DOI: 10.5061/dryad.v41ns1s15
title: Parallel and divergent morphological adaptations underlying the evolution of jumping ability in ants

description: Jumping is a rapid locomotory mode widespread in terrestrial organisms.
 However, it is a rare specialization in ants. Forward jumping has been
 reported within four distantly related ant
 genera: Gigantiops, Harpegnathos, Myrmecia, and
 Odontomachus. The temporal engagement of legs/body parts during jump,
 however, varies across these genera. It is unknown what morphological
 adaptations underlie such behaviors and whether jumping in ants is solely
 driven directly by muscle contraction or additionally relies on elastic
 recoil mechanism. We investigate the morphological adaptations for jumping
 behavior by comparing differences in the locomotory musculature between
 jumping and non-jumping relatives using X-ray micro-CT and 3D
 morphometrics. We found that the size-specific volumes of the trochanter
 depressor muscle (scm6) of the middle and hind legs are 3–5 times larger
 in jumping ants, and that one coxal remotor muscle (scm2) is reduced in
 volume in the middle and/or hind legs. Notably, the enlargement in the
 volume of other muscle groups is directly linked to the legs or body parts
 engaged during the jump. Furthermore, a direct comparison of the muscle
 architecture revealed two significant differences between jumping versus
 non-jumping ants: First, the relative Physiological Cross-Sectional Area
 (PCSA) of the trochanter depressor muscles of all three legs was larger in
 jumping ants, except in the front legs of O. rixosus and M. nigrocincta;
 second, the relative muscle fiber length was shorter in jumping ants
 compared to non-jumping counterparts, except in the front legs of O.
 rixosus and M. nigrocincta. This suggests that the difference in relative
 muscle volume in jumping ants is largely invested in the area (PCSA), and
 not in fiber length. There was no clear difference in the pennation angle
 between jumping and non-jumping ants. However, the hind leg length
 relative to body length was longer in jumping ants. Based on direct
 comparison of the observed vs. possible work and power output during
 jumps, we surmise that direct muscle contractions suffice to explain
 jumping performance, in two species, but elastic recoil is likely
 important in one. We suggest that increased investment in jumping-relevant
 musculature is a primary morphological adaptation that separates jumping
 from non-jumping ants. These results elucidate the common and
 idiosyncratic morphological changes underlying this rare adaptation in
 ants.
description: Material
 We selected one worker
 specimen preserved in ethanol (70–99%) from each genus for which jumping
 behavior has been previously documented, to carry out detailed computed
 tomography (CT) scanning and 3D morphometry: <em>Harpegnathos
 saltator</em> Jerdon, 1851 (unique specimen identifier:
 CASENT0764679), <em>Gigantiops destructor </em>Fabricius, 1804
 (CASENT0709414), <em>Odontomachus rixosus </em>Smith, F., 1957
 (CASENT9741319) and <em>Myrmecia nigrocincta</em> Smith, F.,
 1858 (CASENT0741302). As a control, we imaged a small set of non-jumping
 ant species: <em>Euponera sikorae </em>Forel, 1891
 (CASENT0709898), <em>Formica rufa </em>Linnaeus, 1761
 (CASENT0741323), <em>Odontomachus kuroiwae</em> Matsumura,
 1912 (CASENT0741313) and <em>Nothomyrmecia macrops</em> Clark,
 1934 (CASENT0795539). An
 effort was made to select comparison species as closely related as
 possible to the jumping species considering availability of preserved
 specimens. All specimens
 were stained in iodine solution 3­–7 days prior to the scanning date. The
 length of the leg segments was measured three times within the same
 specimen (in the right and/or left legs). For the specimens for which no
 information on the body mass was available, the whole body was scanned,
 and body volume was used as proxy for body mass via assuming a uniform
 density of 1040
 kg/m<sup>3</sup>: <em>Euponera sikorae
 </em>(CASENT0741359); <em>Formica</em>
 <em>rufa</em> (ANTSCAN, CASENT0709272); <em>Gigantiops
 destructor</em> (CASENT0744574); <em>Nothomyrmecia</em>
 <em>macrops</em> (CASENT0741364).  
 Micro-CT scanning and
 3D-reconstruction Micro-CT scans were generated with a Zeiss
 Xradia 510 Versa 3D X-ray microscope operated with the Zeiss
 Scout-and-Scan Control System software (version 14.0.14829.38124) at the
 Okinawa Institute of Science and Technology Graduate University, Japan.
 Scans were conducted with a 40 kV (75 μA) / 3 W beam strength under a 4x
 magnification. Voxel size and exposure time depended on specimen size
 (Suppl. Table S1). As the mesosoma of ants exceeds the field-of-view of
 the camera at high magnification, vertical stitching of serial scans was
 used. 3D reconstructions of the resulting scan projection data were done
 with the Zeiss Scout-and-Scan Control System Reconstructor (version
 14.0.14829.38124) and saved in txm file format. Postprocessing of txm raw
 data was done with Amira 2019.2 (Visage Imaging GmbH, Berlin, Germany) to
 segment individual structures into discrete tissue volumes. The segmented
 voxels were then exported with the plugin script “multiExport” (Engelkes
 et al., 2018) in Amira 2019.2 as 2D TIFF image stacks. VG-Studio 3.4
 (Volume Graphics GmbH, Heidelberg, Germany) was used to create volume
 renders from the TIFF image series. Muscle architecture was reconstructed
 with Amira 2019.2 XTracing extension, following the workflow presented in
 Katzke, Puchenkov, Stark, & Economo (2022). The accuracy of the
 tracing algorithm in Katzke et al. (2022) was 92% for fiber length
 estimation and 100% for the pennation angle estimation. Muscle identity
 and nomenclature follows Aibekova et al. 2022. Muscles most relevant in
 the movement of the legs were segmented (Ipcm2, Iscm4, I-, II-, IIIscm1,
 II-, IIIscm2, I-, II-, IIIscm3, Ipcm8, II-, IIIscm6, Ipcm4, II-,
 IIIpcm3_4, I-, II-, IIIctm1, I-, II-, IIIctm2, I-, II-, IIIctm3); in
 addition, large muscles including the indirect muscle of the head (Idvm5),
 the levator (IA1), and one of the rotators (IA2) of abdomen were segmented
 for control. We want to mention one caveat related to preparation
 technique. It is possible there may be different degrees of muscle
 shrinkage due to the preservation in high ethanol concentrations (70–99%)
 This could in principle cause spurious differences across species, as the
 level of contraction may vary among species, however we have no evidence
 this was an issue in this case, and it is unlikely to affect the broad
 differences identified in this study.
description: Amira version 2019.2 was used to process Volumetric
 data. Amira XTracing Extension 2019.2 was used to trace
 muscle fibers.