DOI: 10.5061/dryad.gb5mkkwsb
title: Attempting genetic inference from directional asymmetry during convergent hindlimb reduction in squamates

description: Loss and reduction of paired appendages is common in vertebrate evolution.
 How often does such convergent evolution depend on similar developmental
 and genetic pathways? For example, many populations of the Threespine
 Stickleback and Ninespine Stickleback (Gasterosteidae) have independently
 evolved pelvic reduction, usually based on independent mutations that
 caused reduced Pitx1 expression. Reduced Pitx1 expression has also been
 implicated in pelvic reduction in manatees. Thus, hind limb reduction
 stemming from reduced Pitx1 expression has arisen independently in groups
 that diverged tens to hundreds of millions of years ago, suggesting a
 potential for repeated use of Pitx1 across vertebrates. Notably, hindlimb
 reduction based on reduction of Pitx1 expression produces left-larger
 directional asymmetry in the vestiges. We used this phenotypic signature
 as a genetic proxy, testing for hindlimb directional asymmetry in six
 genera of squamate reptiles that independently evolved hindlimb reduction
 and for which genetic and developmental tools are not yet developed:
 Agamodon anguliceps, Bachia intermedia, Chalcides sepsoides, Indotyphlops
 braminus, Ophisaurus attenuatuas and O. ventralis, and Teius teyou.
 Significant asymmetry occurred in one taxon, Chalcides sepsoides, whose
 left-side pelvis and femur vestiges were 18% and 64% larger than
 right-side vestiges, respectively, suggesting modification of Pitx1
 expression in that species. However, there was either right-larger
 asymmetry or no directional asymmetry in the other five taxa, suggesting
 multiple developmental genetic pathways to hindlimb reduction in squamates
 and vertebrates more generally.
description: <em>Micro Computed Tomography (μ-CT) scanning</em>.
 Each specimen was wrapped in plastic and scanned for 4 minutes using a
 Perkins-Elmer Quantum GX2 micro-CT Imaging System. To prevent beam
 hardening, an image artifact, we used either an Al 1.0 mm or an Al 0.5 mm
 + Cu 0.06 mm filter, as needed. Because individual size varied within and
 among taxa, we adjusted voltage, current, and voxel size for specimen size
 (Table 1). We used the smallest voxel size that still contained the pelvis
 and femurs (if present). <em>Image
 processing.</em> We used the open-source program InVesalius v.3.1 to
 reconstruct 3D images from raw DICOM files. We applied a reconstruction
 threshold to reduce image noise. Thresholds varied by voxel size and
 tissue density and were adjusted by eye to create surfaces with reduced
 artifact, maximal bone quality, and high definition. Because these
 criteria are subjective, no single setting optimizes 3D surface
 reconstruction for all specimens. For consistency, the thresholds were
 determined by the same individual (S.S.) across all samples.
 <em>Landmarks</em>. Homologous landmarks were placed
 on each 3D surface using open-source software MeshLab 2016. All landmarks
 were digitized by S.S. for consistency. Each image was rotated during
 landmark placement to minimize parallax, and the images were rotated after
 landmark placement to ensure visually that they had been placed correctly.
 We marked the anterior point of the pubis and the posterior point of the
 ilium on right and left sides to measure the pelvis (Fig. 1). In specimens
 with a less well-developed pubis, the anterior- medial point of the
 epipubis was used as the anterior landmark. In species without femurs, the
 pubis, ilium, and ischium bones of the pelvis were usually
 indistinguishable; we therefore landmarked the anterior and posterior
 points of the pelvic vestige. For species with femurs,
 we placed landmarks on the proximal point of the femur head (at the hip)
 and the most distal point of the femur (at the knee; Fig. 1). CT
 resolution was not fine enough to distinguish distal limb bones in
 <em>B</em>. <em>intermedia</em> due to extensive
 reduction (Fig. 1B). Therefore, proximal landmarks were placed on femur
 head and distal landmarks were placed on the most distal skeletal point of
 the limb, which might include non-femoral elements. However, in mice,
 altered <em>Pitx1</em> expression resulted in reduction in the
 tibia, fibula, and metatarsals, as well as the pelvis and femur (Lanctot
 et al. 1999; Szeto et al. 1999; Marcil et al. 2003). Thus, including
 elements distal to the femur should yield a suitable measure of hind limb
 length in <em>B</em>. <em>intermedia</em>,
 especially since our metric is asymmetry. We exported landmarks from
 MeshLab as picked_points.pp files.
description: R statistical software is all that is required.