound eye is slightly smaller than in Formica and has fewer ommatidia (63, n = 1); it is also slightly less ovoid and located only a short distance behind the dorsal mandibular articulation, close to the anterior head margin. Ocelli are missing. The hypostomal carina (hysc, Figs. 1F, 2F) is broader than in Formica and straight, without a twist in its lateral part; its anterolateral edges are distinctly projecting above the mesal mandibular base (hye, Figs. 1F, 2F); the triangular processes are more rounded and thicker (hysp, Fig. 11B); they are not concave anteriorly and the lateral stipital margin consequently inserts along the edge between the process and the remaining hypostoma (*, Fig. 11B). An additional process forming Fig. 1. Photomicrographs of heads of Formica rufa (A – C) and Brachyponera luteipes (D – F). Note the slight difference in shape between a small (A,B) and a larger head (C). A,D: Dorsal view. B,E: Lateral view. C,F: Ventral view. — Abbreviations: ce – compound eye; cl – clypeus; dma – dorsal mandibular articulation; es – epistomal sulcus; fr – frontal area; frc – frontal carina; fl – frontal line; ga – galea; glo – glossa; hysc – hypostomal carina; hyst – elongated tip of hypostomal carina; lbr – labrum; md – mandible; oc – ocelli; oca – occipital carina; occ – occipital region; pgb – postgenal bridge; pgr – postgenal ridge (visible through the head capsule); plb – labial palp; pmx – maxillary palp; pm – prementum; pocc – postocciput; sca – supraclypeal area; st – stipes; to – torulus; tol – torular lobe; vma – ventral mandibular articulation; vt – area of the vertex. — Symbols: white arrowhead – ventral carina on the postgena; black arrowhead – groove on lateral clypeus. 139 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 the mesal arch of the pleurostomal fossa is missing (compare Fig. 11A,B). The cuticle is largely smooth but covered with small pores with minute setae inserted in them. 3.2. Endoskeleton Formica rufa The long, tube-like anterior tentorial arms (HAO_0001454) (ata, Figs. 5E,F, 9A,B, 11A, 13B) are oval to round in cross section. They extend through the head with a diagonal orientation. The median lamellae form short broad lobes along the middle third of the anterior arms (ml, Figs. 5E,F, 9A,B, 11A); they appear slightly sinuate in sagittal view (Fig. 9B). Additional lateral lamellae are present between the proximal limit of the median lamellae and the anterior tentorial pits (ll, Figs. 9A,B, 13B); they are dorsally oriented at their posterior origin and ventrolaterally at their anterior end, forming a relatively even curve between these points. The posterior arms Fig. 2. SEM micrographs of heads of Formica rufa (A – C) and Brachyponera luteipes (D – F). Note the slight difference in shape between a small (A,B) and a larger head (C) in Formica rufa. A,D: Dorsal view. B,E: Lateral view. C,F: Ventral view. — Abbreviations: al – atala; atp – anterior tentorial pit; ce – compound eye; cl – clypeus; dma – dorsal mandibular articulation; es – epistomal sulcus; fr – frontal area; frc – frontal carina; fl – frontal line; ga – galea; glo – glossa; hysc – hypostomal carina; hyst – elongated tip of hypostomal carina; lbr – labrum; md – mandible; oc – ocelli; oca – occipital carina; occ – occipital region; pgb – postgenal bridge; pgr – postgenal ridge (visible as line of smooth cuticle); plb – labial palp; pmx – maxillary palp; pm – prementum; pocc – postocciput; sca – supraclypeal area; st – stipes; to – torulus; tol – torular lobe; vma – ventral mandibular articulation; vt – area of vertex. — Symbols: white arrowhead – ventral continuation of occipital carina; black arrowhead – groove on lateral clypeus. Richter et al.: Head anatomy of Formicidae 140 (HAO_0001343) are very short (pta, Figs. 5E,F, 9A,B, 11A). The tentorial bridge (HAO_0000998), located shortly before the occipital foramen, is slightly curved upwards (tb, Figs. 5E,F, 9A,B, 11A); it bears a distinct anteromedian process (tba, Fig. 11A) (HAO_0002479) and a small anterior strengthening ridge. Dorsal tentorial arms (HAO_0000275) are present as thin outgrowths shortly anterad the posterior end of the lamellae (dta, Figs. 5E,F, 9A,B, 11A); they are anterodorsally orientated and do not reach the head capsule; the distal end is slightly inflated. A secondary tentorial bridge is missing. Posterior tentorial processes are present as short tubes in the lateroventral postoccipital region (ppt, Figs. 5B,F, 9B,F). The well-developed postgenal ridge extends along the entire ventral midline of the head; its inflated dorsal edge forms a rod-like margin (pgr, Figs. 5F, 9F); posteriorly it splits into two flat ridges, which extend towards the posterior tentorial pits without reaching them. The torolus bears a finger-like apodeme extending into the cephalic lumen (toa, Figs. 5B,F, S1A) (phragma of the antennal acetabulum of richter et al. 2019/ torular apodeme). The epistomal ridge (HAO_0000305) is strongly developed internally (esr, Fig. 5B). Two short ridges originating at the posterior clypeal margin between the antennal foramina (see Fig. S1A) demarcate the triangular supraclypeal area. Brachyponera luteipes The posterior position of the occipital foramen and the elongate shape of the head result in a more horizontal orientation of the tentorium (Figs. 5G,H, 9C,D, 11B, 13H). The median lamellae are present along almost the entire length of the anterior arms (ml, Figs. 9C,D, 11B); a posteriorly deepening concavity forms the area of origin of the extrinsic antennal muscles (Fig. 5H). The lateral lamellae (ll, Fig. 9C,D) are much broader than in Formica; anteriorly they end only shortly before the median lamellae; they are dorsolaterally oriented over most of their length, twisting only slightly dorsad posteriorly and laterad anteriorly. The anterior arms are sharply bent dorsad at their posterior end; the vertical portion is fused to the posterior head capsule (ata, Figs. 5H, 9D); it is connected with the tentorial bridge (tb, Figs. 5G,H, 9C,D), which is shaped like a rounded arch and strengthened by a ridge on its ventral side. The posterior arms (pta, Figs. 9C,D, 11B) are very short and fused with the anterior arms, and thus almost unrecognizable as individual endoskeletal elements. The obsolete dorsal arms, minute short tubes originating at the posterior end of the median lamellae, run anteriorly directly ventrad the brain (not visible in µCT-data; Fig. S2F). Tube-like posterior processes in the postoccipital region are missing. The torular apodeme, a long and flat cuticular extension, reaches into the head capsule; its distal part is bent towards the anterior cephalic margin (toa, Fig. 5C). The epistomal ridge is firmly connected with the closely approximated antennal sockets (esr, Fig. 5D); the left and right branches diverge posterior to the antennal foramina, before flattening and converging in the sagittal plane; in one specimen of B. chinensis the left and right branches of the ridge ridge fuse in front of the antennal insertions; the division of the two branches remains as a Y-shaped tip of the ridge (Fig. 14E); the base of the Y-shaped ridge shortens posterad, resulting in a new separation of the right and left branches, which then converge towards the midline before obliterating completely. The supraclypeal area is not marked by internal ridges. 3.3. Antennae Formica rufa The geniculate 12-segmented antennae (HAO_0000101) are inserted directly behind the clypeus; the distance between the foramina (HAO_0001022) is only slightly shorter than their distance to the lateral margin of the head (Figs. 1A, 2A). The semisperical bulbus (HAO_0000889, radicle) (bb, Fig. 3B) of the scapus (HAO_0000908) (sc, Fig. 3B) is almost completely visible as it rests on the shallow torular acetabulum (ac, Fig. 3F); its lateral margin articulates with a stout, peg-like antennifer (HAO_0001431). The bulbus is connected to the distal main part of the scape by a short, straight constriction (bulbus neck, bbn, Fig. 3B), located on its posterolateral surface. The scapus is almost half as long as the entire antenna. The cylindrical pedicel (HAO_0000706) (pd, Fig. 3B) is straight except for a basal angle resulting in the geniculate antennal shape. The straight and cylindrical flagellomeres (HAO_0000342) are longer than wide, and decrease in length towards the antennal apex. They bear a dense vestiture of thin articulated hairs interspersed with thicker setae (see waLther 1979 for a more detailed documentation). Short straight setae on the bulbus including its neck and the proximal pedicellar region, presumably prorpioreceptors, are only indistinctly visible (Fig. 3B). Musculature (Figs. 5E,F, 12): The four extrinsic muscles are of similar size (smallest ca. 0.1% and largest ca. 0.15% of the head volume). All of them insert on thin tendons (the precise insertion sites are not shown in the 3D-reconstructions; the tendons were not recognizable in the data set but visible on histological sections, see Fig. 12E). M. tentorioscapalis anterior (M. 1/ 0an1): origin (= O): dorsal surface of the anterior tentorial arm, lateral lamella and posteriorly also on mesal lamella; Insertion (= I): on a tendon originating anterolaterally on the bulbus. M. tentorioscapalis posterior (M. 2/ 0an2): smallest of the four muscles, O: mesally on the mesal lamella, posterior to 0an4; I: on a tendon originating posteromesally on the bulbus. M. tentorioscapalis lateralis (M. 3/ 0an3): largest of the four muscles, O: anterior tentorial arm, posterior to the other three muscles, partly on the posterior ends of the lateral and mesal lamellae; I: on a tendon originating laterally/ventrally (as the bulbus is more vertically than horizontally oriented) on the bulbus. M. tentorioscapalis medialis (M. 4/ 0an4): O: anterior tentorial arm and on the anterior mesal and lateral lamellae, anteromesad 0an1; I: on a tendon originating mesal- 141 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 ly/dorsally on the anterior region of the bulbus. Intrinsic muscles, one of them bipartite. M. scapopedicellaris lateralis (M. 5/ 0an6), O: laterodorsally on the scapus at the level of the distal bundle of 0an7; I: on a short tendon originating from the dorsolateral base of the pedicellus. M. scapopedicellaris medialis (M. 6/ 0an7): two distinct bundles O: mesoventrally on the distal half of the scapus; I: on a long tendon originating from the mesoventral base of the pedicellus. Brachyponera luteipes The antennal sockets are closely approximated medially, and also closer to the anterior cephalic margin (Figs. 1D, 2D). The bulbus is sunk into the deeper acetabulum of Fig. 3. SEM micrographs of the antennae and antennal sockets of Formica rufa (B,D,F) and Brachyponera luteipes (A,C,E). A – D: Dorsal view. E,F: Lateral view. — Abbreviations: ac – torular acetabulum; bb – bulbus; bbn – bulbus neck; es – epistomal sulcus; frc – frontal carina; pd – pedicellus; sc – scapus; tol – torular lobe; trl – lateral torular arch. Richter et al.: Head anatomy of Formicidae 148 3.6. Labium and distal hypopharynx Formica rufa A membrane, which is folded inwards and thus not visible externally, connects the proximal part of the labium (HAO_0000453) with the maxillary base; the stipitopremental conjunctivum connects the entire maxillolabial complex with the infrabuccal pouch (HAO_0001563) (spc, Figs. 7B, S1D, S2D,E). The labium is also completely fused to the distal hypopharynx (HAO_0001575), which forms the dorsal surface of the proximal part of the entire structure (the labium and the distal hypopharyngeal part including the infrabuccal pouch [hypopharyngeal wall, HAO_0000409] are described together here); the ventral labial surface is formed by the small postmentum (psm, Fig. 7B, HAO_0000785) and the large prementum (Fig. 7B, HAO_0000804); the anterior upper surface bears the fused glossae (simply referred to as “glossa” in the following, HAO_0000376) (glo, Fig. 7A – C) and paraglossae (HAO_0000686) (pgl, Fig. 7A,B,E). The postmentum, a relatively broad horseshoe-shaped sclerite, is connected to the prementum by a membrane; the distal region of the sclerite and the proximal membrane are covered with small, spine-like microtrichia. The preFig. 7. SEM micrographs of the labium of Formica rufa (A – E) and Brachyponera luteipes (F – H). A,C,D: Dorsal view. B,E,H: Lateral view. F,G: Dorsolateral view. E,H: Close view of the paraglossa. — Abbreviations: bpb – basiparaglossal brush; dhy – distal hypopharynx; glam – anterior glossal margin; glds – dorsal glossal sclerite; glo – glossa; ibp – infrabuccal pouch; pgl – paraglossa; plb – palpus labialis; pmd – premental ditch; spc – stipito-premental conjunctivum; svo – salivary opening. 149 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 mentum is strongly sclerotized; it is divided into a lateral (HAO_0002152) and a ventral face (HAO_0002156) by shallow premental ditches (HAO_0002227) (pmd, Fig. 7B); the distal margin of the oval ventral face appears slightly truncated in ventral view (Figs. 1C, 2C); the lateral premental face bears relatively short, stout premental arms (HAO_0002155) (pma, Fig. 13B) as proximolateral extensions; they are connected to long crescent-shaped lateral hypopharyngeal sclerites (hypopharyngeal rods, HAO_0000408) (hyr, Fig. 13B), which stabilize the distal hypopharynx. The distal hypopharyngeal part (dhy, Fig. 7A,B) is relatively short, broad and raised high above the prementum; the lateral sclerites are anteriorly continuous with a broad, weakly sclerotized conjunctivum, which is bent downwards into the distal tip of the hypopharynx, thus forming the massive hypopharyngeal buttons (HAO_0002234) (hyb, Fig. 13A); these structures are ventrodistally continuous with the salivarial sclerite (HAO_0001682) (sv, Figs. 9F, 12A). The lateral hypopharyngeal surface is mostly smooth, whereas the dorsal surface bears a dense cover of long, hair-like microtrichia (Fig. 7A,B); its anterior apical region has a scale-like surface structure with each scale bearing a single hair-like microtrichium; the distal hypopharynx is limited by the salivary opening (HAO_0000906) (svo, Fig. 7C); proximally it is continuous with the large, bulbous, hemispherical infrabuccal pouch (ibp, Figs. 7A,B,D, 11E, 12A, 13B); only a narrow opening is visible, especially when the ventral mouthparts are retracted; the upper and lower lip of the pouch opening bear minute microtrichia (Fig. 12C); the inner wall is glabrous and has a honeycombed surface (Fig. 9D); from the upper lip of the pouch opening the hypopharyngeal wall continues towards the prepharynx (described in the section “cephalic digestive tract”). The well-developed basiparaglossal brushes (HAO_0002199) (bpb, Fig. 7A – C) on both sides of the salivary opening consist of several rows of blunt setae which are shortest laterally; the lateral base of the brushes is covered with rows of minute finger-like microtrichia; the brushes are anteriorly continuous with membranous folds covered with spines, representing the strongly reduced paraglossae (HAO_0000686) (pgl, Fig. 7A,B,E). The glossa (HAO_0000376) (glo, Fig. 7A – C), which is inserted between these folds, is stabilized dorsally by the broad dorsal glossal sclerites (anterior glossal plate, HAO_0000112) (glds, Fig. 7A – C) and ventrally by the ventral glossal sclerite (posterior glossal sclerite, HAO_0000748) (glvs, Fig. 12A); the middle region of the latter is thick and two lateral arms project anteriorly into the ventral glossal wall; the membranous wall of the glossa is densely covered with hair-like microtrichia (Fig. 7B,C); the distal edge of the glossa (HAO_0002206) (glam, Fig. 7B) and of the prementum each bear rows of long setae. The four-segmented labial palps (HAO_0000450) (plb, Fig. 7A,B) insert immediately proximad the paraglossae; the palpomeres are similar in length and diameter, except for the slightly less wide apical palpomere; palpomere 1 is glabrous except for its distal margin, whereas the entire surface of the other palpomeres is covered with long, thin setae; this fine pubescence is interspersed with thicker, longer, more erect hair-like sensilla on the dorsal surface of especially palpomere 3 and 4, around the distal margins of palpomeres 2 and 3 and the tip of palpomere 5; the articulatory membranes between the palpomeres are covered with small denticles. Musculature (Figs. 9E,F, 12, 13): M. tentoriopraementalis (M. 29/ 0la5): O: posterior postgena close to posterior tentorial pit, ventrad 0hy3; I: the tendons of the paired muscle merge medially as a broad unpaired structure which extends over the dorsal margin of the hypostomal cavity and inserts on the posterior premental margin. M. praementoparaglossalis (M. 31/ 0la11): O: median premental region; I: middle region of the ventral glossal sclerite. M. praementoglossalis (M. 32/ 0la12): O: on the prementum promixad 0la11; I: base of the dorsal glossal sclerites. M. praementopalpalis externus (M. 34/ 0la14): small muscle; O: anteriorly on the premental arms; I: base of palpomere 1. M. palpopalpalis labii primus/ secundus (M. 35/36 0la16/ 17): intrinsic muscles of the labial palp; O: base of palpomere 1/2; I: base of palpomere 2/3. M. tentoriohypopharyngalis (M42/ 0hy3): O: posterior postgena close to the posterior tentorial pit, dorsad 0hy3; I: with long tendon on hypopharyngeal button, close to the salivarium. Brachyponera luteipes The distal part of the postmentum bears only few small denticles instead of spine-like microtrichia (psm, Fig. 8D). The premental ditches are slightly deeper and narrower (pmd, Fig. 8D) than in Formica. The ventral premental face is more oval; (pm, Fig. 8D) the premental arms are thinner and rather short. The distal hypopharynx is also raised high above the prementum; the stabilizing lateral sclerites (hypopharyngeal rods) are very long and thin (hyr, Fig. 14C); the lateral surface of the distal hypopharynx (dhy, Fig. 7F) is set with long scales formed by comb-like microtrichia; the dorsal surface is covered by a complex microtrichial array; the dorsolateral margins are densely set with hair-like microtrichia; the apical part of the hypopharynx has a scale-like surface structure like in Formica, with each scale bearing a minute hair-like projection posteriorly; a dorsal smooth triangular depression of the hypopharynx is sparsely set with long microtrichia; they are more densely arranged proximally; the proximal region of the distal hypopharynx has a scale-like surface structure formed by combs of short microtrichia; the length of the microtrichia decreases further proximad towards the infrabuccal pouch; the membrane adjacent with the pouch is completely smooth. The salivary opening lies distally between the basiparaglossal brushes, directly behind the glossa; a stripe of cuticle with short microtrichia on its ventral side covers the opening (svo, Fig. 7F,G). The infrabuccal pouch is smaller than in Formica (ibp, Figs. 11H, 12B); the opening is also narrow; very distinct folds are present dorsally (similar position in all examined individuals); a strongly folded surface is displayed when the pouch is extruded, with widely opened mouthparts (Fig. 8A,C). The lateral sur- Richter et al.: Head anatomy of Formicidae 150 face of the basiparaglossal brushes is covered with rows of minute finger-like microtrichia. Reduced paraglossae are likely represented by folds of smooth cuticle with two minute distal sensilla (pgl, Fig. 7F,H). The broad ventral glossal sclerite is formed by a thin layer of cuticle close to the distal premental margin; it narrows towards the glossa, forming a dorsoventral flat sheet that fans out ventrally before it splits into two arms; the area between the distal premental margin and the glossa is larger than in Formica (Fig. 8D); most of the glossal surface is densely covered with hook-shaped microtrichia, which are thinner towards the lateral margins; the distal edge of the glossa is smooth (glam, Fig. 8D). The labial palps (plb, Figs. 7F, 8A,D) are three-segmented, with a very thin palpomere 2; the apical palpomere is club-shaped; palpomere 1 bears two ventral setae (Fig. 8D), palpomere 3 three long distal setae, a dorsal seta at about midlength, and a short terminal cone-like sensillum (Fig. 7F). Musculature (Figs. 9G,H, 12, 14): M. tentoriopraementalis (M. 29/ 0la5): like in Formica. M. praementoparaglossalis (M. 31/ 0la11): O: proximolaterally on the prementum; I: further laterad compared to Formica. M. praementoglossalis (M. 32/ 0la12): O: central premental area, distad 0la11, covering larger area of prementum than in Formica; I: dorsal glossal sclerites. M. praemento pal palis externus (M. 34/ 0la14): small muscle, very difficult to distinguish from 0la11 but otherwise like in Formica. M. palpopalpalis labii primus/ secundus (M. 35/36 0la16/ 17): only the first intrinsic muscle recognizable with the applied techniques. M. tentoriohypopharyngalis (M42/ 0hy3): O: very close to the posterior tentorial pit, laterad 0la5; I: hypopharyngeal button. 3.7. Salivarium and salivary duct Formica rufa The wall of the salivary duct (HAO_0002236) is formed by a thick layer of tissue throughout most of its length, with the diameter decreasing towards the labium (svd, Fig. 8. SEM micrographs of the head and details of mouthparts of Brachyponera luteipes. A – C,E: Anteroventral view. D: Ventral view. — Abbreviations: al – atala; cl – clypeus; ep – epipharynx; ga – galea; glam – anterior glossal margin; glo – glossa; hysc – hypostomal carina; lbr – labrum; md – mandible; plb – palpus labialis; pm – prementum (ventral face); pmd – premental ditch; pmx – palpus maxillaris; psm – postmentum; st – stipes; vma – ventral mandibular articulation. Fig. 9. Volume renderings of the head of Formica rufa (CASENT0790267: A,B,E,F) and Brachyponera luteipes (CASENT0709409: C,D,G,H). A,C,E,G: Upper part dorsal view, lower part ventral view. B,D,F,H: Sagittal view. A – D: Maxillary musculature and gland. E – H: Labial musculature and salivary duct. — Abbreviations: 0hy3 – M. tentoriohypopharyngalis; 0hy7 – M. praementosalivarialis; 0hy12 – M. hypopharyngosalivarialis; 0la5 – M. tentoriopraementalis; 0la11 – M. praementoparaglossalis; 0la12 – M. praementoglossalis; 0la14 – M. praementopalpalis externus; 0la16 – M. palpopalpalis labii primus; 0la17 – M. palpopalpalis labii secundus; 0mx1 – M. cranio- 151 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 cardinalis externus; 0mx3 – M. tentoriocardinalis; 0mx4 – M. tentoriostipitalis anterior; 0mx6 – M. stipitolacinialis; 0mx7 – M. stipitogalealis; 0mx8 – M. stipitopalpalis externus; 0mx12 – M. palpopalpalis maxillae primus; 0mx13 – M. palpopalpalis maxillae secundus; ata – anterior tentorial arm; dta – dorsal tentorial arm; lb – labium; ll – lateral lamella; ml – mesal lamella; mx – maxilla; mxg – maxillary gland; pta – posterior tentorial arm; ppt – posterior process of tentorium; sv – salivary sclerite; svd – salivary duct; tb – tentorial bridge. — Colors: beige / brown – mouthparts; grey – cuticle; orange / red – muscles; purple – glands and ducts. Richter et al.: Head anatomy of Formicidae 152 Figs. 9E,F, 12A, 13E); the duct forms a loop anterior to the brain (Fig. 9E,F); it opens into the sclerotized salivarium (HAO_0000906) (sv, Figs. 9F, 13E) between the distal hypopharynx and the basiparaglossal brushes. The long, U-shaped and sclerotized salivarium encloses the salivary duct ventrally; it fuses with the hypopharyngeal button proximad its opening and to the basiparaglossal brushes distally. Musculature (Figs. 9E,F, 12A): M. hypopharyngosalivarialis (M. 37/ 0hy12): two closely adjacent bundles, O: dorsolaterally from the distal hypopharynx, mostly from the stabilizing sclerites (hypopharyngeal rods); I: mesal bundle dorsally on the distal salivary duct, lateral bundle laterally on the salivarium. M. praementosalivarialis anterior & (or) posterior (M. 38, 39/ 0hy7): a well-developed muscle; O: prementum proximad 0la12; I: ventrally on the sclerotized salivarium, and a few fibers ventrally on the distal salivary duct. Brachyponera luteipes The salivary duct is also thick-walled, especially the portion forming the loop (svd, Figs. 9G,H, 12B, 14A,B), which is largely restricted to the horizontal plane in contrast to Formica, bending only very slightly in vertical direction (Fig. 9H). The salivarium is broader and shorter. Musculature (Fig. 9G,H): M. hypopharyngosalivarialis (M. 37/ 0hy12): like in Formica. M. praementosalivarialis anterior & (or) posterior (M. 38, 39/ 0hy7): largely reduced, no fibers originating on the prementum and inserting on the salivarium are visible in B. luteipes or B. chinensis. 3.8. Labrum Formica rufa The anterior surface of the trapezoid labrum (HAO_ 0000456) forms a distinct hump (Fig. 10A); the distal margin is bilobed, with a median notch; proximolateral processes are posteriorly directed (not visible in frontal view, lbrp, Fig. 10A,B), cone-shaped and slightly bent downwards; the bases of the maxillary palps fit below the processes when the mouthparts are retracted (Fig. S1E). The labral surface displays a scale-like cuticular surface structure, and a minute cone-shaped sensillum is inserted on the posterior surface of each distal lobe (Fig. 10B’). Musculature (Figs. 11C, 13B): M. frontoepipharyngalis (M. 9/ 0bl2): a moderately sized muscle; O: frontal area shortly posterad the antennal bases, laterad 0bu1; I: upon a thin, long tendon attached mesad the base of the proximolateral processes. Brachyponera luteipes The labrum has an even surface, without an anterior hump (Fig. 10C). The proximolateral processes are welldeveloped, hook-shaped and project laterad, beyond the margins of the labrum proper (lbrp, Fig. 10C,D); they fit behind the laterodistal margin of the stipes when the maxillolabial complex is retracted (Fig. S1F). Musculature (Figs. 10D, 11F): M. frontoepipharyngalis (M. 9/ 0lb2): O: frontal area shortly posterad the antennal bases, laterad 0bu1, just mesad the frontal carina; I: like in Formica. Fig. 10. SEM micrographs of the labrum of Formica rufa (A,B,B’) and Brachyponera luteipes. (C,D,D’). A,B,B’: Frontal view. C,D,D’: Caudal view. — Abbreviations: ep – epipharyngeal wall; 0lb2 – M. frontoepipharyngalis; ep – epipharynx; lbrp – proximolateral labral processes. 153 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 3.9. Distal epipharynx Formica rufa The distal part of the epipharyngeal wall (HAO_0000300), the unsclerotized inner wall of the labrum and the laterally free portion of the epipharyngeal wall distad the prepharynx, forms the semimembranous upper wall of the laterally open buccal cavity (ep, Figs. 11E, 12A,C); the surface is smooth, including the upper lip of the functional mouth opening (Fig. 12C). Musculature: epipharyngeal muscles function as part of the cephalic digestive tract and are treated in that section. Brachyponera luteipes The epipharyngeal surface (ep, Figs. 8A,B, 11H, 12B,D) is covered with slightly wavy rows of short finger-like microtrichia; the dorsal lip of the functional mouth opening bears a brush of very long hair-like microtrichia (white arrowhead, Fig. 12D), expanded as a roughly triangular patch in the middle region of this structure (Fig. 8B). Musculature: see above. 3.10. Cephalic digestive tract Formica rufa The cephalic digestive tract is divided into three functional subunits: the buccal cavity (HAO_0000670), the prepharynx (pph, Figs. 11C – E, 12A, 13A,B) (cibarium, HAO_0000201), and the pharynx (ph, Figs, 11A,C – E, 12A) (HAO_0001740). The buccal cavity is the space containing the mouthparts. Within this compartment, a narrow pre-oral chamber is limited by the labium and distal hypopharynx on the ventral side, the maxillae laterally, by the distal epipharynx dorsally, and by the labrum anterodorsally when the mouthparts are retracted (Fig. 11E). The distal hypopharynx posteriorly expands, forming the infrabuccal pouch (ibp, Figs. 11E, 12A). The lateral margins of the proximal epiand hypopharynx fuse, thus forming an extensive prepharyngeal tube (pph, Figs. 11C – E, 12A, 13A,B), which anteriorly opens into the buccal cavity via the broad, fissure-shaped functional mouth (HAO_0000361) (fmo, Figs. 11D,E, 12B); the anteriormost section of the prepharynx, the buccal tube (bt, Figs. 11D,E, 12A,B), appears like a slightly flattened crescent in cross section, and is distinctly bent backwards when the mouthparts are in a retracted position (Fig. 11E); its cuticle is thick both on the anterior (epipharyngeal) and posterior (hypopharyngeal) side, and displays a peculiar banding pattern (Fig. 12A,C); the epipharyngeal side is densely covered with minute microtrichia, whereas the hypopharyngeal side display a sparse pattern of much longer hair-like microtrichia (Fig. 12C); both types of hairs are oriented towards the functional mouth; the main part of the prepharynx appears broadly crescent-shaped to oval in cross section (Fig. 13A,B); it runs parallel to the clypeus and anterior frontal region, ending posteriorly with the anatomical mouth opening (amo, Fig. 11E), marked by the frontal ganglion (fg, Fig. 11C,E) and the insertion sites of the muscles 0bu2 and 0hy1. The anterior roof of the prepharynx is sclerotized, with a broad hump (Figs. 11E, 12A), and appears finshaped in sagittal view (possibly representing the buccal lobe, HAO_0002412); the sclerotized prepharyngeal floor forms the sitophore plate (HAO_0000939) (sp, Fig. 13A); this structure bears two stabilizing elements, the oral arms (oa, Figs. 11C, 13B, S1G), which originate anterolaterally and are ventrally directed; they form an even curve towards the dorsal side of the prepharynx and are dorsally arranged around the anatomical mouth opening, with the dorsal portion reaching into the anterior pharynx; the posterior ends of the arms are bent outwards and form an intricate sheath-like structure which accommodates the openings of the pharyngeal gland. The pharyngeal opening, i.e. the anatomical mouth (amo, Fig. 11E), is not sclerotized except for the oral arms; at the level of the anatomical mouth opening the digestive tube forms an angle of about 60° (n = 2), with the pharynx running straight towards the occipital foramen; its dorsal wall is deeply folded (Fig. S2C), resulting in a crescent to Yshape in cross section. The width of the posterior pharynx decreases and it is flattened at the attachment area of 0ph2; the pharyngeal wall is finely wrinkled throughout most of its length, especially in the attachment area of 0bu2 and 0bu3 (Fig. 12A). Musculature (Figs. 11C,E, 12, 13): M. frontohypo pharyngalis/ M. frontooralis (M. 41/ 0hy1): well-develop ed paired muscle (Fig. 11C,E): O: centrally on the frontal area, laterad 0bu3 (Fig. S1A); I: broadly on the posterior oral arms, on the posterior/dorsal side of the sheath surrounding the pharyngeal gland opening (Fig. 11C). M. clypeopalatalis (M. 43/ 0ci1) two subcomponents (Figs. 11E, 12A,E, 13A); M. 43a/ 0ci1a: unpaired, relatively small muscle, O: mesally on the anterior clypeus, flanked by 0ci1b (Fig. S1A); I: broadly on the anterior wall of the buccal tube, close to the functional mouth opening; M. 43b/ 0ci1b: very strongly developed paired muscle; asymmetric, with stronger bundle on one side; O: along the midline of almost the entire clypeus, intersecting with 0ci1a (Fig. S1A); I: anterior dorsal prepharyngeal wall, especially on the hump. M. clypeobuccalis (M. 44/ 0bu1): distinctly developed but much smaller than 0ci1b; paired muscle (Figs. 11E, 12A, 13B), O: posterior clypeus close to the epistomal ridge; I: dorsal prepharyngeal wall, behind the hump amd anterior to the frontal ganglion. M. frontobuccalis anterior (M. 45/ 0bu2): well-developed, paired but closely adjacent along the midline (Figs. 11E, 12A), O: frontal area, distinctly posterad the supraclypeal area (Fig. S1A); I: mesally on the dorsal side of the pharynx, directly posterior to the frontal ganglion. M. frontobuccalis posterior (M. 46/ 0bu3): strongly developed, flat and unpaired muscle with two subunits (Figs. 11E, 12A), O: anterior subunit directly posterad 0bu2, posterior subunit distinctly separated from it, shortly anterad the median ocellus (Fig. S1A); I: dorsomesally on the pharynx between the dorsal longitudinal folds, posterad 0bu2. M. frontobuccalis lateralis/ M. tentoriooralis (M. 47/ 0hy2): a relatively small, Richter et al.: Head anatomy of Formicidae 154 paired muscle (Figs. 11C, 13B), O: on the proximal part of the torular apodeme; I: anteriorly on the posterior oral arm, opposite to 0hy1. M. tentoriobuccalis anterior (M. 48/ 0bu5) (possibly together with M.50/ 0bu6): strongly developed unpaired muscle, constricted anteriorly due to the limited space between the large infrabuccal Fig. 11. Volume renderings of heads of Formica rufa (CASENT0790267: A,C – E) and Brachyponera luteipes (CASENT0709409: B,F – H). A,B: Frontal view of head capsule. C,F: Dorsal view of cephalic digestive tract with glands and parts of the musculature, 0lb2, and the central nervous system. D,G: Ventral view of cephalic digestive tract including glands, and the central nervous system. E,H: Sagittal view of cephalic digestive tract including glands, musculature and buccal cavity, and the central nervous system. — Abbreviations: 0bu1 – M. clypeobuccalis; 0bu2 – M. frontobuccalis anterior; 0bu3 – M. frontobuccalis posterior; 0bu5 – M. tentoriobuccalis posterior; 0ci1a – M. clypeopalatalis, unpaired portion; 0ci1b – M. clypeopalatalis, paired portion; 0hy1 – M. frontooralis; 0hy2 – M. tentoriooralis; 0hy9 – M. oralis transversalis; 0lb2 – M. frontoepipharyngalis; 0ph2 – M. tentoriopharyngalis; ala – atalar acetabulum; amo – anatomical mouth opening; ata – anterior tentorial arm; atp – anterior tentorial pit; br – brain; bt – buccal tube; clf – ventral clypeal projection / flange; cc – cardinal condyle; dhy – distal hypopharynx; dta – dorsal tentorial arm; dma – dorsal mandibular articulation; ep – epipharynx; epk – epipharyngeal keel; fc – frontal commissure; fg – frontal ganglion; fmo – functional mouth opening; frc – frontal carina; hyc – hypostomal cavity; hysc – hypostomal carina; hysp – hypostomal process; ibp – infrabuccal pouch; lb – labium; lbr – labrum; mped – M. pharyngoepipharyngalis, dorsal portion; mpel – M. pharyngoepipharyngalis, lateral portion; nan – antennal nerve; no – optical nerve; oa – oral arm; ocn – ocellar nerve; opl – optic lobes; pgr – postgenal ridge; ph – pharynx; phg – pharyngeal gland; pph – prepharynx; pphg – prepharyngeal gland; pta – posterior tentorial arm; sog – suboesophageal ganglion; tb – tentorial bridge; to – torulus; tol – torular lobe; trl – lateral torular arch; vma – ventral mandibular articulation. — Colors: beige / brown – mouthparts; green – cephalic digestive tract (prepharynx and pharynx); grey – cuticle; orange / red – muscles; purple – glands; yellow – nervous system. — Symbols: white arrowhead – hypostomal hump contacting mandibular groove; * – anterior surface of triangular hypostomal process receiving lateral stipital margin. 155 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 pouch and the prepharynx (Figs. 11E, 12A, 13B), O: anterior process of the tentorial bridge with a long tendon. I: broadly on the sitophore plate. M tentoriopharyngalis (M. 52/ 0ph2): relatively small, paired muscle (Fig. 11E), O: tentorial bridge and posterior tentorial arm; I: ventral side of the pharynx above and slightly posterad the suboesophageal ganglion. M. transversalis buccae (M. 67)/ M. oralis transversalis (0hy9): a well-developed layer of transverse muscles between the dorsal oral arms, directly anterior to the frontal ganglion, and an additional layer of transverse fibers connecting the arms on the ventral side posterior to the frontal ganglion (Figs. 11C,E, 12A, 12B). M. annularis stomadaei (M. 68/ 0st1): a thin layer of ring muscles around the pharynx behind the frontobuccal muscles. M. longitudinalis stomadaei (M. 69/ 0st2): weakly developed layer of longitudinal muscles below the ring musculature layer. M. pharyngoepipharyngalis (Mpe): very strongly developed longitudinal muscles connecting the anterior pharynx and the epipharynx: a thin unpaired median bundle on the dorsal side of the prepharynx connecting the dorsal prepharyngeal hump and the pharynx at the insertion site of 0bu3 (Mped, Figs. 11C,E, 12A); paired large lateral bundles connect the posterior oral arms with the epipharynx directly before it connects with the buccal tube (Mpel, Figs. 11C, 13A,B). Brachyponera luteipes In its general features the cephalic digestive tract is similar to that of Formica. The bend of buccal tube is less distinct (bt, Fig. 11H), and almost completely straigthened when the mouthparts are extruded (Fig. 12B). The lumen of the posterior prepharynx and pharynx is narrower (relative volume of cephalic digestive tract 1,2 % in Formica, 0,5 % in Brachyponera). The microtrichia on the hypopharyngeal side of the buccal tube are more densely arranged than in Formica (Fig. 12D). The dorsal sclerotization of the prepharynx is more pronounced and forms a short sclerotized cuticular keel on top of the more elongated prepharyngeal hump (epk, Figs. 11H; 12B). The oral arms (oa, Figs. 11F, 12B, 14B) anteriorly originate on the ventrolateral part of the sitophore plate and are bent inwards; they are not evenly curved like in Formica but abruptly bent in front of the frontal ganglion; their dorsal portions form nearly straight, elongate wall-like structures (Fig. S1H), which are strongly constricted at the level of the frontal ganglion (fg, Fig. 11F,H); posterior to this constriction, the posterior ends of the arms are strongly bent laterad and form a sheath around the openings of the pharyngeal gland as in Formica. The pharynx forms a slightly less distinct angle (40 – 50°, n = 2) with the prepharynx (Fig. 11H); its lumen is very narrow and moderately flattened throughout most of its length. Musculature (Figs. 11F,H, 12, 14): M. frontohypo pharyngalis/ M. frontooralis (M. 41/ 0hy1): smaller than in Formica (Figs. 11F, 12B); O: centrally on the frontal area, slightly posterolaterad 0bu3 (Fig. S1B); I: narrower and concentrated on the posterior side of the short posterior end of the oral arm (Fig. 11F). M. clypeopalatalis (M. 43/ 0ci1), two components (Figs. 11H, 12B,C, 14A,D): M43a/ 0ci1a: O: similar to Formica (Fig. S1B), I: narrower than in Formica. M43b/ 0ci1b: not as asymmetric as in Formica; O: anterior, broad portion of the clypeus, narrowing posteriorly (Fig. S1B): I: only on the sclerotized keel of the dorsal prepharyngeal wall. M. clypeobuccalis (M. 44/ 0bu1): very flat but longer (and with slightly larger relative volume) than in Formica (Figs. 11H, 12B); O: along the thin posterior clypeal portion between the antennal sockets (Fig. S1B). I: dorsal prepharyngeal wall posterad the sclerotized keel and anterad the frontal ganglion. M. frontobuccalis anterior (M. 45/ 0bu2): overall similar to Formica, but with a different attachment angle (Fig. 11H), originating around the middle dorsal cephalic region (Fig. S1B). M. frontobuccalis posterior (M. 46/ 0bu3): not recognizable as a separate bundle, likely very closely adjacent to 0bu2 or reduced. M. frontobuccalis lateralis/ M. tentoriooralis (M. 47/ 0hy2): similar to that of Formica, but longer due to the anteriorly shifted antennal sockets (Figs. 11F, S1B); insertion area narrower due to the short posterior ends of the oral arms (Fig. 11F). M. tentoriobuccalis anterior (M. 48/ 0bu5) (possibly together with M.50/ 0bu6): Appears more massive, with much larger relative volume (0.9% vs. 1.7%); not constricted by the infrabuccal pouch; origin and insertion as in Formica (Figs. 11H, 12B, 14B). M tentoriopharyngalis (M. 52/ 0ph2): very similar to that of Formica (Fig. 11H). M. transversalis buccae (M. 67)/ M. oralis transversalis (0hy9): a well-developed layer of transverse muscles between the straight portion of the dorsal arms of the sitophore plate, directly anterior to the frontal ganglion (Fig. 11F,H), and an additional thin layer of transverse fibers connecting the arms on the ventral side posterior to the frontal ganglion. M. pharyngoepipharyngealis (Mpe): very strongly developed longitudinal muscle bundles connecting the anterior pharynx and the epipharynx; the median portion of three thin dorsal bundles (Mped, Figs. 11F,H, 12B) connects the posterior end of the epipharyngeal sclerotized keel with the insertion site of 0bu2; the lateral bundles attach close to the sclerotized keel; additionally, two large paired lateral bundles are present (Mpel, Figs. 11F, 14A), one of them originating laterally, the other one mesally on the dorsal wall formed by the oral arms; the mesal bundle inserts on the sclerotized keel and the lateral bundle on the epipharynx, where it bends into the buccal tube. 3.11. Cephalic glands Formica rufa The mandibular gland (HAO_0000509) (mdg, Figs. 5A,B, 13B) is composed of ca. 50 large cells forming a flat, cup-shaped layer, adjacent to a roughly spherical reservoir (mdr, Fig. 13B), which is connected to the mandalus at the dorsal mandibular base by a thin duct. From the membranous wall of the mandalus, a moderately sclerotized fold reaches towards the duct, result- Richter et al.: Head anatomy of Formicidae 156 ing in an anchor-like shape in cross-section (Fig. 13A). The flat maxillary gland (HAO_0000514) is formed by a group of about 50 slightly smaller gland cells, each of them connected to an individual minute duct. The ducts collectively open in the membranous area laterad the cardinal base (mxgo, Fig. 13C). The well-developed Fig. 12. Histological sections of head of Formica rufa (A,C,E) and Brachyponera luteipes (B,D,F). All sections longitudinal. A,B: Parasagittal region, overwiew. C,D: Parasagittal region, detail of buccal area. E,F: Details of antennal insertion. — Orientation: ← anterior region in all images except E: ↓ anterior region. — Abbreviations: 0an1 – M. tentorioscapalis anterior; 0an3 – M. tentorioscapalis lateralis; 0an4 – M. tentorioscapalis medialis; 0bu1 – M. clypeobuccalis; 0bu2 – M. frontobuccalis anterior; 0bu3 – M. frontobuccalis posterior; 0bu5 – M. tentoriobuccalis posterior; 0ci1a – M. clypeopalatalis, unpaired portion; 0ci1b – M. clypeopalatalis, paired portion; 0hy1 – M. frontooralis; 0hy3 – M. tentoriohypopharyngalis; 0hy7 – M. praementosalivarialis; 0hy9 – M. oralis transversalis; 0hy12 – M. hypopharyngosalivarialis; 0la5 – M. tentoriopraementalis; 0la11 – M. praementoparaglossalis; 0la12 – M. praementoglossalis; 0md3 – M. craniomandibularis externus; bb – bulbus of scapus; br – brain; bt – buccal tube; dhy – distal hypopharynx; ep – epipharynx; epk – epipharyngeal keel; fmo – functional mouth opening; ga – galea; gl – glossa; glvs – ventral glossal sclerite; hyb – hypopharyngeal button; ibp – infrabuccal pouch; lb – labium; lbr – labrum; lc – lacinia; md – mandible; Mped – M. pharyngoepipharyngalis, dorsal portion; oa – oral arm; ph – pharynx; phg – pharyngeal gland; pph – prepharynx; pphg – prepharyngeal gland; sog – suboesophageal ganglion; sv – salivarium; svd – salivary duct; toa – torular apodeme. — Symbols: white arrow – fringe of long microtrichia along functional mouth opening. 157 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 prepharyngeal (/ propharyngeal) gland laterad the prepharynx (pphg, Figs. 11D, 13B,D) is of rather irregular shape; mesally it extends into the space between the buccal tube and the main prepharyngeal part; it opens with numerous very thin ducts (pphgd, Fig. 13D) on a sieve plate at the lateral edges of the buccal tube (pphgo, Fig. 13A,D). The pharyngeal (“postpharyngeal”) gland (phg, Figs. 11A – E, 12A), by far the largest in the head (ca. 3.5% of the head volume), opens dorsolaterally into the pharynx at the level of the anatomical mouth; its openings are stabilized by the posterior ends of the oral arms (phgo, Fig. S2B); it is composed of more than 40 tightly packed tubes surrounded by thick gland epithelium; it completely encloses the brain dorsally and anterolaterally; anteriorly, about four ventrally directed tubes on each side almost completely enclose the anterior pharynx; the tubes coalesce close to the openings, thus forming more extensive reservoirs. Fig. 13. Histological sections of head of Formica rufa. A,B,C: Cross sections. D,E: Frontal sections. — Orientation: A,B,C: ↓ ventral; D: ↓ anterior region; E: ← anterior region. — Abbreviations: 0an4 – M. tentorioscapalis medialis; 0bu1 – M. clypeobuccalis; 0bu5 – M. tentoriobuccalis posterior; 0ci1b – M. clypeopalatalis, paired portion; 0hy2 – M. tentoriooralis; 0hy3 – M. tentoriohypopharyngalis; 0hy7 – M. praementosalivarialis; 0hy9 – M. oralis transversalis; 0la5 – M. tentoriopraementalis; 0la11 – M. praementoparaglossalis; 0la12 – M. praementoglossalis; 0la14 – M. praementopalpalis externus; 0md3 – M. craniomandibularis externus; 0mx3 – M. tentoriocardinalis; 0mx7 – M. stipitogalealis; 0mx8 – M. stipitopalpalis externus; 0la14 – M. praementopalpalis externus; al – atala; ata – anterior tentorial arm; bpb – basiparaglossal brush; bt – buccal tube; cd – cardo; dhy – distal hypopharynx; ep – epipharynx; hyb – hypopharyngeal button; hyr – hypopharyngeal rod; ibp – infrabuccal pouch; lc – lacinia; ll – lateral tentorial lamella; md – mandible; mda – mandibular apodeme; mdgd – mandibular gland duct; mdgr – mandibular gland reservoir; Mpel – M. pharyngoepipharyngalis, lateral portion; mxg – maxillary gland; mxgo – maxillary gland opening; oa – oral arm; plb – palpus labialis; pma – premental arm; pmx – palpus maxillaris; pph – prepharynx; pphg – prepharyngeal gland; pphgo – prepharyngeal gland opening; sc – scapus; sp – sitophore plate; spc – stipito-premental conjunctivum; st – stipes; sv – salivarium; svd – salivary duct; svo – salivary opening. Richter et al.: Head anatomy of Formicidae 164 hashimoto’s coding for the subfamily. This indicates that the character shows more variation than previously suggested. The labial muscles are largely conserved like those of the maxilla. While 0la5 originates on the posterior tentorium in A. aspersus, its usual area of origin is apparently the postgenal area close to the occipital foramen. Other differences concern mostly the size and sarcomere length (PaUL et al. 2002), potentially interesting in a functional context, but likely phylogenetically uninformative. The infrabuccal pouch of ants has been studied with respect to its function (e.g. eisner & haPP 1962; qUinLan & cherret 1978; feBvaY 1981). The morphology and ultrastructure were recently investigated (wanG et al. 2019). Though some of the morphological interpretations of wanG et al. (2019) are questionable (e.g. labelling the galeolacinial complex as “hypopharyngeal plate”, fig. 3B), they provide a detailed documentation of different kinds of microtrichia and the cuticular surface structure of different areas of the pouch. The results generally conform with our observations. Groups of microtrichia on cuticular scales are visible on the surface of the distal part of the pouch, whereas honeycomb-like plates with more or less distinct folds are present proximally, with a transition area between the two patterns. While the infrabuccal pouch is usually referred to as a “filtering device”, actual filtering is achieved by microtrichia and setae of the mouthparts and on the epiand hypopharynx (including the buccal tube), with the pouch functioning as a storage device for the processed material. wanG et al. (2019) demonstrated that the size of the pouch varies depending on the amount of material it contains. This makes comparisons between species difficult, even though distinct differences among taxa do occur. In Formica, for instance, the pouch is unusually large, thus displacing parts of muscle 0bu5/ 6. Another interesting variation is the presence of three folds on the dorsal side of the pouch in Brachyponera, which were observed in all investigated species and specimens of the genus. These structures probably facilitate the enlargement of the pouch when it contains a large mass of substrate. The position of such “reserve folds” is possibly conserved, at least at species level. A structure rarely described in detail is the salivary duct. Even though this structure is apparently rather uniform, certain differences can be found within ants. A similar condition is found in Formica and Brachyponera, where the duct runs through the ventral region of the head capsule, forming a loop anterad the brain and finally connecting to the sclerotized salivarium. The wall is formed by a thin cuticle and a layer of tissue cells with a decreasing thickness at the level of the labium. The loop is partly vertically oriented in Formica, which is not the case in Brachyponera, probably due to the limited space between the brain and infrabuccal pouch. A slightly different condition is found in Wasmannia affinis: the duct appears thin throughout its entire length (although different protocols for histological sections may play a role) and a loop is missing completely (richter et al. 2019). The loop probably facilitates the extension of the duct when the maxillolabial complex (including the salivarium) is protracted. However, this raises the question why it is absent in some species. Further investigations of the salivary duct may clarify functional issues and possibly provide phylogenetic information. Another interesting structure linked with the salivarium is muscle 0hy7. It is generally reduced in Aculeata according to Zimmermann & viLheLmsen (2011), but a muscle originating on the prementum and inserting on the salivarium was identified in Wasmannia (richter et al. 2019) and now also observed in Formica. It is missing in Brachyponera, but at least a very small muscle in this position is present in some other ponerines we examined. The current data suggest that this muscle may be absent in the groundplan of Aculeata, with reversal in ants. It is probably used to open the salivarium, indicating that an active opening of this cavity is important in many ant species, but apparently not in all of them (see Brachyponera). More work is required to assess the precise functional background of this muscle and its potential phylogenetic significance. The labrum of ants is an interesting character complex for different reasons. In most ants it forms different interlocking mechanisms with the maxillolabial complex, and various modifications have evolved with different functional backgrounds. The rectangular, bilobed shape found in generalist species investigated here is conserved in most groups (see also GotwaLd 1969). As this shape also occurs in outgroup taxa (e.g. the crabronid Pison spinolae Shuckard, 1837, cowLeY 1959), it is most likely a groundplan condition. The labrum of some of the trap-jaw ants of the myrmicine Attini underwent profound transformations to act as latch mechanism for their power amplified mandible strike (e.g. GronenBerG 1996; BoLton 1999). Interestingly, the modifications in Strumigenys F. Smith, 1860 and Daceton Perty, 1833 were considered as homologous when these genera were still assigned to the same tribe (e.g. BoLton 2003). However, it is now known that Strumigenys is not closely related to the “dacetines” (ward et al. 2015), and closer scrutiny revealed differences in the labral modifications (J. Katzke unpubl. observation), even within the genus Strumigenys (E. Economo unpubl. observation). Very diverse labral shapes have evolved in the Basiceros genus group (e.g. LonGino & BoUdinot 2013), closely related to Strumigenys (ward et al. 2015). This has recently been investigated in detail for Basiceros (ProBst et al. 2019), revealing adaptions of the labrum for prey capture and a distinct phylogenetic signal in the character system within the genus. The authors discuss the possibility of a second pair of labral muscles (“anterior frontolabral muscle”) based on the presence of a second pair of tendons on the labrum (ProBst et al. 2019: fig. 2G), without presenting direct evidence. The presence of such a muscle appears highly unlikely considering its absence in all other ants and in fact Aculeata (Zimmermann & viLheLmsen 2016; richter et al. 2019). This underlines the importance and potential of detailed anatomical documentation. 165 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 Some noteworthy differences of the epipharynx were observed in the present study. In most ants the membranous main part of this structure, i.e. the part not fused to the hypopharynx to form the prepharyngeal tube, is covered with rows of minute microtrichia (see Formica, Wasmannia, richter et al. 2019). Additionally, the epipharynx of Brachyponera bears a band of much longer microtrichia that extends along the anterior margin of the functional mouth opening and expands into a more or less triangular patch around the epipharyngeal midline. The microtrichia of the preoral cavity are usually interpreted as part of a filter apparatus preventing solid particles from entering the digestive tract (e.g. eisner & haPP 1962; wanG et al. 2019). A varying vestiture might thus be associated with the preferred food, which is probably mainly arthropod prey in the case of species of Brachyponera (schmidt & shattUcK 2014). It is noteworthy in this context that B. luteipes (ZhoU et al. 2007) and also B. senaarensis (deJean & LachaUd 1994) belong to the few ponerine species also feeding on seeds. However, whether the specific armature of microtrichia is related to the preferred diet, cannot be clarified with the currently available data. It is also important in this context that recent studies show that ponerine ants rely more on plant materials as food than was previously assumed (hanisch et al. 2019). Therefore, possible correlations between structures involved in feeding and carnivorous habits should be carefully considered in each case. 4.5. Cephalic digestive tract, glands and nervous system The prepharynx of ants (pph, Figs. 11C – H, 12A – D, 13A,B, 14A,D,E, S1G,H, S2A,B), previously interpreted as the pharynx (ph, Figs. 11C – H, 12A, 14B, S1G,H, S2C), was only recently correctly homologized (richter et al. 2019). Many features of this part of the digestive tract have not been investigated in detail so far. The buccal tube (bt, Figs. 11D,E,G,H, 12A – D, S1G,H) has been considered as a distinctive prepharyngeal subunit based on a sharp bend separating it from the remaining prepharyngeal tube. This is more strongly pronounced in ants than in other groups of Aculeata, where the anteriormost part of the cephalic digestive tract is also more or less distinctly curved (Zimmermann & viLheLmsen 2016). It is evident, however, that the sharp bend in ants is associated with the deep retraction of the maxillolabial complex, as it almost completely disappears when this structure is in a protracted position (see Fig. 12B,D). Comparisons with other aculeates that strongly retract the maxillolabial complex (e.g. Mutillidae, osten 1982) could help to clarify whether this condition is unique to ants, or whether a sharp bend in the prepharynx is generally associated with strongly retracted ventral mouthparts. Another prepharyngeal character complex is the sclerotization pattern. The sitophore plate, the sclerotized ventral (hypopharyngeal) ventral wall of the prepharynx (Fig. 12A,B), is a groundplan apomorphy of Hymenoptera (viLheLmsen 1996; BeUteL & viLheLmsen 2007). However, the epipharyngeal dorsal wall of the prepharynx also displays distinct sclerotized elements in ants, just posterior to the buccal tube. In Wasmannia this is a simple flat sclerite. In Brachyponera this structure additionally bears a sclerotized keel along its midline (epk, Figs. 11H, 12B, 14A,D). In Formica this area is rather weakly sclerotized and hump-shaped. In all three species this is the insertion area of a part of M. clypeopalatalis (0ci1) and also of the longitudinal pharyngo-epipharyngeal muscle, which is generally very large in Hymenoptera (BeUteL & viLheLmsen 2007). Even though the presently available information is insufficient for a reliable evolutionary interpretation, this character system should be carefully considered in future studies. A possible explanation for the presence of a sclerotized keel in Brachyponera is the strong median constriction of the clypeus, which results in a very narrow M. clypeopalatalis (0ci1). The keel provides additional surface for the insertion of its fibers. The most diverse sclerotized prepharyngeal structures are the oral arms (oa, Figs. 1C,D,F, S1G,H). These hypopharyngeal sclerites stabilize the prepharynx and their posterior parts are insertion sites of the oral muscles 0hy1 and 0hy2. Additionally, these posterior parts of the arms enclose the opening of the pharyngeal gland, indicating that the oral muscles are likely involved in opening and closing the aperture of this gland (Verhaegen unpubl. observations). The distinct differences in the shape of these arms may contain phylogenetic signal and are potentially relevant in the context of dietary specializations. The only detailed comparative study on these structures by Porto et al. (2019) was focused on Apoidea. The authors could demonstrate that some of the changes in the shape of the oral arms (pharyngeal rods in their terminology) and the general configuration of the hypopharynx are useful characters on higher and/or lower taxonomic levels (Porto et al. 2019). A character system not investigated by Porto et al. (2019) but certainly of interest in ants is the prepharyngeal and pharyngeal musculature (Figs. 11 C – H, 13, 14). In addition to minor variations in shape and size of the individual muscles, some characters may have distinct functional implications and also contain phylogenetic signal. A seemingly unusual feature of ants is the subdivision of M. clypeopalatalis (0ci1) into an unpaired subcomponent inserting at the functional mouth opening (part a, retractor of buccal tube of PaUL et al. 2002) and one part inserting on the epipharyngeal sclerite discussed above (part b). In other groups of Aculeata, the anterior component was interpreted as 0ci1 and no posterior part was described (Zimmermann & viLheLmsen 2016). The only muscle inserting on the dorsal prepharyngeal wall described by these authors is M. clypeobuccalis (0bu1), which is sometimes subdivided into two individual bundles (Zimmermann & viLheLmsen 2016). It is conceivable that these seemingly different configurations are rather due to homologization problems than to evolutionary changes in the character system. BeUteL & viLheLm- Richter et al.: Head anatomy of Formicidae 166 sen (2007) described M. clypeopalatalis (M. 43/ 0ci1) as consisting of two subunits in basal hymenopterans, compatible with the presence of two separate bundles in most insects (v. KéLer 1963). This suggests that our interpretation of the anterior unpaired muscle and of the anterior paired bundle as two subunits of 0ci1, and the posterior paired bundle as 0bu1 is consistent with the general interpretation in the literature (e.g. BeUteL et al. 2014). Another interesting case is M. tentoriooralis (0hy2). It was previously interpreted as a subunit of M. frontooralis (0hy1) by richter et al. (2019), even though a possible interpretation as 0hy2 (M. 47) was discussed in that study. Our investigations show that the frontoclypeal ridge, which is the area of origin of this muscle in some other aculeate goups (Zimmermann & viLheLmsen 2016), is very closely adjacent (Formica) or even fused (Brachyponera) to the antennal acetabulum, which also carries the torular apodeme serving as area of origin of the muscle in question in ants. Considering this configuration, a shift from the frontoclypeal ridge to the torular apodeme, consistent with a homologization as 0hy2, is much more likely, than a shift from the middle frontal region, which would be implied by a homologization with 0hy1. It is an interesting question whether the muscle shifted its origin first and the torular apodeme evolved after this to optimize the attachment site, or whether the muscle shifted to a preformed torular apodeme, which evolved for a different reason in the first place? Comparisons with other poneroid (e.g. Amblyoponinae) or more basal formicid lineages (e.g. Leptanillinae) plus closely related aculeate groups (e.g. Scoliidae and spheciform Apoidea) will likely help to clarify the functional and evolutionary background of the involved structural transformations. A muscle affected by size reduction or even complete loss is 0bu3. It is completely reduced in Brachyponera and very small in Wasmannia and other examined ponerines. In clear contrast, it is large and subdivided into two bundles in Formica and also in Lasius (Janet 1906), suggesting that this may be an apomorphy of Formicinae. The absence of M. verticopharyngalis (0ph1) is very likely an apomorphy of the entire Formicidae, as this muscle is usually present in Aculeata (Zimmermann & viLheLmsen 2016), but has never been found in ants so far. One possible reason for this reduction is the prognathous condition, which results in a shift of different cephalic structures. The brain usually largely fills out the posterior lumen of the head, resulting in very limited space for dorsal pharyngeal muscles in this region. Two large glands are associated with the prepharyngeal/pharyngeal sucking pump, the prepharyngeal and the pharyngeal gland. The shape of both may be phylogenetically relevant. The prepharyngeal gland almost entirely encloses the wall of the infrabuccal cavity, whereas most cells are located behind it in the myrmicine Monomorium pharaonis (Linnaeus, 1758) (Boonen & BiLLen 2016), a condition also found in the myrmicine Wasmannia affinis (richter et al. 2019). A placement of two gland cell clusters laterad the prepharynx and infrabuccal pouch was observed in Formica and also in Camponotus pennsylvanicus (De Geer, 1773) (forBes et al. 1961). For ponerine species only few descriptions of this gland are available, but it is apparently concentrated in the space between the buccal tube and the remaining prepharynx in different Brachyponera species (BiLLen & aL-KhaLifa 2015). Different shapes and positions of the gland are likely related to the spatial configuration of internal structures, for instance the extension of the infrabuccal pouch. The pharyngeal gland also varies considerably in shape in different groups of ants. In most described species it appears “glove-like”, with several tubes connecting to a reservoir that opens near the anatomical mouth (e.g. forBes 1938; PereGrine et al. 1973; BiLLen & aLKhaLifa 2015; BiLLen et al. 2015; richter et al. 2019). This also applies to Formica, where the gland is formed by more than 40 individual tubes. In clear contrast to this, the gland of Brachyponera (including B. sennaarensis BiLLen & aL-KhaLifa 2015) is sack-shaped, with about five more or less separated lobes instead of individual tubes. Additionally, the epithelial cells are more globular. As this general gland shape was also observed in other ponerine species (Gama & crUZ Landim 1981; schoeters & BiLLen 1997) (also in Pseudomyrmecinae, Gama & crUZ Landim 1981), this is arguably a plesiomorphic condition for the group. The groundplan condition of ants is most likely the glove-shaped configuration, which was also found in leptanillines (BiLLen et al. 2013) and other groups of Aculeata (e.g. herZner et al. 2013). Further study may reveal additional phylogenetically informative differences. Some features of the brain are very easy to correlate with the behavior and lifestyle of the two investigated species. The large optic neuropils are certainly linked with the importance of visual orientation for F. rufa (e.g. nichoLson et al. 1999). Brachyponera on the other hand has about ten times less ommatidia and seems less reliant on visual cues, showing distinctly reduced optic neuropils and lacking ocelli and ocellar nerves. Interestingly, the distinct anterior shift of the eye causes the optic nerve to run at a distinct angle through the fibers of 0md1. Due to the anterior placement of the eyes, fibers of this muscle can occupy regions that are occupied by the optic lobes and the compound eye in Formica. This suggests a possible tradeoff between the visual sense and additional space for attachment of mandibular muscle bundles. Another interesting aspect is the general size of the brain relative to the head volume. It is well known that relative brain size increases in smaller species in ants and other groups of organisms (e.g. wehner et al. 2007; seid et al. 2011; LiLico-oUachoUr et al. 2018). The brain of Brachyponera looks proportionally very large, occupying most of the posterior lumen of the head. This is even more pronounced than in Wasmannia affinis (richter et al. 2019), which is even smaller, and thus obviously not only a result of miniaturization (PoLiLov 2015). Even though specific neuroanatomical features of ants were already treated in several studies (e.g. KeLBer et al. 2009; mcKenZie et al. 2016), there is probably still potential in 167 ARTHROPOD SYSTEMATICS & PHYLOGENY — 78 (1) 2020 comparing size and shape of the brain in different ant lineages. As the brain limits the space for other head structures, its shape is also influenced by the presence and extension of other organs (see optic nerve and 0md1). Comparative studies could reveal tradeoffs between the complexity of the brain and the mechanical efficiency of different functional elements of the head. The tracheal configuration is apparently a highly conserved character system. The general branching pattern described here was also found in Wasmannia affinis (richter et al. 2019), Camponotus pennsylvanicus (Keister 1963) and even in representatives of the basal hymenopteran family Xyelidae (BeUteL & viLheLmsen 2007). In summary, our morphological investigations revealed a broad spectrum of characters relevant in the context of the evolution of ant head structures. Some of them have received very little attention before, like the tentorium and other endoskeletal elements, or also the salivarium and associated structures. Another neglected character system is the cephalic digestive tract, with a broad spectrum of variation likely relevant in a functional and phylogenetic context. The mandibles and the labrum have already been investigated in some detail. Nevertheless, more structural information on these key elements of the head of ants will likely lead to a better understanding of important transformations in the evolution of the group. 5. Acknowledgements Our very great thanks are due to Brendon Boudinot (UC Davis, Department of Entomology and Nematology) for providing a very thorough and detailed review within very short time. This helped greatly to improve our study. Many helpful comments made by the editor in chief Dr. Klaus-Dieter Klass (Senckenberg Museum für Tierkunde Dresden) are also gratefully acknowledged. We also thank the OIST Imaging Section for providing access to the SEM and µCT scanner, especially Sasaki Toshiaki who helped with sample preparation and explained the SEM facilities, and Komoto Shinya for general support with the µCT scanner. We are grateful to An Vandoren for making the serial histological sections. 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Electronic Supplement Files at http://www.senckenberg.de/arthropod-systematics ASP_78-1_Richter_Electronic_Supplements.zip DOI: 10.26049/ASP78-1-2020-06/1 File1: richter&al-formicidaehead-asp2020-electronicsupple ment-1.pdf. — Fig. S1: Volume renderings of heads of F. rufa (CASENT0790267: A,C – E,G) and B. luteipes (CASENT0709409: B,F,H). A,B: ventral view of the head capsule, cut open to reveal the dorsal endoskeleton and the insertions of the dorsal head muscles. C: posterior view, showing the functionless muscle X. D: lateral view of the anterior head, showing the connection of the stipito-premental conjunctivum with the maxilla and hypopharynx/ infrabuccal pouch. E,F: lateral view of the anterior head, showing the interaction of the labrum with the maxillolabial complex. G,H: lateral view of anterior cephalic digestive tract, showing the oral arms. — Abbreviations: 0bu1 – M. clypeobuccalis; 0bu2 – M. frontobuccalis anterior; 0bu3 – M. frontobuccalis posterior; 0ci1a – M. clypeopalatalis, unpaired portion; 0ci1b – M. clypeopalatalis, paired portion; 0hy1 – M. frontooralis; 0hy2 – M. tentoriooralis; 0lb2 – M. frontoepipharyngalis; 0md1 – M. craniomandibularis externus; 0mx1 – M. craniocardinalis externus; 0mx3 – M. tentoriocardinalis; 0mx4 – M. tentoriostipitalis anterior; ant – antennifer; bt – buccal tube; cd – cardo; epk – epipharyngeal keel; esr – epistomal ridge; ga – galea; lc – lacinia; Ibp – infrabuccal pouch; lbr – labrum; lbrp – labral process; MX – functionless muscle X; mxg – maxillary gland; oa – oral arm; ph – pharynx; pmx – palpus maxillaris; pph – prepharynx; spc – stipito-premental conjunctivum; st – stipes — Colors: beige/ brown – mouthparts; green – cephalic digestive tract (prepharynx and pharynx); grey – cuticle; orange/ red – muscles; purple – glands. — Fig. S2: Histological sections of heads of F. rufa (A – E) B. luteipes (F,H) and B. chinensis (G). All sections transverse. A: Section at the level of the maxillolabial complex showing especially the interaction of the stipes with the hypostomal triangular process. B: Section on the level of the pharyngeal gland opening. C: Section on the level of the eyes, showing the Y-shaped pharynx. D,E: Sections showing the stipito-premental-conjunctivum, D further posterior than E. F: Section at the level of the posterior brain, showing the tiny dorsal tentorial arm. G,H: Section at the level of the anterior mouthparts, showing the mandibular pit gland. — Abbreviations: ata – anterior tentorial arm; dhy – distal hypopharynx; dta – dorsal tentorial arm; hyr – hypopharyngeal rod; ibp – infrabuccal pouch; lcs – lacinial sclerite; md – mandible; mdp – mandibular pit; mdpg – mandibular pit gland; oa – oral arm; ph – pharynx; phg – pharyngeal gland; phgo – pharyngeal gland opening; pph – prepharynx; pma – premental arm; spc – stipito-premental conjunctivum; stis – stipes internal sclerite; svd – salivary duct; — Symbols: white arrowhead – interaction of lateral stipes with anterior concavity of hypopharyngeal triangular process. File 2: richter&al-formicidaehead-asp2020-electronicsupple ment-2.xlsx. — Volume measurements. File 3: richter&al-formicidaehead-asp2020-electronicsupple ment-3.docx. — Table S1: Comparison of morphological features between B. luteipes, F. rufa and W. affinis, data on the latter are based on richter et al. (2019).