A peer-reviewed open-access journal ZooKeys 693: 33-93 (2017) RS Re #ZooKeys http:/ / ZOO keys -pen soft. net Launched to accelerate biodiversity research Next-generation morphological character discovery and evaluation: an X-ray micro-CT enhanced revision of the ant genus Zasphinctus Wheeler (Hymenoptera, Formicidae, Dorylinae) in the Afrotropics Francisco Hita Garcia', Georg Fischer', Cong Liu', Tracy L. Audisio', Evan P. Economo' | Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son 904-0495, Japan Corresponding author: Francisco Hita Garcia (fhitagarcia@gmail.com) Academic editor: B. Fisher | Received 3 April 2017 | Accepted 12 July 2017 | Published 23 August 2017 http.//zoobank.org/E670A42A- 1 1 F8-437E-90B6-C880DC2D8E2F Citation: Hita Garcia F, Fischer G, Liu C, Audisio TL, Economo EP (2017) Next-generation morphological character discovery and evaluation: an X-ray micro-CT enhanced revision of the ant genus Zasphinctus Wheeler (Hymenoptera, Formicidae, Dorylinae) in the Afrotropics. ZooKeys 693: 33-93. https://doi.org/10.3897/zookeys.693.13012 Abstract New technologies for imaging and analysis of morphological characters offer opportunities to enhance revisionary taxonomy and better integrate it with the rest of biology. In this study, we revise the Afrotropi- cal fauna of the ant genus Zasphinctus Wheeler, and use high-resolution X-ray microtomography (micro- CT) to analyse a number of morphological characters of taxonomic and biological interest. We recognise and describe three new species: Z. obamai sp. n., Z. sarowiwai sp. n., and Z. wilsoni sp. n. The species delimitations are based on the morphological examination of all physical specimens in combination with 3D scans and volume reconstructions. Based on this approach, we present a new taxonomic discrimina- tion system for the regional fauna that consists of a combination of easily observable morphological characters visible at magnifications of around 80-100 x, less observable characters that require higher magnifications, as well as characters made visible through virtual dissections that would otherwise require destructive treatment. Zasphinctus are rarely collected ants and the material available to us is comparatively scarce. Consequently, we explore the use of micro-CT as a non-invasive tool for the virtual examination, manipulation, and dissection of such rare material. Furthermore, we delineate the treated species by pro- viding a diagnostic character matrix illustrated by numerous images and supplement that with additional evidence in the form of stacked montage images, 3D PDFs and 3D rotation videos of scans of major body Copyright Francisco Hita Garcia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 34 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) parts and full body (in total we provide 16 stacked montage photographs, 116 images of 3D reconstruc- tions, 15 3D rotation videos, and 13 3D PDFs). In addition to the comparative morphology analyses used for species delimitations, we also apply micro-CT data to examine certain traits, such as mouthparts, cuticle thickness, and thoracic and abdominal muscles in order to assess their taxonomic usefulness or gain insights into the natural history of the genus. The complete datasets comprising the raw micro-CT data, 3D PDFs, 3D rotation videos, still images of 3D models, and coloured montage photos have been made available online as cybertypes (Dryad, http://dx.doi.org/10.5061/dryad.4s3v1). Keywords 3D model, cuticle, cybertype, micro-CT, morphology, mouthparts, new species, taxonomy Introduction The primary goal of taxonomic science is to organize life by developing hypotheses delimiting species and higher groups (Winston 1999; Wagele et al. 2011), but a sec- ondary goal is to generate and curate information about species that can be useful for future taxonomic work as well as the broader fields of biology (Wheeler and Valdecasas 2007). The moment of description is particularly important, at which time an account of the species, supported and illustrated with information of some nature, is put on the record for posterity (Wheeler et al. 2004). The emergence of new technologies offers new opportunities for enhancing taxonomic descriptions and broadening their utility for other biological disciplines (La Salle et al. 2009; Schlick-Steiner et al. 2010; Faulwetter et al. 2013). Here, we continue a recent series of taxonomic works (Fischer et al. 2016; Sarnat et al. 2016; Hita Garcia et al. 2017a) exploring the use of three-di- mensional (3D) data generated from X-Ray microcomputed tomography (micro-CT) for ant taxonomy, with a revision of the Afrotropical species of the genus Zasphinctus Wheeler. In our treatment of this group, we focus on exploring the potential for using micro-CT data for new character discovery and evaluation, enhancing the way de- scriptions themselves are organized and presented given the new data, and identifying anatomical characters that can be linked to the biology and ecology of the organism. Micro-CT is a powerful imaging technology that enables the generation of high- resolution, virtual, and interactive 3D reconstructions of whole specimens or parts thereof. Such reconstructions can be virtually rotated, sectioned, measured, and dis- sected, thus allowing a comprehensive 3D analysis of the anatomy and morphology of the studied organisms (e.g. Faulwetter et al. 2013; Friedrich et al. 2014). Since its initial use for the study of insect cephalic morphology (Hérnschemeyer et al. 2002), micro-CT has gradually gained popularity as a fundamental tool for a variety of re- search fields that rely on the exact examination of animal morphology. It has been pri- marily employed for comparative and functional morphology (e.g. Beutel et al. 2008 2010; Zimmermann et al. 2011; Wojcieszek et al. 2012; Lipke et al. 2015), but also for the study of insect fossils in amber (Dierick et al. 2007; Barden and Grimaldi 2012), forensic entomology (Richards et al. 2012), and developmental biology (Metscher 2009). Surprisingly late, micro-CT has also been applied for invertebrate taxonomy Next-generation morphological character discovery and evaluation... 35 of myriapods (Stoev et al. 2013; Akkari et al. 2015), spiders (Michalik and Ramirez 2013), earthworms (Fernandez et al. 2014), and flatworms (Carbayo et al. 2016; Car- bayo and Lenihan 2016). Despite its great potential for the taxonomy of extant insects, it has so far only been used in lepidopterans (Simonsen and Kitching 2014) and ants (Fischer et al. 2016; Sarnat et al. 2016; Hita Garcia et al. 2017a, b). Compared to traditional methods like histology, the use of micro-CT provides the means for a quick and non-invasive generation of almost artefact-free morphological raw data for visualisation in 3D (Faulwetter et al. 2013; Friedrich et al. 2014). The non-destructive nature of the technique is of crucial importance for systematic research since it permits the scanning of very rare species and/or museum material, and it can be very well applied to type material. One drawback of modern collections-based sys- tematics is that often important reference or type material is not available or accessible for examination, thus effectively slowing taxonomic progress (Smith and Blagoderov 2012; Wheeler et al. 2012; Faulwetter et al. 2013). One recent development with the potential to improve this situation is the establishment of virtual natural history collections that provide rapid access to anatomically correct and permanent digital reconstructions of type material. Based on the idea of Godfray (2007) to create virtual types, Faulwetter et al. (2013) introduced the concept of “cybertypes” and proposed a workflow to generate such virtual collections. Shortly afterwards Stoev et al. (2013) and Akkari et al. (2015) used micro-CT scanning for the description of new myriapod species and presented the first invertebrate cybertypes. Recently, Hita Garcia et al. (2017a, 2017b) critically explored the use of micro-CT for ant taxonomy and pro- posed the first ant cybertypes. Another great advantage is the application of micro-CT for virtual dissections and character identification, which has proven successful for a variety of invertebrates, such as myriapods (Blanke and Wesener 2014), Neuroptera (Zimmermann et al. 2011), and dragonflies (Simonsen and Kitching 2014). Compared to other insect groups, ant taxonomy is thoroughly founded on the morphology of the very simplified worker caste. Despite that several authors also ex- amine reproductive castes (e.g. Boudinot 2014; Yoshimura and Fisher 2014; Probst et al. 2015), this is often not possible due to the sheer absence of reproductives in collec- tions and unknown associations between castes. Consequently, ant taxonomy predom- inantly focuses on phenotypical differences between workers of different species (Ward 2010). Due to the simplified female morphology of the worker caste, the vast majority of studies use very basic characters for species delimitations, mostly setation, surface sculpture, size, and shape differences of few body parts, especially eyes, mandibles, pro- podeal spines and the waist segments (e.g. Fernandez 2004; Sarnat 2008; Bolton 2007; Fischer et al. 2012; Branstetter 2013; Hita Garcia et al. 2014). This approach offers the advantage that taxonomic studies of ants are relatively easy to perform. However, such a simplified approach increasingly often reaches its limits and cannot provide discriminatory evidence for species delimitations. Integrative taxonomy approaches including morphometrics (e.g. Csész and Schulz 2010; Csész and Fisher 2015), mo- lecular phylogenetics (e.g. Branstetter 2012; Blaimer 2012), next generation sequenc- ing (Fischer et al. 2015; Jesovnik et al. 2017), or combinations of multiple data sources 36 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) (e.g. Schlick-Steiner et al. 2010; Csész et al. 2014), have proven efficient to resolve the relationships within morphologically challenging ant genera. Nevertheless, in recent years, there have been very few approaches of advancing and improving traditional worker-based character sets used for species diagnostics (e.g. Bolton and Fisher 2012; Yoshimura and Fisher 2014). The ant genus Zasphinctus Wheeler is a moderately small genus distributed in the Afrotropical, Indomalayan, and Australasian regions. Currently, 20 valid species are recognised (Bolton 2017), of which the vast majority occur in the Australasian region (15 spp. from Australia, one from New Caledonia, and one from New Guinea). By contrast, there is only one species known from South East Asia and two from the Afrotropical region. These ants are rarely collected and the material housed in natural history collections is somewhat limited. Perhaps due to its moderate species richness and relative rarity of collections, knowledge about the biology of Zasphinctus is rather incomplete. Wilson (1958) and Brown (1975) provided field notes about the biology of Z. steinheili (Forel) and laboratory observations of Z. caledonicus (Wilson). Both species turned out to be myrmecophagous feeding on larvae and adults of a variety of ant species captured during nest raids. Later, Buschinger (1989) confirmed this behav- iour in Z. steinheili under laboratory conditions. Based on data from Z. steinheili and Z. caledonicus, colonies are found in soil and range in size from 100 to 500 workers. However, whether or not this is true for other Zasphinctus species, especially outside Australia, remains unknown. A taxonomic problem commonly encountered in doryline ants is the existence of two or even three parallel taxonomic systems: a female-based one, which often splits into worker-based and queen-based, and a male-based one (e.g. Wilson 1964; Jaitrong and Yamane 2011; Borowiec 2016). Workers and reproductives are rarely collected together, and usually only one caste is available for taxonomic evaluation, which cre- ates great difficulties for the association of workers with queen and/or males. ‘This situation is especially problematic in, but not restricted to, the army ant genera Ae- nictus Shuckard and Dorylus Fabricius (Wilson 1964; Gotwald 1995). Prior to this study the taxonomy of Zasphinctus in the Afrotropical region was solely based on two male-based species described more than a hundred years ago from West African savan- nahs (Santschi 1915). Since then, no further taxonomic studies on Zasphinctus were published and the scarce male-based or worker-based material in collections has been tentatively assigned to one of these male-based species without evidence of any associa- tion. Recent collections in Kenya (Hita Garcia et al. 2009), Mozambique, and Uganda have yielded worker-based material without any males, thus not associable to any valid species name. Consequently, in order to use the genus for ant diversity inventories or conservation studies, it is imperative to create a taxonomic system founded on the worker caste. In this study, we provide a taxonomic revision of the genus for the Afrotropical region on the basis of the worker caste. All three species treated herein are newly de- scribed. The taxonomic decision-making was founded on the examination of all physi- cal specimens, as well as on 3D volume reconstructions of high-resolution micro-CT Next-generation morphological character discovery and evaluation... 37 scanning data from several specimens per species, if available. Based on that approach, our newly developed taxonomic discrimination system consists of a new character set, which is unusual in ant taxonomy. The backbone of it is still based on easily observable morphological characters visible at magnifications of around 80 to 100 x. On the basis of micro-CT scanning data, we also present less perceivable characters that require higher magnifications, previously only achieved through scanning electron microscopy (SEM), as well as characters that are usually hidden or partly obscured and would require destructive treatment of the physical material. Through virtual dissections of 3D reconstructed specimens, we recovered several of these hidden characters. Further- more, we present our results in a different way compared to previous ant taxonomy re- visions by including numerous stacked montage images, micro-CT microphotographs, 3D PDFs, and 3D rotation videos of relevant body parts in addition to full specimens. We argue that such a wealth of illustrative power obviates the need for lengthy descrip- tions and a traditional identification key. Instead, we opt for a thorough genus descrip- tion and brief species accounts supplemented by a detailed diagnostic species character matrix with high quality illustrations for all characters. Finally, we use micro-CT data to examine traits, such as mouthparts, cuticle thickness, thoracic and abdominal mus- cles, and the sting in order to gain insights into the natural history of the genus. The complete datasets comprising the micro-CT raw data, 3D PDFs, 3D rotation videos, and coloured montage photos have been made available online as cybertypes (Hita Garcia et al. 2017c). Material and methods Abbreviations of depositories Institutional museum collection abbreviations follow Evenhuis (2017). The material on which this study is based is located and/or was examined at the following institutions: BMNH. The Natural History Museum, London, U.K. CASC California Academy of Sciences, San Francisco, U.S.A. MCZC —- Museum of Comparative Zoology, Harvard University, Cambridge, U.S.A. NMKE National Museums of Kenya, Nairobi, Kenya ZFMK Zoological Research Museum Alexander Koenig, Bonn, Germany Material examined and terminology The general terminology for ant morphology predominantly follows Keller (2011) and to a lesser extent Bolton (1990) and Borowiec (2009, 2016). For the description of mouthparts, we used the terminology of Gotwald (1969) and Keller (2011). The ter- minology for the description of surface sculpturing follows Harris (1979). 38 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Montage images and line drawings All raw images were taken with a Leica DFC450 camera attached to a Leica M205C microscope and Leica Application Suite (version 4.1). The raw photo stacks were then processed to single montage images with Helicon Focus (version 6). All montage im- ages used in this publication are available online and can be seen on AntWeb. Vector illustrations were created with Adobe Illustrator (version CS 5) by tracing specimen photographs. Measurements and indices We measured 17 physical workers with a Leica M125 equipped with an orthogonal pair of micrometers under magnifications of 80 to 100 x. Measurements and indices are presented as minimum and maximum values with arithmetic means in parenthe- ses. In addition, measurements are expressed in mm to two decimal places. Since the workers of all three species treated herein are eyeless we omit any eye measurements and do not generate an ocular (or eye) index. We refrain from using total length since it is dificult to measure in already dry-mounted specimens that are not orientated in a straight line. The standard measurements HW and WL provide sufficient information about general body size dimensions. The following measurements and indices partly follow Bolton and Fisher (2012), Hita Garcia and Fischer (2014) and Hita Garcia et al. (2014) or are used here for the first time (Fig. 1): HL Head Length: maximum distance from the midpoint of the anterior clypeal margin or from a line spanning the anterior-most points of the frontal lobes (depending on which projects farthest forward) to the midpoint of the pos- terior margin of head, measured in full-face view (Fig. 1C). HW Head Width: the maximum width of the head capsule, measured in full-face view (Fig. 1C). SL Scape Length: the maximum straight-line length of the scape, excluding the basal constriction or the neck (Fig. 1C). PH Pronotal Height: the maximum height of the pronotum in profile (Fig. 1A). PW Pronotal Width: the maximum width of the pronotum in dorsal view (Fig. 1B). DML Dorsal Mesosoma Length: maximum length of mesosomal dorsum from antero- dorsal margin of pronotum to dorsal margin of propodeal declivity (Fig. 1B). WL Weber's Length of Mesosoma: the maximum diagonal length of the meso- soma in profile, from the angle at which the pronotum meets the cervix to the posterior basal angle of the metapleuron (Fig. 1A). MFL Metafemur Length: the maximum straight-line length of the metafemur, measured in dorsal view (Fig. 1D). PTL Abdominal Segment II (petiole) Length: the maximum length of abdominal segment II (petiole), measured in dorsal view (Fig. 1B). Next-generation morphological character discovery and evaluation... a) PH PTH a3y A4W ABW AGW MFL Figure I. Schematic line drawings illustrating the measurements used in this study. A Body in profile with measuring lines for PH, PTH, and WL B Mesosoma and metasoma in dorsal view with measuring lines for A3L, A3W, A4L, A4W, ASL, ASW, AGL, AGW, DML, PW, PTL, and PTW C Head in full- face view with measuring lines for HL, HW, and SL D Metafemur in dorsal view with measuring line for MEL. 40 PTH DMI DMI2 LMI LPI DPI DA3I DA4I DASI DAGI Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Abdominal Segment II (petiole) Height: the maximum height of the petiolar tergum in profile view, including laterotergite, excluding petiolar sternum (Fig. 1A). Abdominal Segment II (petiole) Width: the maximum width of abdominal segment II (petiole), measured in dorsal view (Fig. 1B). Abdominal Segment II Length: the maximum length of abdominal seg- ment III, measured in dorsal view (Fig. 1B). Abdominal Segment HI Width: the maximum width of abdominal segment III, measured in dorsal view (Fig. 1B). Abdominal Segment IV Length: the maximum length of abdominal seg- ment IV, measured in dorsal view (Fig. 1B). Abdominal Segment IV Width: the maximum width of abdominal segment IV, measured in dorsal view (Fig. 1B). Abdominal Segment V Length: the maximum length of abdominal segment V, measured in dorsal view (Fig. 1B). Abdominal Segment V Width: the maximum width of abdominal segment V, measured in dorsal view (Fig. 1B). Abdominal Segment VI Length: the maximum length of abdominal seg- ment VI, measured in dorsal view (Fig. 1B). Abdominal Segment VI Width: the maximum width of abdominal segment VI, measured in dorsal view (Fig. 1B). Cephalic Index: HW / HL x 100 Scape Index: SL/ HL x 100 Dorsal Mesosoma Index: PW / WL x 100 Dorsal Mesosoma Index 2: DML / WL x 100 Lateral Mesosoma Index: PH / WL x 100 Metafemur Index: MFL / HW x 100 Lateral Petiole Index: PTL / PTH x 100 Dorsal Petiole Index: PTW / PTL x 100 Dorsal Abdominal Segment HI Index: A3W / A3L x 100 Dorsal Abdominal Segment IV Index: A4W / A4L x 100 Dorsal Abdominal Segment V Index: ASW / A5L x 100 Dorsal Abdominal Segment VI Index: AGW / AGL x 100 Micro X-ray computed tomography Micro-CT scans were performed using a ZEISS Xradia 510 Versa 3D X-ray micro- scope and the ZEISS Scout and Scan Control System software (version 10.7.2936). The scanned specimens were left attached to their paper point, which was clamped to a holding stage. Scan settings were selected according to yield optimum scan quality: optical magnification of 4 x, exposure times of 1-3 s, binning of two by two pixels, source filter “air”, voltage of 35-85 keV, power of 3—7.5 W, current of 71-88 yA, and Next-generation morphological character discovery and evaluation... 4] Table |. Data summary for micro-CT scanning giving an overview of the specimens and body parts scanned for the three species and presenting specimen data, scan settings, and voxel sizes for the resulting scans (all specimens are workers and all files are in DICOM format). Specs | al ‘ae oe Z_obamai__| full body 40 75 Z. obamai head 70 85 Z. obamai mesosoma CASENT0764127 5 oS 82 Z. obamai metasoma CASENT0764127 4 50 80 Z. sarowiwai | full body CASENT0764650 4 50 81 Z. sarowiwai | mouthparts | CASENT0764652 6 65 84 Z. sarowiwai | full body CASENT0764654 3 40 76 Z. sarowiwai head CASENT0764654 5 60 83 Z. sarowiwai | mesosoma CASENT0764654 4 50 80 Z. sarowiwai | metasoma CASENT0764654 4 45 78 Z. wilsoni full body | MCZ-ENT-00512764 3 55 71 Z. wilsoni head MCZ-ENT-00512764 3 6 70 86 Z. wiloni___|_mesosoma 55 82 Z. wilsoni metasoma 2g? 2 4 45 78 field mode “normal”. ‘The combination of voltage, power and exposure time was set to yield intensity levels of between 10,000 and 15,000 across the whole specimen. Scan- ning duration varied from 1.2 to 2.2 h, depending on exposure time. Full 360 degree rotations were done with a number of 1601 projections. The resulting scans have reso- lutions of 1013 x 1013 pixels and voxel sizes are in range of 0.94—4.6 um. The original file size was 3113.577 MB for all scans. We scanned a varying number of specimens per species, depending on specimen availability and character suitability. An overview of the specimens used and scanning settings is provided in Table 1. Virtual reconstruction and post-processing of raw data 3D reconstructions of the resulting scans were done with XMReconstructor (version 10.7.2936) and saved in DICOM file format (default settings; USHORT 16 bit out- put data type). Post-processing of DICOM raw data was performed with Amira soft- ware (version 6.1.1). Virtual examinations of 3D surface models were performed by using either the “volren” or “volume rendering” functions. ‘The desired volume render- ings were generated by adjusting colour space range to a minimum so that the exterior surface of specimens remained visible at the highest available quality. The 3D models were rotated and manipulated to allow a complete virtual examination of the scanned specimens. Images of shaded surface display volume renderings were made with the “snapshot” function at the highest achievable resolution (usually at around 1500 by 893 pixels). Volumetric surface rendering rotational videos of head, mesosoma, meta- soma, and full body scans were created with the “camera path” object (5—10 keyframes, 42 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) constant velocity for constant rotation speed) and “movie maker” function (param- eters: MPEG format, AntiAlias2, total of 1200 frames at 60 frames per second, and resolution of 1920 x 1080 pixels). Character recognition and virtual dissections In addition to the traditional morphological examination of the physical specimens under a light microscope with magnifications up to 100 x, we virtually examined the full external morphology of the treated species in Amira. For this we compared more than 50 morphological characters potentially significant for dorylines (Bolton 1990; Keller 2011; Borowiec 2016), especially the genera previously grouped as Cerapachyi- nae, in the 3D models and made more than 350 snapshots to assess which characters have diagnostic value. A series of characters were hidden or obscured by other body parts, thus not observable by light microscopy, or only after destructive dissection. Due to the severe lack of material the latter was not an option. In order to examine such characters and explore a wider range of morphology, we used the segmentation function in Amira to deselect body parts obscuring segmented body parts. By doing so we were able to expose every desired structure. We examined the head capsule from all sides, which is usually ventrally and posteriorly obscured by antennae, legs, or anterior mesosoma, as well as ventral metasomal characters usually hidden between propode- um, legs, and different abdominal segments. Based on more than 110 character images per species, we chose to highlight 24 characters of high diagnostic significance for our newly developed species delimitation system (see Table 2 for complete list of examined characters). Furthermore, the volume reconstructions of the mouthparts and muscula- ture were generated by using the segmentation function in Amira. Targeted structures were first visualised by adjusting density and contrast and then segmented by manually tracing their outline slide by slide. Virtual measuring of cuticle thickness In addition to the taxonomic standard measurements of external morphology given above, we also measured the thickness of the exoskeleton cuticle of the cephalic cap- sule, the pronotum, and abdominal segments II (petiole) and III. Measuring was per- formed with Amira by using the 2D measuring tool on slices representing sagittal sec- tions along the median axis of the chosen body parts. For each body part, we measured five times over a defined area (Fig. 2) and calculated the average thickness. Based on Peeters et al. (2017) we put the measurements in context to body size by using the fol- lowing measurements and indices: Cephalic capsule cuticle thickness (CCC): thickness of cuticle of head measured in profile a short distance posterior of torulo-posttorular complex (Fig. 2A). Next-generation morphological character discovery and evaluation... 43 Table 2. List of all important characters examined in this study with assessment of diagnostic potential and information on usage in this study (characters marked with * were used for species delimitations). Characters examined Head characters Shape of head in full-face view Shape of head in profile * Diagnostic assessment and usage none, no significant interspecific variation observed, not used in this study high, used in this study Shape of mandibles none, no significant interspecific variation observed, not used in this study Mandibular dentition none, no significant interspecific variation observed, not used in this study Shape of clypeus Presence of median clypeal tooth * Cuticular apron of clypeus low, no significant interspecific variation observed, not used in this study high, used in this study none, no significant interspecific variation observed, not used in this study Torulo-posttorular complex * high, used in this study Antennal bulbus none, no significant interspecific variation observed, not used in this study Antennal scapes * Antennal pedicel and funiculus Anterior tentorial pits Parafrontal ridges * Eyes high, used in this study none, no significant interspecific variation observed, not used in this study none, no significant interspecific variation observed, not used in this study high, used in this study none, absent in the worker caste Vertex * high, used in this study Occipital margin in posterodorsal view * Occiput in posterior view * Occipital margin in posteroventral view * high, used in this study high, used in this study high, used in this study Hypostoma * high, used in this study Mouthparts (maxillae, labium, labrum) unclear, none in closed in condition; described in open condition for Z. sarowiwai, but needs further investigation with better preserved alcohol material for wCT scanning Tentorium (internal) Mesosoma characters Mesosoma in profile * unclear, tentatively examined in this study and appears species-specific, but needs further investigation with better preserved alcohol material for wCT scanning high, used in this study Endosternum (internal) Transverse mesopleural groove Propleuron unclear, tentatively examined in this study and appears species-specific, but needs further investigation with better preserved alcohol material for wCT scanning moderately variable among species, not used in this study none, no significant interspecific variation observed, not used in this study Pleural endophragmal pit * high, used in this study Mesopleuron moderately variable among species, not used in this study Metapleuron Mesosoma dorsal * Probasitarsus Calcar of strigil Metasoma characters low, no significant interspecific variation observed, not used in this study high, used in this study low, no significant interspecific variation observed, not used in this study low, no significant interspecific variation observed, not used in this study Levator of petiole unclear, not examined in this study, very difficult to virtually dissect Petiolar tergum in profile * high, used in this study Laterotergites low, no significant interspecific variation observed, not used in this study Subpetiolar process of petiolar sternum in profile * high, used in this study 44 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Characters examined Diagnostic assessment and usage Petiolar tergum in dorsal view * | high, used in this study Disc of petiole none, no significant interspecific variation observed, not used in this study Subpetiolar process in ventral hioh, used in this study view * Helcium unclear, not examined in this study pe punal segment III in dorsal ish bused tnahisaatly view Abdominal segment II] in high, used in this study ventral view * Posterior end of abdominal high, used in this study segment III in ventral view * Prora in anteroventral view * high, used in this study Abdominal segment IV in dorsal . ; Bich ; hap : moderate, relatively variable within species, not used in this study view Abdominal segment IV in ; moderate, relatively variable within species, not used in this study ventral view Abdominal segment V in dorsal view Abdominal segment V in ventral low, no significant interspecific variation observed, not used in this study ; low, no significant interspecific variation observed, not used in this study view Abdominal segment VI in dorsal view * Abdominal segment VI in ventral view high, used in this study high, not used in this study Girdling constrictions abdominal segments IV, V, VI * Pygidium low, no significant interspecific variation observed, not used in this study high, used in this study Hypopygium low, no significant interspecific variation observed, not used in this study Spiracles abdominal segments I-VI General surface sculpture * high, used in this study Cuticle thickness (internal) none, no significant interspecific variation observed, not used in this study unclear, examined in this study but needs further investigation with more specimens Dorsal pronotum cuticle thickness (PRC): thickness of cuticle of pronotum measured in profile a short distance posterior of anterodorsal margin (Fig. 2B). Dorsal abdominal segment II (petiole) cuticle thickness (ASHIC): thickness of cuticle of abdominal segment II measured in profile a short distance posterior of antero- dorsal margin (Fig. 2C). Dorsal abdominal segment III cuticle thickness (ASIIIC): thickness of cuticle of ab- dominal segment III measured in profile a short distance posterior of anterodorsal margin (Fig. 2D). Cephalic capsule cuticle thickness index (CCCI): CCC / HW x 1000 Dorsal pronotum cuticle thickness index (PRCI): PRC / HW x 1000 Dorsal abdominal segment II (petiole) cuticle thickness index (ASIICI): ASIIC / HW x 1000 Dorsal abdominal segment III cuticle thickness index (ASIIICI): ASIUC / HW x 1000 Next-generation morphological character discovery and evaluation... 45 Figure 2. Microtomographic slides showing cuticle thickness measurements (with measuring lines in white). A Head in profile B Mesosoma in profile C Petiole (abdominal segment I) in profile D Abdomi- nal segment II in profile. 3D PDFs The first step to creating unicoloured 3D PDFs was to make 3D renderings of ant speci- mens in Amira using the Isosurface function (deselect compactify) for exporting surface meshes in the STL file format. These were imported into Meshlab (version 1.3.3) where the number of vertices per specimen was reduced in three steps to decrease total file size and before importing into Adobe Acrobat. First, the scan files were cleaned from isolated vertices (Filters > Cleaning and Repairing > Remove isolated pieces (wrt diameter) [set max diameter: 0.05—1%]) and the paper tips on which the ants are mounted were digitally removed as much as possible. The next step removed all internal vertices so that only the exoskeleton remained (1. Filters > Color Creation and Processing > Ambient Occlusion Per Vertex; 2. Filters > Selection > Select Faces By Vertex Quality (min = 0, max = 0.001); 3. Remove Selected Faces). In the last step, the number of total vertices was reduced to the final number of <750,000 (Filters > Remeshing, Simplification and Reconstruction > Quadratic Edge Collapse Decimation) in order to get a manageable resolution resulting in 3D PDF files of approximately 20 MB in size for supplementary files (the final step was omitted for files uploaded to Dryad). The processed STL files were annotated and exported as 3D PDFs in Adobe Acrobat Pro DC (version 2015.006.30119) using the Tet- ra4D Converter plug-in (version 5.1.2). When viewing the 3D PDFs with Adobe Acrobat 46 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Reader (version 8 or higher), trusting the document by clicking on the image will activate the interactive 3D-mode and allows rotating, moving and zooming into the 3D model. To generate the coloured 3D PDF of the mouthparts, we first segmented each mouthpart (maxillae, labium, labrum) independently and labelled each with a dif- ferent colour. A surface mesh of the combined segmentation data was then generated using Generate Surface function in Amira with Unconstrained Smoothing (Smoothing Extent set to 1.5). We exported the surface data into Open Inventor Format, where it was converted to U3D format using IvTuneViewer plugged in Amira. Finally, the 3D PDF was generated by importing the U3D file to Adobe Acrobat Pro DC (version 2015.006.30119) with Tetra4D plugged in (version 5.1.2). Data availability All specimens used in this study have been databased and the data is freely accessible on AntWeb (http://www.antweb.org). Each specimen can be traced by a unique specimen identifier attached to its pin (e.g. CASENT0764125). The Cybertype datasets provided in this study consist of the full micro-CT original volumetric datasets, 3D PDFs, 3D rotation video files, all light photography montage images, and all image plates including all important images of 3D models for each species. In addition to the cybertype data- sets, we also provide high-resolution 3D videos and/or 3D PDFs of the mouthparts and musculature reconstructions, as well as the full micro-CT original volumetric dataset of the mouthpart scan. All data have been archived and are freely available from the Dryad Digital Repository (Hita Garcia et al. 2017c, http://dx.doi.org/10.5061/dryad.4s3v1). In addition to the cybertype data at Dryad, we also provide freely accessible 3D surface models of type material on Sketchfab (https://sketchfab.com/arilab). Results Taxonomy of Zasphinctus in the Afrotropical region Notes on the genus in the region At the beginning of our study we encountered a situation in which the only two valid species from the region were described from males from West Africa (Santschi 1915), whereas the material available to us consisted of three worker-based species not as- sociated to any males. A DNA-based association is currently not possible since the two male-based species are only known from their respective type material, thus not available for any molecular analysis. There are additional males available from Nigeria and Uganda, but they are also not associated with any workers and their conspecificity with the other male-based species is uncertain. Since Zasphinctus is one of the rarest ant genera in the region, it is not likely that more specimens than currently available will be collected anytime soon, which means that the lack of male-worker associa- Next-generation morphological character discovery and evaluation... 47 tion problems will remain. Furthermore, the two male-based species were collected in relatively arid savannah areas in West Africa while two of the worker-based species are from humid equatorial rainforests. The third worker-based species is from a savannah in Mozambique, thus geographically distant from the two male-based species. These discrepancies led us to describe the three worker-based species independent- ly from the already known male-based species and create a comprehensive worker- based taxonomic system for the genus in the Afrotropical region. With this approach, we follow Wilson (1964) who suggested temporarily ignoring the male-based names and establishing a sound worker-based taxonomy until males are found together with workers and the different taxonomic names can be harmonised. More recent authors concur with that approach and also opine that male-based names will eventually be matched with worker-based names using molecular data (Jaitrong and Yamane 2011; Liu et al. 2015; Staab 2015). Diagnosis of Afrotropical Zasphinctus (workers) The following diagnosis is based on Borowiec (2016) with modifications and additions to encompass just the Afrotropical species: HEAD: Antennae with 12 segments and relatively short (SI 47-57), far from ap- proaching posterior head margin. Apical antennal segment conspicuously enlarged, longer than two preceding segments combined. Head distinctly longer than broad (CI 78-86). Clypeus with cuticular apron. Lateroclypeal teeth absent. Parafrontal ridges pre- sent and well developed. Torulo-posttorular complex vertical. Antennal scrobes absent. Labrum with median notch or concavity. Proximal face of stipes not projecting beyond inner margin of sclerite, prementum exposed when mouthparts fully closed, even though only slightly so. Maxillary and labial palps 3-segmented (see section on mouthparts be- low). Mandibles elongate triangular, masticatory margin with 4 or 5 small denticles on basal half, denticles usually strongly reduced and inconspicuous. Eyes and ocelli absent. Head capsule with weakly to well differentiated vertical posterior surface above occipital foramen. Ventrolateral margins of head without lamella or ridge extending towards man- dibles and beyond carina surrounding occipital foramen. Posterior head corners dorsolat- erally immarginate. Carina surrounding occipital foramen ventrally present. MESOSOMA: Mesosoma in profile relatively low and elongate to moderately high and stocky (LMI 34-41). In dorsal view usually slightly more than twice as long as broad (DMI2 49-58). Pronotal flange separated from collar by distinct ridge. Prome- sonotal connection with suture completely fused. Pronotomesopleural suture absent. Mesometapleural groove not impressed or weakly impressed. Transverse groove divid- ing mesopleuron absent. Pleural endophragmal pit concavity present, weakly to well developed. Mesosoma dorsolaterally immarginate. Metanotal depression or groove on mesosoma absent. Propodeal spiracle situated low on sclerite. Propodeal declivity with distinct dorsal edge or margin and rectangular in posterior view. Metapleural gland without bulla visible through cuticle. Propodeal lobes present and well developed. 48 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) LEGS: Mesotibia with single pectinate spur. Metatibia with single pectinate spur. Metabasitarsus not widening distally, circular in cross-section. Posterior flange of hind coxa not produced as raised lamella. Metatibial gland an oval patch of whitish cuticle. Metabasitarsal gland absent. Pretarsal claws of metatibia simple. Metafemur short to moderately long (MFI 75-100). METASOMA: Abdominal segment II (petiole) sessile without peduncle and peti- olar node well developed. In profile petiolar tergum between 1.0 to 1.2 times longer than high (LPI 102—123). Petiole anterodorsally marginate, dorsolaterally rounded, and laterally above spiracle weakly marginate. Laterotergites well developed and clearly demarcated. Sternum of petiole well developed with strongly anteroventrally project- ing subpetiolar process, process with or without fenestra. Helcium axial and in rela- tion to tergosternal suture placed at posttergite. Prora simple, not delimited by carina. Prora forming a U-shaped margin with median ridge. Spiracle openings of abdominal segments IV—VI circular. Abdominal segment II anterodorsally immarginate and dor- solaterally immarginate. In profile view abdominal segment III distinctly larger than succeeding segment IV, in dorsal view abdominal segment II longer than segment IV. Cinctus of abdominal segment IV not impressed. Girdling constrictions of segments IV, V, VI present and distinct, either unsculptured or cross-ribbed. Abdominal tergite IV not folding over sternite, and anterior portions of sternite and tergite equally well visible in lateral view. Pygidium large, with weakly impressed medial field. SETATION: Most of body with numerous short to moderately long, appressed to suberect (very rarely erect) setae. Pygidium armed with modified, thick, and often peg-like setae. Hypopygium armed with modified setae. COLOURATION: All known species predominantly dark brown to black with often lighter appendages. Synoptic list of Afrotropical Zasphinctus Zasphinctus chariensis Santschi, 1915 * [Chad] Zasphinctus sarowiwai Hita Garcia sp. n. [Cameroon, Democratic Republic of Congo, Ghana, Ivory Coast, Uganda] Zasphinctus obamai Hita Garcia sp. n. [Kenya] Zasphinctus rufiventris Santschi, 1915 * [Benin, Mali] Zasphinctus wilsoni Hita Garcia sp. n. [Mozambique] * Only known from males and not treated in this study. Diagnostic treatment Based on a thorough examination of external morphology and character evaluation, we provide the following character matrix (Table 3) that contains 24 morphological characters of high diagnostic value. 49 Next-generation morphological character discovery and evaluation... (OS 3) BIISOUD} PoIeNUdIAYIP Moy WM AIAPIUOD Yeo pur sursreur yenuaa pur Jorolue pousyory) YIM (NG “St]) Brssua} pore -NuarayIp ym AqAvouod podopaaap ]Jom pue SUISICUT [eIUIA PUL JOLIOUe PoUdyOTy? YIM (ING “SEJ) esouay porenusrayip yam AyAvou0s padojaaap [Jam pur SUIGICU TeIJUDA PUP JOTIOIUPS PIUOYSI TMA. (Srew [NUA pur JOLIE pouDdypIy Up! ayyoid ur umnuse)s Jeyo -nod Jo ssao0sd sejonadqng (OS “t) (711 Id‘) Ysry ueY) JoSuC] sou JT OysTY APoaneyor (1S Sty) (ES TING ‘0% ING) 2erpouaiur Suresdde ([¢ “S1q) snonstdsuosur pue podoyaaap Apyeam AISA (NS ‘8hq) (ZLI-ZOT Id) 43ty uey? Josuoy sawn [*] 01 QT soysry APaaneyor (IS “Stq) (8S-€¢ TING “69-1 ING) 299xP!p Sureodde (HS ‘814) doap pue podojaaap Ajsuons (NS “81q) (EZI-LIT (IS “Stq) (LE JWT) 2eSuoye pur samoy APaaneyor (4G ‘SLJ) suze perarey avefn3ue Ajsuons pure xpi Ajarezopour yim Surssoarp Asuons (9¢ 314) peuyep Ayrepnsoi pue dreys AjoresJopour suTpno (HS ‘Sty) (1¥—-0F INT) 2edwoo pur Joysry Ajoreropou (4 ‘Sig) suze [eroxe] popunos Ajsuons pue ypryp Ar YM SuIssoarp AjSuons (a¢ “3tq) peuyop Aprejnsar Aaa pue dreys ourpno Id 7) Ysry uerp ro8u0y sou 7°] -Jomoy Apanryar apgosd uy wimn319) Jejonag oe SJOP BUIOSOSII/ (€6-6F TIN ‘0F-8¢ IW) Puump Suresdde Vite (D¢ “Btq) d|QISIA INq MoTTeYs pue podojaaap Apyeam ad yeusesydopua jemeyg (D¢ “Btq) (9€-HE IWTD aresu0]e pue Jomo] Appaneyas apyoud Ul PUTOSOSIIA] (1g ‘St) sure Pesoxe] popunos Apsour pur ury) Ajaaneyar yim Burssaarp ss2y vuroysod Ay : AAOTA JEIJUDA Gy ‘2ig), PPULPp apenas pure dyeqs SuyANO. || Ad uy uIseus Jed (WP ‘31q) Surpnnosd Ayerpour Apyeom ursreur yen -UDA SUTSTeUT yeIUDA UTP Japeorg Ayres] JoLaysod (OF ‘S1q) 3urpnnoid Ayerpour Jou urssreur [eUA ‘peosg Aprepruuts SUISIeUT [eNUIA pue JopoIsod ‘prosdiyjo arour (dy ‘81q) Surpnnosd ATerpour ursreuwr PeNuSA MIA JOLIOISOd ut yndI99G ‘peosq Ayreyrums sursreur yemusA pure JoraIsod re ae : (NF 314) ; MOLE [PSIS POTS (OF ‘S1]) pouyop AprepnSoim pue Apyeom ouTpNO SON Gna eNnuy ee AenO (WP “81q) pouyop AyrepnSait pue dreys surpno Denier opto (OF ‘SI-J) podojaaap (NP ‘StJ) pedopasp Apeaa (WP “81q) podojaaap Aysuons Aj8uons pray Jo a0vj Jorosod pur ursrew pexowoa| peoy Jo sovy JOLAIsod pure UISIeUT [eXoVIA peoy Jo c0vy Joraysod pure ursreur [exovoA ere (Ty Bq) (L9Z TIS) (Mh Bid) (197-8ET TIS) Xode ze peor ueyp ([h td) (CHEST TIS) xode 3e Udioe eater xode ye peorg uey} Josuoy sou /°7 sJouUTUA adeos | Ja3u0] sou 9°7 01 HZ =yory? Apave9pour adeds (Iv ‘31J) jouoys pur Joxpryy Ajaanereduros ([p “S1.J) pouaxprya Afsnonords (HP ‘S1]) JoSuo] pue Jouury) Ajoanereduroo (HP “S1-) pousxorm Aysnonoids peoig uey) Josuoy sou F°7 01 7°7 sJaypTy adeos AIA [eSIOP UT (Dp “S1J) Jowoys pur Joyory) Apoanereduroo Sed uies fens OnaeE (Dp ‘B1q) poueypryp ATsno -U09 puke xoauod Aprepn3ar Apsour suTPNO JesIop | -UOd JOU puUe XoAUOD ATIe;NSaI suTPNO [esIop | -nordsuood pue xaauoo Ayre;NSait suTPNO Tesrop soSpii [ewuospereg (dv ‘SI]) qi00) uerpour snonoidsuos ynoyuM (ay ‘3IJ) yoo} uerpour snonoidsuos YAM (dy ‘31]) q100) uerpour snonoidsuos ynoy WM vare yead Ay) (Dp ‘B1q) JouUTTP pue Josuoy Sureadde (qp ‘S1-J) Joyo pur sJoaoys Surresdde (VP “SLJ) JoUUT) pur Jdsu] Sureadde ayyord ur peop] quogumn °F IVMLMNODS "7 1wUulygo "7 saisodg ‘smgouigdsyy Tenidonorisy Jo waisds uoneiuTap saidads paseq ~JOyIOM JOfF pesn SToJOeIEYS onsouseip Ife SUIMOYsS XTIJEU JooeIey) "€ 31geL Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) 50 sammound snosopyid uomadeiour uo juepungqe YM Sururys pue yoours AjUeuTWOpard amaidynos ayeiound yim saumouros ‘samniound uimrsAdoddy pue ‘gjonad yesorey Jo sour ‘umnspodosd yesorey “uoI -ngjdosour ‘ummiouosd soroyue ‘umssop sT7eydao Ae J so anit Soin recy fe eee snozayipid daop Appaneyar ‘posoaeds YIM jo vare uerpowrosaque ayeound-syenonay IOj SUSIE EHS [Eames See ae pel ae Nel ae ae Sururys A194 pue yloours AJaIs7duro9 asouuye ydaoxa ‘samnaound snosayyid daap Ajaaneyar -ososour ‘aso8nJ-ayepnonar Apsour umnssop stpeydas Se cerca aortas ie trosuie acon (09 “SHq) IA IA ‘A AI Stusursas feurarop (49 Fig) pomadinosm 29 A Ue? AJ UO JoyxeaM YONUT ‘paqq!-ssoI9 (9.2) poradposan -qe SUOTITNsUOD SuTTpsIF) (09 “Btq) (661 INVA) (N9 “3k) (Z61-98T I9VC) Buoy weTp Joprorq (9 “8tq) (EZI-€91 I9VC) AAaTA [esTOp BuO] UY) Jopeosg sou 9°] :Ja8u0] APOuNstp sou 7 01 6'[ punore :so1Jo0ys Apounstp Su] UeYp Jopeorg sou /*[ sa3u0, ApOUNsIp UI [A JUourses [euTMIOpqy (79 ‘S17) (319 “Stq) sursreur penusaosiaey podeys SUISIEUT [eNUIAOIOI] JUSSqe Isowye 01 Yeam AoA | Aprepnsar Ara pur Aydreys ym podopaap-]foma ({9 “31.J) Ausosns pue soaoors yea YIM praisur Guasqe dA00I3 dsIoAsUeN (H9 “S1]) 2a00738 asiaasueN pourpno Apr] -ngor Ajaaneyor pur Aydreys ‘dasp Jouury) yi ((9 “S1J) sursreur pemUsAOJoIe] popunoy pure podeys AprepnSarm Spry yt podoyaaap-am (59 “B1J) 20038 asiaasueN) pourpno Ajrejnsoim pue Aydreys ‘dosp syrup yA MOTA [VIJUDAOIOIUL UT CIOL AASTA [RIJUIA UT [I] WoUTsas [eurmopgqe Jo pus JoLIA}sog ({9 “81q) ero1d spremoi Surmor (H9 “814) erord spremo} SuIMoI (9 ‘31]) erord spremoi Surmoreu AASTA [EITUIA -reu Ajayeopour pure ioys ‘peorg Ajaaneseduroo | -seu Asuons pur yoys ‘peosg Apanereduroo | puss ATuo pure Tosuoy Jouuty ApPanereduros UI [J] JuewZes FeurMOpqy (49 “SLJ) orepnsue a1our (q9 ‘S1J) pepunos ssour ATfensn (19 “S1q) aepNsue sJour ATA TeSIOp uIsreu JoLaVUe YT yeprozoden srowr Suresdde | ursreur Joyowe YUM popunos ssow Burresdde | ursreur Joro Ue YUM yeprozoden ssow Surresdde | —_ uy [J] JuourSes peurwsopqy (q9 ‘Stq) 40Ys (V9 “SIq) ANOTA [ETTUDA (9° 2) SOL Pee UTUp Mle Ra eas TEES} pure yortp Ajoyeropour UISTeUT [eIIUIA ‘OAIPPIOF (US BI) (€6 Id) Peorq (OS “Seq) (TII-10T Id) SU] uetp Jopeorq uey} JoSUO] Sou [*] puNore sOUUTY ApPAANepAI quosun ‘7 sou *[*[ 01 OT punore sJoxpIyp Apoaneyos IWMIMNOLS °F qoys pure yoru AOA UISIEUT [IIUIA ‘OAIPIOF (dS 314) (S8-Z8 Id) peorq uey) Josuoy sown Z| punore srouuTyp ApPaneps 1ULgo "7 ur ssas0sd sejonadqng ee Jessop url uMS39) Jejoneg saisodg Next-generation morphological character discovery and evaluation... 51 Identification key to Afrotropical Zasphinctus species (workers) 1 With head in full-face view median clypeal area with conspicuous tooth (Fig. 4E, H) and torulo-posttorular complex comparatively long (Fig. 4H); in posterodorsal view vertexal margin and posterior face of head weakly de- veloped! (Fig..4IN) Soak Stes eae cetera eceeciaecette ne cea taeee cbaeoacesare Z. sarowiwai - With head in full-face view median clypeal area without any tooth (Fig. 4D, 4F, G, I) and torulo-posttorular complex comparatively short (Fig. 4G, I); in posterodorsal view vertexal margin and posterior face of head strongly devel- Oped (Pity AME, Oya. a sou sus alec Bsentatiaeest iadadhds ae suidh sasilea chanel nubdintatesaseltats 2 2 With head in full-face view parafrontal ridges with irregularly shaped dorsal outline (Fig. 4G); petiolar tergum in profile relatively lower, ca. 1.2 times longersthan highs (PI 117-123) (Figs 5M jak. oe e Se ates Z. obamai - With head in full-face view parafrontal ridges with regularly shaped dorsal outline (Fig. 41); petiolar tergum in profile relatively higher, ca. 1.1 times longerthan: hiphi(U RL 2 Pio DO cesses ue sieiredivonnacuatt sce edieses Z. wilsoni Zasphinctus obamai Hita Garcia, sp. n. http://zoobank.org/2B973F61-641C-436D-89AC-5955B519563A Figs 3, 4A, D, G, J, M, P, 5A, D, G, J, M, P, 6A, D, G, J, M, PB, 7, 8, 13A, 16, Video 1 Type material. Holotype, pinned worker, KENYA, Western Province, Kakamega For- est, Buyangu, 0.35222, 34.8647, 1640 m, secondary rainforest, leaf litter, collection code FHG00001, VII.-VII.2004 (F Hita Garcia) (NMKE: CASENT0764125). Paratypes, seven pinned workers: two with same data as holotype (BMNH: CASENT0764126; MCZC: CASENT0764127); one from KENYA, Western Province, Kakamega For- est, Isecheno, equatorial rainforest, sifted litter and soil under Morus mesozygia, 0.34, 34.85, 1550 m, 6.X1.2002 (W Okeka) (LACM: CASENT0178218; ZFMK: CASENT0764648); two from KENYA, Western Province, Kakamega Forest, Kisere Forest Fragment, 0.38505, 34.89378, 1650 m, rainforest, ex leaf litter, Transect 11, col- lection code FHG00036, 16.VII.2007 (F Hita Garcia) (NMKE: CASENT0764128; NMKE: CASENT0764129); and one from KENYA, Western Province, Kakamega For- est, Bunyala Forest Fragment, 0.37889, 34.69917, 1448 m, Winkler leaf litter extraction, collection code ANTC39476, VIII.2008 (G. Fischer) (ZFMK: CASENT0764647). Cybertypes, the cybertype dataset consists of all volumetric raw data in DICOM format, 3D PDFs and 3D rotation videos of scans of head, mesosoma, metasoma, and the full body of the physical holotype (NMKE: CASENT0764125) and/or one para- type (MCZC: CASENT0764127) in addition to montage photos illustrating head in full-face view, profile and dorsal views of the body of both specimens. ‘The data is de- posited at Dryad and can be freely accessed as virtual representation of both types (Hita Garcia et al. 2017c, http://dx.doi.org/10.5061/dryad.4s3v1). In addition to the cyber- 59 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Table 4. Morphometric data of the three species treated in this study. Z. obamai (N=6) Z. sarowiwai (N=11) Z. wilsoni (N=1) Min Max Mean Min Max Mean HL 0.55 0.59 0.56 0.60 HW 0.44 0.47 0.45 0.64 0.77 i ye) 0.49 SL 0.26 0.31 0.28 0.41 0.50 0.48 O92: SW 0.12 0.14 0.13 0.17 0.21 0.19 QZ PH 0.26 0.29 0.27 0.32 PW 0.28 0.33 0.30 0.35 DML | 0.53 0.65 0.59 0.85 0.99 0.95 0.66 WL 0.73 0.81 0.77 0.87 MEL 0.33 0.37 0.35 0.49 PTL 0.27 0.29 0.28 0.40 0.47 0.44 0.29 PTH 0.22 0.24 0.23 0.26 PTW 0:23 0.26 0.24 0.41 0.50 0.47 0.27 A3L 0.33 0.39 0.36 0.50 0.59 0.55 0.48 A3W 0.38 0.43 0.41 0.56 0.67 0.63 0.43 A4L 0.26 0.29 0.28 0.31 A4W 0.46 0.52 0.49 0.54 ASL 0.25 0.29 0.27 0.40 0.49 0.45 0.32 ASW 0.47 0.52 0.49 0.55 A6L 0.26 0.30 0.28 0.36 0.41 0.30 0.32 AGW 0.45 0.49 0.47 0.67 0.78 Ovo 0.51 cl 78 80 80 82 SI 47 53 50 53 57 55 53 sI2 215 242 228 238 261 247 267 DMI 38 40 39 Al 44 42 40 DMZ | 49 53 51 53 LMI 34 36 36 40 Al 40 37 MFI 75 79 77 88 91 89 100 LPI 117 123 120 102 105 112 DPI 82 93 88 101 111 105 93 DA3I 108 115 112 112 114 90 DAI 170 181 176 145 173 159 174 DASI 174 188 180 167 181 177 172 DAGI 163 173 169 186 197 189 159 type data at Dryad, we also provide a freely accessible 3D surface model of the holotype at Sketchfab (https://sketchfab.com/models/dfe15a585 14c4be89cdeff7f9713091c). Differential worker diagnosis. See Table 3. Worker measurements and indices. See Table 4. Next-generation morphological character discovery and evaluation... a0 Species A Zasphinctus obamai sp. n. @ Zasphinctus sarowiwai sp. n. Zasphinctus wilsoni sp. n. Figure 3. Map of sub-Saharan Africa showing the known distribution of the Z. obamai sp. n., Z. sarowiwai sp. n., and Z. wilsoni sp. n. Video I. 3D rotation video of Zasphinctus obamai sp. n. holotype worker (CASENT0764125) based on shaded volumetric surface rendering of full body. Etymology. ‘This species is named in honour of Barack Hussein Obama, the 44th President of the United States of America. We want to acknowledge his important efforts undertaken for the conservation of fragile natural habitats around the globe. Also, the type locality of Z. obamai is geographically close to the hometown of Obama's paternal family in Western Kenya. 54 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Zasphinctus obamai Zasphinctus sarowiwai Zasphinctus wilsoni YF rea ee 7 SES * Ne f\ pf wy EGO PRE PSE a oe S Wi — iin = Figure 4. Illustrated diagnostic character matrix based on micro-CT images used for species delimita- tions (Z. obamai = left column, Z. sarowiwai = middle column, Z. wilsoni = right column). A, B, C Ce- phalic capsule in profile (virtually dissected) D, E, F Clypeus and torulo-posttorular complex in anterior view G, H, I Anterior head (antennae virtually removed) showing parafrontal ridges (orange) and torulo- posttorular complex (green) J, K, L Antennal scape in dorsal view (virtually dissected) M, N, O Head in posterodorsal view showing vertexal margin (orange), posterior face, and occipital margin (green) P, Q, R Head in posterior view showing occiput and occipital foramen (virtually dissected) (ventral head facing upwards). Next-generation morphological character discovery and evaluation... 55 Zasphinctus obamai_ i Zasphinctus sarowiwai Zasphinctus wilsoni TRE ag Le”E fj Sa POT: a Fae: Figure 5. Illustrated diagnostic character matrix based on micro-CT images used for species delimita- tions (Z. obamai = left column, Z. sarowiwai = middle column, Z. wilsoni = right column). A, B, C Pos- terior head in ventral view showing ventral occipital margin (virtually dissected) D, E, F Head in ventral view showing mouthparts and hypostoma (virtually dissected). G, H, | Mesosoma in profile (orange) with pleural endophragmal pit (green) J, K, L Mesosoma in dorsal view M, N, O Petiole in profile showing pet- iolar tergum (green) and petiolar sternum (orange) with subpetiolar process P, Q, R Petiole in dorsal view. 56 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Zasphinctus wilsoni Figure 6. Illustrated diagnostic character matrix based on micro-CT images used for species delimitations (Z. obamai = \eft column, Z. sarowiwai = middle column, Z. wilsoni = right column). A, B, C Subpetiolar process of petiolar sternum in ventral view (virtually dissected) D, E, F Abdominal segment II in dorsal view G, H, | Abdominal segment III (orange) in ventral view with posterior end (green) J, K, L Abdominal segment III in anteroventral view showing prora (virtually dissected) M, N, O Abdominal segment VI in dorsal view P, Q, R Abdominal segments II, IV, V, and VI in ventral view showing girdling constrictions. Next-generation morphological character discovery and evaluation... 57 I -. = ~~. Figure 7. Zasphinctus obamai sp. n. holotype worker (CASENT0764125). A Body in profile B Body in dorsal view C Head in full-face view D Abdominal segments IIJ—VU in dorsal view. Distribution and biology. Zasphinctus obamai is only known from the type lo- cality, the Kakamega Forest in Western Kenya, which is a tropical equatorial rainfor- est. Despite a thorough ant inventory (Hita Garcia et al. 2009), Z. obamai was only sampled four times making it one of the rarest ant species of the Kakamega Forest. It was only found in the leaf litter layer of primary or near-primary forest habitats. Considering the rarity of this species in the type locality it is possible that it might also be encountered in other rainforest localities westwards of Kakamega, but eluded collections in the past. However, presently, Z. obamai appears to be endemic to this one forest. 5S Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Figure 8. Shaded surface display volume renderings of 3D models of Zasphinctus obamai sp. n. paratype worker (CASENT0764127). A Head in full-face dorsal view B Head in anterodorsal view C Anterior cephalic dorsum and mandibles in anterodorsal view D Head in ventral view E Occiput in posterior view (ventral head facing upwards) F Head in posterodorsal view G Mesosoma in profile H Mesosoma in dorsal view | Abdominal segment II (petiole) in profile J Abdominal segment II (petiole) in dorsal view K Abdominal segment II (petiole) in ventral view L Abdominal segments III—VII in profile M Abdominal segments HI and IV in dorsal view N Abdominal segments V—VII in dorsal view O Abdominal segments HI-VII in ventral view. Next-generation morphological character discovery and evaluation... a) Diagnostic comments. Zasphinctus obamai appears to be morphologically closer to Z. wilsoni than to Z. sarowiwai. Among other important differences, Z. obamai and Z. wilsoni are significantly smaller, lack a median clypeal tooth, and have a clearly defined vertexal margin compared to Z. sarowiwai. Zasphinctus obamai and Z. wilsoni can be easily separated by the characters provided above in Table 3. On the basis of the type series, there is no observable intraspecific variation. Zasphinctus sarowiwai Hita Garcia sp. n. http://zoobank.org/ DB20AFDC-3644-44A5-AA74-9B53249B5COD Figs’, 4B; EEU-KN)©, 5B, EEU-KG.N, ©, 6B; E. Hi, Kk, N, Q;-9; 10, 13B, 14,15, Videos 2, 4, 5 Type material. Holotype, pinned worker, CAMEROON, Centre Province, Mbal- mayo, 3.4597, 11.4714, ca. 600 m, rainforest, XI.1993 (NV. Stork) (BMNH: CASENT0764654). Paratypes, three pinned workers with same data as holotype (BMNH: CASENT0764646; CASENT0764649; CASENT0764650). Cybertypes, the cybertype dataset consists of all volumetric raw data in DICOM format, 3D PDFs and 3D rotation videos of scans of head, mesosoma, metasoma, and the full body of the physical holotype (BMNH: CASENT0764654) and/or one para- type (BMNH: CASENT0764650) in addition to montage photos illustrating head in full-face view, profile and dorsal views of the body of both specimens. ‘The data is de- posited at Dryad and can be freely accessed as virtual representation of both types (Hita Garcia et al. 2017c, http://dx.doi.org/10.5061/dryad.4s3v1). In addition to the cyber- type data at Dryad, we also provide a freely accessible 3D surface model of the holotype at Sketchfab (https://sketchfab.com/models/3e5a54cb8ea94028a49f0722bd5eefe8). Non-type material. DEMOCRATIC REPUBLIC OF CONGO: Epulu, 1.38333, 28.58333, 750 m, rainforest, 1.X1.1995 (S.D. Torti); GHANA: Wiawso, 6.2158, -2.485, ca. 160 m, 25.IV.1969 (D. Leston); WORY COAST: Tai Forest, 5.75, -7.12, ca. 250 m, rainforest, 18.—20.V.1977 (I Diomande); UGANDA: Western, Kabarole, Kibale National Park, Kanyawara Biological Station, 0.56437, 30.36059, 1510-1520 m, rainforest, 6.-16. VII.2012 (different independent collectors: FA. Esteves, F Hita Garcia & PG. Hawkes). Differential worker diagnosis. See Table 3. Worker measurements and indices (N=11). See Table 4. Etymology. The name of the new species is a patronym in honour of the famous Nigerian writer, environmentalist, and human rights activist Kenule Beeson “Ken” Saro-Wiwa. By naming a species from threatened rainforest habitats after him, we want to acknowledge his environmental legacy and draw attention to the often-problematic conservation situation in most Afrotropical rainforests. Distribution and biology. The new species has a comparatively wide distribution ranging from Ivory Coast to Uganda, even though it is not known from all countries in-between. However, this is likely based on a sampling artefact considering the rarity of Zasphinctus in general and the poor sampling in most African countries. Therefore, 60 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) |) ee SY ee _ Seas — —_ = | Figure 9. Zasphinctus sarowiwai sp. n. paratype worker (CASENT0764650). A Body in profile B Body in dorsal view C Head in full-face view D Abdominal segments [II-VI in dorsal view. we expect future collections in all countries in-between. All samples are from rainforest habitats at elevations from 250 to 1510 m. Based on the available collection data, the species lives in soil and leaf litter. Diagnostic comments. Zasphinctus sarowiwai differs in most diagnostic characters from the other two Afrotropical species. Most obviously, it can be separated from the other species by its much larger body size, the prominent median clypeal tooth, and the almost complete lack of surface sculpture. Despite its wide distribution range, there is very little observable variation. Most notably, the colour appears to be generally darker in the specimens from Uganda and Cameroon, which are uniformly very dark Next-generation morphological character discovery and evaluation... 61 Figure 10. Shaded surface display volume renderings of 3D models of Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). A Head in full-face dorsal view B Head in anterodorsal view © Anterior ce- phalic dorsum and mandibles in anterodorsal view D Head in ventral view E Occiput in posterior view (ven- tral head facing upwards) F Head in posterodorsal view G Mesosoma in profile H Mesosoma in dorsal view I Abdominal segment II (petiole) in profile J Abdominal segment II (petiole) in dorsal view K Abdominal seg- ment II (petiole) in ventral view L Abdominal segments II-VI in profile M Abdominal segments III and IV in dorsal view N Abdominal segments V—VII in dorsal view O Abdominal segments III—VII in ventral view. brown to black, while the specimens from West Africa tend to have a much lighter abdomen and often relatively bright legs. Furthermore, we observed some variation in the material from Uganda. In some specimens, the subpetiolar process of the petiolar 62 Francisco Hita Garcia et al. / ZooKeys 693: 33-93 (2017) Video 2. 3D rotation video of Zasphinctus sarowiwai sp. n. paratype worker (CASENT0764650) based on shaded volumetric surface rendering of full body. sternum had a slightly weaker, but still distinct, fenestra compared to the material from other localities, and the ventral margin of the process had a posteroventral tooth-like projection. In addition, the anterodorsal margin of abdominal segment III was slightly more angulate in a few specimens while in several other specimens the metapleuron had some weak punctate sculpture. Overall, we consider this variation as geographic and very well within the intraspecific range of such a widespread species. Zasphinctus wilsoni Hita Garcia sp. n. http://zoobank.org/355B3D80-3029-4C8A-B48C-939C1 1914552 Figs 3,4; Fe nO, RSC) Bol, EO; Ro GGAr TL: Beall ed 2s1 36 Video 3 Type material. Holotype, pinned worker, Mozambique, Sofala, Gorongosa National Park, 2 km S Chitengo, -18.99472, 34.35769, 1 m, secondary forest, leaf litter, collec- tion code ANTC37418, 30.V.2012 (G.D. Alpert) (MCZC: MCZ-ENT-005 12764). Cybertype, the cybertype dataset consists of the volumetric raw data in DICOM format, as well as 3D PDFs and 3D rotation videos of scans of the head, mesosoma, metasoma, and the full body of the physical holotype (MCZC: MCZ-ENT-005 12764) in addition to montage photos illustrating head in full-face view, profile and dorsal views of the body. The data is deposited at Dryad and can be freely accessed as virtual representation of the holotype (Hita Garcia et al. 2017c, http://dx.doi.org/10.5061/ dryad.4s3v1). In addition to the cybertype data at Dryad, we also provide a freely ac- Next-generation morphological character discovery and evaluation... 63 Figure Il. Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-00512764). A Body in profile B Body in dorsal view C Head in full-face view D Abdominal segments III—-VII in dorsal view. cessible 3D surface model of the holotype at Sketchfab (https://sketchfab.com/models /36bab7ecaa8d45b18013ea679b7ca54a). Differential worker diagnosis. See Table 3. Worker measurements and indices (N=1). See Table 4. Etymology. This new species is dedicated to the renowned scientist, author, and conservationist Edward O. Wilson from Harvard University in honour of his more than six decades of accomplishments to the fields of myrmecology, sociobiology, bio- diversity, and conservation. 64 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Figure 12. Shaded surface display volume renderings of 3D models of Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). A Head in full-face dorsal view B Head in anterodorsal view C Anterior ce- phalic dorsum and mandibles in anterodorsal view D Head in ventral view. E Occiput in posterior view (ven- tral head facing upwards) F Head in posterodorsal view G Mesosoma in profile H Mesosoma in dorsal view I Abdominal segment II (petiole) in profile J Abdominal segment II (petiole) in dorsal view K Abdominal seg- ment II (petiole) in ventral view L Abdominal segments [II-VI in profile M Abdominal segments III and IV in dorsal view N Abdominal segments V—VII in dorsal view O Abdominal segments II-VI] in ventral view. Distribution and biology. Currently, Z. wikoni is only known from its type locality, the Gorongosa National Park where it was collected in the leaf litter of a secondary dry forest. Considering how generally undersampled south-eastern Africa is, it is likely that Z wilsoni might be encountered in other woodland localities in Mozambique, Tanzania, or Zimbabwe. Next-generation morphological character discovery and evaluation... 65 Video 3. 3D rotation video of Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764) based on shaded volumetric surface rendering of full body. Diagnostic comments. Zasphinctus wilsoni is morphologically closer to Z. obamai than to Z. sarowiwai. It shares the smaller body size, the lack of median clypeal tooth, and a clearly defined vertexal margin with Z. obamai, separating both from Z. sarowiwai. However, the conspicuous surface sculpture on the cephalic dorsum and the sides of mesosoma and petiole clearly distinguishes Z wilsoni from the other two species. Since Z. wilsoni is only known from the holotype there is no available information about intraspecific variation. 3D mouthparts morphology (excluding mandibles) The small number and the preservation conditions of the specimens available for this study provided some limitations for the examination of mouthparts. It was not possible to dissect in vivo or micro-CT scan the open mouthparts of Z. obamai, nor of Z. wilsoni. Fortunately, the mouthparts of one pinned specimen of Z. sarowiwai were open and mostly exposed, thus available for superficial examination under the light microscope and for micro-CT scanning. Consequently, we were unable to test mouthpart morphol- ogy in detail for species delimitation. However, based on the limited information observ- able in closed condition, there appears to be no significant difference between the three species (Fig. 13). In the following we briefly describe the open mouthparts of Z. sarowi- wai based on a 3D reconstruction of segmented micro-CT data (Fig. 14 and Video 4): Labrum: distal margin conspicuously cleft medially; median area from anterior cleft to proximal articulation very thin, dividing labrum into two lobes; each lobe bulging medially; lateroventrally with two conspicuous hook-like labral arms projecting parallel to remainder of labrum; row of ten to twelve setae (1 very long pair plus four/five shorter 66 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Figure 13. Shaded surface display volume renderings of 3D models of mouthparts (excluding mandi- bles) in closed configuration (green=maxillae; yellow=labrum; orange=labium). A Zasphinctus obamai sp. n. (CASENT0764127) B Zasphinctus sarowiwai sp. n. (CASENT0764654) C Zasphinctus wilsoni sp. n. (MCZ-ENT-005 12764). Video 4. 3D rotation video of segmented surface reconstructions of the mouthparts of Zasphinctus sarowiwai sp. n. (CASENT0764652) in open configuration (green= maxillae; yellow=labrum; orange=labium). pairs) on basal third of exterior face; row of four to six setae (1 very long pair plus one/ two shorter pairs) on exterior face close to distal margin; labral tubercles absent. Maxillae: maxillary palp three-segmented with second segment being greatly en- larged, third segment with very long seta, second with two long setae; deep and conspic- uous diagonal, transverse stipital groove present dividing stipes into proximal external face and distal external face; articulation of labrum with maxillae of labro-stipital type via lateral extension/shoulder; proximal faces projecting beyond inner margin of stipites, thus almost completely concealing prementum; galea with well-developed galeal crown and maxillary brush, galeal comb apparently absent; lacinial comb not observable. Labium: labial palp three-segmented with first segment being greatly enlarged, first and second segment with one long seta, third segment with three long setae; premental shield with several moderately long setae; shape of glossa not observable (structure collapsed); subglossal brush present and conspicuous with numerous long and thick setae; paraglossae absent. Next-generation morphological character discovery and evaluation... 67 Figure 14. Volumetric 3D model of segmented surface reconstructions of the mouthparts of Zas- phinctus sarowiwai sp. n. (CASENT0764652) in open configuration (green=maxillae; yellow=labrum; orange=labium). A Frontal view B Lateral view C Posterior view D Dorsal view. Cuticle thickness The results of our cuticle thickness data are provided in Table 5. The measurements show absolute values of 13-22 um in Z. obamai, 20-30 um in Z. sarowiwai, and 14-21 um in Z. wilsoni, and by putting these in relation to body size the thickness indices range between 31—44. On the basis of cuticle thickness data throughout nine subfamilies of ants (Peeters et al. 2017), the cuticle thickness values of our three Zas- phinctus species are at the upper range meaning that these species possess among the thickest cuticles measured so far. Thoracic and abdominal musculature Based on our virtually reconstructed and segmented data, we can show that the meso- soma and metasoma both contain high degrees of musculature (Fig. 15; Video 5). The propodeum is tightly packed with dorsal and ventral muscles moving the abdominal segment II (petiole) and stabilizing the weight of the abdominal segments III to VII. Due to the massive size of the latter, the volume of the propodeal muscles attach- ing to the anterior petiole is high and comparable to that of the neck muscles in the pronotum. The petiole and the following segments also have a high muscle density, which is prominently visible in lateral (Fig. 15A, 15B), dorsal (Fig. 15C, 15D), and posterodorsal views (Fig. I5E, 15F) in the segmented 3D models of the metasoma. While the muscles in abdominal segment II — and to a lesser extent in segment III — evenly fill almost the entire segment, those in segments IV to VII are mostly limited to 68 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Table 5. Morphometric data generated from 3D measuring cuticle thickness. For each species the five raw measurements with corresponding calculations into indices are given, as well as mean values and standard deviations (SD). Species Z. wilsoni CCCI CCCI CCCI CEG 2 0.018 0.023 0.014 29 CCC 4 0.021 0.024 0.017 34 CCC 5 0.022 0.023 0.015 30 MEAN 0.019 0.023 0.015 31 7 eset Srsi te inmm PRCI inmm PRCI in mm PRCI PRE 0.017 0.027 0.020 7 PRC 4 0.016 0.029 0.020 4l MEAN 0.016 0.027 0.020 40 inmm ASIICI in mm ASIICI in mm ASIICI ASIIC 2 0.014 0.026 0.019 39 ASIIC 4 0.013 0.026 0.020 42 MEAN 0.014 0.027 0.019 Bp : oe es inmm ASIITCI inmm ASIITCI inmm ASHITICI ASIIIC 1 0.013 0.022 0.018 37 ASIIIC 2 0.015 0.029 0.018 37 ASITIC 4 0.016 0.022 0.017 35 MEAN re Ll ae 36 SD | 0.001 001 0. | 0.003 | | 0.001 001 1 positions along the lateral and ventral walls. Finally, attached to the sting apparatus are two separate muscle sets, the protractors and retractors of the sting shaft. The former set is responsible for extending the sting from the tip of the abdomen during attack or defence and the latter for retracting it back to its resting position within the abdomen (visible in dorsal view in Fig. 15F). Next-generation morphological character discovery and evaluation... 69 Figure 15. Still images of 3D model of full body of Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). False-colour volume rendering of segmented mesosoma and metasoma musculature (red) and sting apparatus (green) superimposed on semi-transparent surface model (A, C, E) or stand- alone (B, D, F). A, B Body in profile view C, D Body in dorsal view E, F Body in posterodorsal view. Discussion Virtual recovery of morphology Almost all previous studies that used micro-CT for invertebrate taxonomy encoun- tered problems with the achievable voxel resolution in relation to body size resulting in a poor recovery of certain, very fine or small structures, such as setae, ommatidia, and microsculpture (Faulwetter et al. 2013, 2014; Fernandez et al. 2014; Carbayo et al. 2016; Fischer et al. 2016). In the case of ant taxonomy this was intensively discussed by Hita Garcia et al. (2017a) who achieved voxel sizes of around 5 um for full body scans of the two treated species. Based on these results and in order to improve the voxel resolution and present better resolved 3D reconstructions, we scanned the head, the mesosoma, and the metasoma separately in addition to a full body scan for each species. As a consequence, we attained smaller voxel sizes for the 70 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Video 5. 3D rotation video of full body of Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). False-colour volume rendering of segmented mesosoma and metasoma musculature (red) and sting apparatus (green) superimposed on semitransparent surface model. 3D models of the single body parts (0.95—2.83 ym versus 3.00—4.61 pm) resulting in a much higher resolution, and significantly reduced or eliminated the problems encountered by Hita Garcia et al. (2017a) (Fig. 16), except for ommatidia that are absent in Zasphinctus workers. While setae were poorly recovered by Hita Garcia et al. (2017a), they are very well visible in our 3D models of single body parts presented in this study. However, due to the higher voxel size, our full body scans of the Zasphinc- tus species have a weaker resolution of setae. Furthermore, compared to the physical specimens, surface sculpture was recovered with high morphological accuracy in the 3D models of single body parts, whereas it was only poorly noticeable in the full body scans. Surprisingly, fine surface sculpture on some body parts was even more observ- able in the 3D models than in the physical material, due to a limited magnification of our light microscope to 100 x. The visualised reconstruction of the mouthparts provides a comparatively adequate and detailed 3D model of the maxillae, labium, and labrum, but also presents some important limitations. The general morphology of maxillae, labium and labrum are well recovered, and they are very similar to the mouthparts of Z. steinheili that were described by Gotwald (1969). The vast majority of setae are well visible, as is the surface sculpture of most structures. More importantly though, for the first time it is possible to examine these structures in their natural position in 3D, as well as their configuration with respect to each other. This is a significant advantage compared to traditional histological dissections that always remove all parts from the head and then separate each structure for separate examination. Our 3D volume reconstruction Next-generation morphological character discovery and evaluation... Jal i‘. { BS ANG a ES Figure 16. Comparison of full body scan versus single body part scans based on Zasphinctus oba- mai sp. n. holotype (CASENT0764125) and paratype worker (CASENT0764127). A Full body scan (CASENT0764125) B Scan of head (CASENT0764125) € Scan of mesosoma (CASENT0764127) D Scan of metasoma showing abdominal segments III to VII in profile (CASENT0764127). allows detailed examinations from all possible angles and the segmented mouthparts can be observed independently or in combination with each other. Nevertheless, there are some problems with our 3D reconstructed model. The most problematic structure is the glossa. As already pointed out by Gotwald (1969), due to its highly membranous nature it is deformed in most dead specimens independently of preservation agent. In our specimen, the glossa was already collapsed to a crater-like appearance prior to micro-CT scanning, thus not available for any shape examination. Another important limitation is that not all structures could be satisfactorily out- lined during the segmentation process. This was especially difficult for the delineation of some components, such as the cardo, the lacinial comb, the regions where labium and maxillae meet, and generally everywhere where membranous and chitinous tissues are in contact. These problems are caused by scanning a dry mounted specimen, in which most internal structures have undergone desiccation, shrinkage, and deforma- tion. In such specimens, the dissimilarities in density and contrast between different tissues or components are minimal to zero, thus causing significant problems for the proper recognition and subsequent outlining of borders between structures. In general, our 3D reconstructed model provides fewer details compared to histological dissec- tions. However, these problems might be solvable in future studies if specimens are preserved and prepared in a way more suitable for micro-CT scanning and virtual reconstruction. Based on unpublished data, the use of freshly killed material or speci- mens in alcohol combined with the use of potassium hydroxide (KOH) and iodine A. Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) staining provides much better resolution of internal structures than the use of dry material. This allows a much more sophisticated recovery of mouthparts morphology. The application of micro-CT scanning to obtain information about cuticle thick- ness is novel. Based on our data however, we refrain from using it for taxonomic diag- nostics at the moment. There are some differences in the cuticle thickness among the three species, most notably the very thick head of Z. obamai (CCCI 44 vs. CCCI 31 in Z. sarowiwai and Z. wilsoni). It also appears that the head of Z. obamai is thicker than the pronotum and abdominal segments I and IH, whereas the heads of the other two species are thinner than or as thin as most other body parts (see Table 5). However, these results could be based on measuring the cuticle at a wrong angle resulting in a dis- torted result. Even though we have tried to find the best possible sagittal section slides for virtual measuring, it cannot be ruled out that we did not measure the thickest part of the cuticle. For future use, we recommend measuring more slides and more body parts in order to achieve a more complete picture of cuticle thickness. Even though we do not use it for taxonomic diagnostics at the moment, we believe there is potential for such use (unpublished data). However, this should be investigated with more taxa and more specimens first. Nevertheless, our results permit to place the treated species in an evolutionary context and make interpretations about natural history. Currently, the use of micro-CT data for virtual cuticle measuring has rather limited applicability due to the sparse availability of scanning resources in the myrmecological community. Nevertheless, we believe such data provides important information that might be valu- able for future systematic and evolutionary studies. Cybertypes As pointed out in previous studies, a crucial advantage of using 3D models based on micro-CT data is its potential application as cybertypes (Faulwetter et al. 2013, 2014; Akkari et al. 2015; Hita Garcia et al. 2017a, 2017b). The concept of a cybertype is to present a detailed and as complete as possible virtual reconstruction of a physical type specimen that is freely accessible. Hita Garcia et al. (2017a) critically assessed the usefulness of cybertypes for ant taxonomy, and due to the limitations in voxel resolu- tion in their scan data suggested to use a combination of micro-CT data (raw data, 3D PDE and 3D rotation video) and montage photos (three standard view: head in full-face view, full body in profile and dorsal view) as a minimal cybertype for ants. Al- though we have achieved much higher quality 3D reconstructions compared to previ- ous ant studies using micro-CT and strongly reduced the limitations discussed by Hita Garcia et al. (2017a), we still believe that a minimum ant cybertype should include optical montage photographs. The main reason for this is the lack of natural colour in the micro-CT, which can only be shown with visible light photography. However, in this study we improve the previous ant cybertypes by providing micro-CT scans of single body parts and scan data for the holotype and one paratype, if available, thus increasing the usefulness of the cybertype datasets. Next-generation morphological character discovery and evaluation... 73 Virtual character evaluation and presentation One aim of this study was to evaluate new taxonomic characters for species level tax- onomy on the basis of traditional morphological analysis and virtual examination of 3D reconstructions. Unfortunately, only dry mounted material was available for this study. As pointed out in Hita Garcia et al. (2017a), most internal anatomical structures of such specimens have undergone significant desiccation, shrinking and deforma- tion. Consequently, micro-CT data from dry specimens provides much less useful information for comparative examination. Based on our initial investigation, however, some internal sclerotized structures, such as the tentorium, several apodemes, and the endosternum appear to have significant potential for comparative morphology among species. However, due to the poor recovery of these structures in our raw data, we could not examine these in detail and focused our character evaluation on external morphology, with the exception of cuticle thickness. For future taxonomic studies us- ing micro-CT, we propose to examine internal characters in more detail by using mate- rial preserved in ethanol. Initially, our intention was to omit a species identification key, which may appear counterintuitive and substandard. However, there are several problems with dichoto- mous identification keys that lead us to take a different approach in this study. Identifi- cation keys for well-studied regions, such as Japan and Central Europe, generally work well and are very stable since new species are rarely encountered and nomenclatorial benchmarking is rare. This is certainly not the case for most tropical and subtropical regions because our knowledge of the local and regional diversity is fragmentary to non-existent. One major limitation of keys for such regions is that they usually only work for the known species at the time of publication. Later discoveries of new spe- cies render keys less useful and often less reliable for identification purposes. To avoid this, it is necessary to update older keys in additional publications after new species are discovered, as done by Hita Garcia and Fischer (2014) to update Hita Garcia et al. (2010). However, this is comparatively work-intensive and most authors of revisionary taxonomy studies are hesitant to revisit previously “finished” groups. ‘This situation is especially problematic in hyperdiverse genera, for which there is no obvious solution except for continuously revising the taxa until most or “all” species are known and described. However, for genera with small or moderate species richness there might be alternatives to traditional identification keys with a few characters per key couplet. In the case of Zasphinctus it is very likely that future collecting in the Afrotropi- cal region will reveal additional species, even though not too many. This assessment is based on the apparent rarity of these ants and the fact that the region is largely under- sampled. Accordingly, any identification key that covers only the three species treated here is doomed to obsolescence with the discovery of additional species, especially if only a few characters are listed per key couplet. Instead of simply providing a short key, we decided to present an illustrated matrix with numerous characters, in which we only present the ones that have proven to be diagnostic. Future users of our iden- tification system can check multiple character illustrations and compare them with 74 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) their specimens at hand. Nevertheless, despite our concerns with a short key with few diagnostic characters, we understand that some users would still prefer to use a short dichotomous key and we still provide one in this study. Furthermore, the characters chosen are suited for diverse audiences with different interests and resources. For users with limited microscopy resources or little taxonomic training, we have included many easy-to-examine characters that are visible at lower magnifications, such as the shapes of head, mesosoma, or abdominal segment II in profile (e.g. Figs 4A—F, J—L, 5G—R). Based on that, we have added numerous fur- ther characters that are not completely necessary for simple identification purposes of faunistic or ecological studies, but target a more taxonomically oriented audience with better microscopy resources and deeper knowledge of ant morphology. We present many characters that require detailed examinations, such as structures on the posterior or ventral head (Figs 4P—R, 5A—F) and the ventral metasoma (Fig. GA—C, G—L, P—R). These are intended to provide important comparative data for future systematic stud- ies, and will very likely improve delimitations of any additional species. As outlined above, compared to the taxonomy of most insect groups, the character sets used in the field of ant worker taxonomy are very often rather limited and rely heavily on setation, sculpture, body size, and colour. These are often problematic since they can be highly variable within species and prone to geographic variability, such as shown for the Neotropical Tatuidris Brown & Kempf (Donoso 2012), Malagasy Tetramorium Mayr (Hita Garcia and Fisher 2011) and Malagasy Crematogaster Lund (Blaimer 2012). Against the background of a resilient taxonomic impediment with continuously declining taxonomic resources and funding (e.g. Wheeler et al. 2004; Ebach et al. 2011), it is imperative to deliver taxonomic works that provide high qual- ity species delimitations at a more accelerated speed that offer a stable taxonomic foun- dation upon which future discoveries can be based. We believe that the application of 21* century taxonomic tools, such as molecular phylogenetics/phylogenomics and 3D next-generation morphology techniques, can strongly improve ant taxonomy. For the revision of Afrotropical Zasphinctus, we have evaluated every single char- acter that could be of diagnostic importance based on the literature record (Bolton 1990; Keller 2011; Borowiec 2016) through a combination of examination of physical material under the light microscope and virtually reconstructed 3D models of micro- CT scans. The latter is of crucial importance since it provides numerous advantages. The most important turned out to be the use of micro-CT data for virtual character examinations and dissections. As noted above, the available material was too scarce to perform physical dissections or dangerous manipulations of specimens. Fortunately, we were able to examine the virtual specimens from all imaginable angles by rotating the 3D models. In addition, in order to examine characters that were hidden behind other body parts, we virtually removed any obstructing structure. By doing this we were able to observe and reveal characters that are challenging to see in most dry- mounted specimens, thus rarely used for ant taxonomy. In particular, we found that the ventral and posterior head possesses a series of useful diagnostic characters, such as the hypostoma (Fig. 5D—F), the vertex (Fig. 4M—O), the occiput (Fig. 4P—R), and Next-generation morphological character discovery and evaluation... 75 several margins around these structures (Fig. 4M—O, 5A—C). The same applies for the ventral metasoma since we found that abdominal segments I] and III in ventral view provided some valuable characters, such as the subpetiolar process (Fig. 5 M—O, 6A—C) or the prora (Fig. 6J—L). Physical examination of most of these would require damag- ing the specimens by removing legs, moving the head, separating abdominal segments, or in order to examine the occiput, detaching the head from the pronotum. By using micro-CT based 3D models, we were able to accomplish this with valuable type mate- rial without any damage. Furthermore, the use of virtually reconstructed 3D models permits a quick and ef- fective use of time and resources. Dissections and manipulations of physical specimens are usually very time-consuming, especially if histology and SEM are involved. By con- trast, the application of micro-CT scanning enables highly accelerated examinations of morphology compared to these methods (Faulwetter et al. 2013; Friedrich et al. 2014). The initial data generation with a powerful scanner and subsequent 3D reconstructions can be done quickly with minimal effort, if the necessary scanning and visualisation resources are available (see limitations below). The virtual examination of characters is easy and very straightforward. The 3D models can be manipulated in many ways to observe the targeted morphological structures and once a character is in focus one can generate high-quality images within seconds. It is also easy to make images of structures from different angles in order to find the best one for presentation purposes. In our study, we generated several hundred character snapshots within a few days, thus allow- ing us to choose the most suitable ones for the illustrations used in this study. Generat- ing so many images by using montage photography, SEM, or histology would require much more time and significantly slow down the speed of the publication process. As already discussed in previous studies employing micro-CT data (Faulwetter et al. 2014; Carbayo et al. 2016; Hita Garcia et al. 2017a), the most important weakness is the limited access to scanning resources since most universities or museums do not have their own micro-CT scanners. Currently, the acquisition of scanners and access to external scanning facilities require substantial economic resources, and this situation will remain unchanged for some time. Some natural history museums and universi- ties already have or are in the process of establishing scanning resources, but presently only a small minority of the taxonomic community has access to the technology. In addition, generating, post-processing, and handling the usually rather large data re- quires time and technical skills. Nonetheless, as with all new technologies, it is likely that technological and computational developments will reduce the costs of scanning, increase the availability for the taxonomic community, and simplify data management (Faulwetter et al. 2014; Hita Garcia et al. 20174). Functional morphology and lifestyle As outlined above, there is no knowledge of the natural history of Afrotropical Zas- phinctus, except that they might live in leaf litter since most specimens were collected 76 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) in litter samples. Against the background that they are dorylines and that their Aus- tralian congeners are predators of other ants, it is likely that the species treated in this study pursue a similar lifestyle. The examination of the micro-CT data generated dur- ing this study allows some inference about the lifestyle of the studied species. As mentioned above, all three Afrotropical Zasphinctus species possess a very thick cuticle. Peeters et al. (2017) found the species with the thickest cuticle in relation to body size to be predominantly large ponerine genera, such as Diacamma Mayr, Odon- toponera Mayr, Leptogenys Roger, and Ectomomyrmex Mayr. However, the thickest cuticle was observed in the species Ooceraea biroi (Forel), which is a doryline in relative close phylogenetic proximity to Zasphinctus. Based on that result, it is not that surprising that African Zasphinctus display such thick cuticle. Notwithstanding that Peeters et al. (2017) found that the best predictors for thick cuticle were body size (larger ants have thicker cuticle) and phylogeny (poneroid ants have thicker cuticle), the thick cuticle of Afri- can Zasphinctus is likely related to a predatory lifestyle. Based on observations of other Zasphinctus species mentioned above, it is highly probable that African Zasphinctus are top predators that feed predominantly on other ants, which is also the case in the clonal raider ant Ooceraea biroi, which feeds primarily on ant brood. Furthermore, Peeters et al. (2017) concluded that cuticle thickness was also negatively correlated with larger colony size in more phylogenetically derived ant lineages (formicoid clade). Despite a severe lack of observation and natural history data, it appears that African Zasphinctus live in small colonies, which is well in accordance with the findings of Peeters et al. (2017). In Zasphinctus, the relative amount of muscles responsible for moving the abdo- men seems to be largely increased compared to other ants from the formicoid clade, e.g. Pheidole Westwood & Terataner Emery, studied in previous publications (Sarnat et al. 2016; Hita Garcia et al. 2017a). It may be tempting to think that the genus Zasphinctus — as compared to the majority of ant lineages which have (evolved) greatly reduced abdominal musculature — has retained a more ‘primitive’ and wasp-like inter- nal morphology in its abdomen. Yet, its species have a morphology that makes them rather special among ants: their relatively long abdomen is serially constricted between individually rotating presclerital plates. This apparent adaptation may have gained Zas- phinctus additional functionality during predation and defence by increasing overall flexibility of use for its well-developed sting apparatus (Fig. 15) in the apical abdominal segment. It seems that other doryline genera such as for example Eusphinctus Emery, Sphinctomyrmex Mayr, and to a lesser degree possibly some Cylindromyrmex Mayr, and Leptanilloides Mann have evolved very similar features independently from Zasphinc- tus. It may be interesting to investigate the evolution of these specialised morphologies with respect to the different army ant lifestyles. Since studies on internal ant morphol- ogy are generally rare, we have little opportunity to compare our present results with those of others. Hashimoto (1996) for example found that, in some ant subfamilies, the muscles in abdominal segment II and III (petiole and postpetiole) show ‘positional and functional modifications’. Although he also gives an anatomically based discussion on the functional morphology of these modifications, there is no information given Next-generation morphological character discovery and evaluation... Tal on related behavioural or ecological functions nor on musculature modifications in the posterior abdominal segments. With our main focus on the taxonomy of the three newly described species we refrain from further speculations and more detailed analy- ses in this publication and defer to future internal functional ant morphology studies using micro-CT scanning technology. Conclusions Our study highlights the potential of in-depth comparative morphology analyses for taxonomy founded on a combined investigation of physical specimens under light mi- croscopy and virtual 3D models generated from micro-CT data. Our approach reveals a wealth of morphological characters with high diagnostic potential that we use to successfully delimit species within Afrotropical Zasphinctus. Even though the worker caste of ants is highly simplified and the presence of cryptic species in many ant genera is increasingly recognised (e.g. Schlick-Steiner et al. 2006; Seifert 2009), we believe that in many cases the whole range of comparative morphology for alpha taxonomy has not been fully explored yet (Keller 2011). Virtual and interactive examination of morphology and anatomy in 3D can fill the gap and improve our understanding of functionality and homology of characters and provide the means for the discovery of new diagnostic characters (Zimmermann et al. 2011; Blanke and Wesener 2014). Furthermore, considering the lack of material and apparent rarity of Afrotropical Zasphinctus, our study also emphasises the strength of micro-CT scanning as a tool for the non-destructive virtual examination of valuable and scarce type material. Based on our results, micro-CT scanning opens up promising possibilities for the integration of very rare type (and non-type) material into systematic studies, as demonstrated here with the singleton holotype of Z. wilsoni. In general, even though it often appears as if the modern era of molecular system- atics has dwarfed the importance of morphology-based systematics, we strongly concur with previous authors that by embracing and employing new technologies, such as micro-CT scanning, the study of morphology can still have a significant impact and remain a strong field in systematic and evolutionary biology (Giribet 2010; Keller 2011; Friedrich et al. 2014). Perhaps the most interesting aspect of using micro-CT for ant taxonomy, however, is the potential to bridge different fields of research. Our study and previous ones for spiny Pheidole (Sarnat et al. 2016; Sarnat et al. 2017) show that by examining morphology and anatomy in detail insights about potential behavioural adaptations can be gained. By including new and internal morphological characters in our taxonomic studies we can draw conclusions about and make a connection with functional morphology and ecology. In more holistic approaches combined with statis- tical analyses and controlled for phylogenetic relationships we can study the evolution of morphological adaptations and learn about the mechanisms that make ants so suc- cessful in their respective environments. 78 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Acknowledgments First, we want to thank Eli M. Sarnat for his suggestion at the initial stage of this study to present taxonomic data in a different way, which strongly influenced the direction of this study, and for providing very helpful comments and points of critique to a previ- ous version of this manuscript. We thank Kenneth Dudley for the generation of the map used in this study. We are very thankful to Peter G. Hawkes from Pretoria, South Africa, Suzanne Ryder from BMNH, and Gary D. Alpert from the MCZ for providing material without which this study would not have been possible. 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Arthropod Structure & De- velopment 40: 571-582. https://doi.org/10.1016/j.asd.2011.06.003 84 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Supplementary material | 3D PDF 1 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus obamai sp. n. holotype worker (CASENT0764125). 3D PDF of volumetric surface rendering model of full body. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl1 Supplementary material 2 3D PDF 2 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus obamai sp. n. paratype worker (CASENT0764127). 3D PDF of volumetric surface rendering model of head (most of antennae virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl2 Supplementary material 3 3D PDF 3 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus obamai sp. n. paratype worker (CASENT0764127). 3D PDF of volumetric surface rendering model of mesosoma (head and metasoma mostly virtually removed). Next-generation morphological character discovery and evaluation... 85 Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl3 Supplementary material 4 3D PDF 4 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus obamai sp. n. paratype worker (CASENT0764127). 3D PDF of volumetric surface rendering model of metasoma (virtually separated from mesosoma and most of legs virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl4 Supplementary material 5 3D PDF 5 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D PDF of volumetric surface rendering model of full body. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl5 86 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Supplementary material 6 3D PDF 6 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D PDF of volumetric surface rendering model of head (most of antennae virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl6 Supplementary material 7 3D PDF 7 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D PDF of volumetric surface rendering model of mesosoma (head and metasoma mostly virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl7 Supplementary material 8 3D PDF 8 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D PDF of volumetric surface rendering model of metasoma (virtually separated from mesosoma and most of legs virtually removed). Next-generation morphological character discovery and evaluation... 87 Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl8 Supplementary material 9 3D PDF 9 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D PDF of volumetric surface rendering model of full body. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl9 Supplementary material 10 3D PDF 10 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D PDF of volumetric surface rendering model of head (most of antennae virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl10 88 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Supplementary material | | 3D PDF 11 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D PDF of volumetric surface rendering model of mesosoma (head and metasoma mostly virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl11 Supplementary material 12 3D PDF 12 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D PDF of volumetric surface rendering model of metasoma (virtually separated from mesosoma and most of legs virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl12 Supplementary material 13 3D PDF 13 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Adobe PDF file Explanation note: Zasphinctus sarowiwai sp. n. worker (CASENT0764652). 3D PDF of volumetric surface rendering of segmented mouthparts excluding mandibles (green= maxillae; yellow=labrum; orang=labium). Next-generation morphological character discovery and evaluation... 89 Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl13 Supplementary material 14 Video 1 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus obamai sp. n. paratype worker (CASENT0764127). 3D rotation video of volumetric surface rendering of head (most of antennae virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl14 Supplementary material 15 Video 2 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus obamai sp. n. paratype worker (CASENT0764127). 3D rotation video of volumetric surface rendering of mesosoma (head and metasoma mostly virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl15 90 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Supplementary material 16 Video 3 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus obamai sp. n. paratype worker (CASENT0764127). 3D rotation video of volumetric surface rendering of metasoma (virtually separated from mesosoma and most of legs virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl16 Supplementary material 17 Video 4 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D rotation video of volumetric surface rendering of head (antennae virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl17 Supplementary material 18 Video 5 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D rotation video of volumetric surface rendering of mesosoma (head and meta- soma mostly virtually removed). Next-generation morphological character discovery and evaluation... 91 Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl18 Supplementary material 19 Video 6 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus sarowiwai sp. n. holotype worker (CASENT0764654). 3D rotation video of volumetric surface rendering of metasoma (virtually separated from mesosoma and most of legs virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl19 Supplementary material 20 Video 7 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus sarowiwai sp. n. paratype worker (CASENT0764650). 3D rotation video of volumetric surface rendering of full body. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl20 92 Francisco Hita Garcia et al. / ZooKeys 693: 33—93 (2017) Supplementary material 2 Video 8 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D rotation video of volumetric surface rendering of head (antennae virtually re- moved). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl21 Supplementary material 22 Video 9 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D rotation video of volumetric surface rendering of mesosoma (head and meta- soma mostly virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl22 Next-generation morphological character discovery and evaluation... 93 Supplementary material 23 Video 10 Authors: Francisco Hita Garcia, Georg Fischer, Cong Liu, Tracy L. Audisio, Evan P. Economo Data type: Video File (.mov) Explanation note: Zasphinctus wilsoni sp. n. holotype worker (MCZ-ENT-005 12764). 3D rotation video of volumetric surface rendering of metasoma (virtually separated from mesosoma and most of legs virtually removed). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.693.13012.suppl23