The use of volume data and digital three-dimensional (3D) surface models in biology has increased quickly and steadily. Various methods are available to acquire 3D data, among them episcopic imaging techniques. Based on the episcopic microscopy with on-block staining protocol of Weninger et al. (1998), we describe a simple and versatile setup for episcopic microtomy. It is composed of a consumer DSLR digital camera combined with standard histology equipment. The workflow of block surface staining and imaging, image processing, stack alignment, surface generation (including a custom Amira ® macro), and 3D model editing is described in detail. For our sample specimen (Alytes obstetricans; Amphibia: Anura) we obtained images with a pixel size of 5.67 x 5.67 µm 2. The generated image stacks allowed distinguishing different tissues and were well-suited for creating a 3D surface model. We analyzed the alignment quality achieved by various selections of specimen and fiducial marker spots. The fiducial spots had a significant positive effect on the alignment quality with the best alignment having a maximum mean alignment error of about 44.7 µm. We further tested the APS-C camera with combinations of macro lens, extension tube or teleconverter. The macro lens and extension tube yielded the smallest pixel size of 2.53 x 2.53 µm 2. Considering data quality and resolution, and depending on object sizes and research goals, DSLR captured episcopic microtomy can be an alternative to other techniques, such as traditional histological sectioning or micro-computed tomography.

Computed‐tomography‐derived (CT‐derived) polymesh surfaces are widely used in geometric morphometric studies. This approach is inevitably associated with decisions on scanning parameters, resolution, and segmentation strategies. Although the underlying processing steps have been shown to potentially contribute artefactual variance to three‐dimensional landmark coordinates, their effects on measurement error have rarely been assessed systematically in CT‐based geometric morphometric studies. The present study systematically assessed artefactual variance in landmark data introduced by the use of different voxel sizes, segmentation strategies, surface simplification degrees, and by inter‐ and intra‐observer differences, and compared their magnitude to true biological variation. Multiple CT‐derived surface variants of the anuran (Amphibia: Anura) pectoral girdle were generated by systematic changes in the factors that potentially influence the surface geometries. Twenty‐four landmarks were repeatedly acquired by different observers. The contribution of all factors to the total variance in the landmark data was assessed using random‐factor nested permanovas. Selected sets of Euclidean distances between landmark sets served further to compare the variance among factor levels. Landmark precision was assessed by landmark standard deviation and compared among observers and days. Results showed that all factors, except for voxel size, significantly contributed to measurement error in at least some of the analyses performed. In total, 6.75% of the variance in landmark data that mimicked a realistic biological study was caused by measurement error. In this landmark dataset, intra‐observer error was the major source of artefactual variance followed by inter‐observer error; the factor segmentation contributed < 1% and slight surface simplification had no significant effect. Inter‐observer error clearly exceeded intra‐observer error in a different landmark dataset acquired by six partly inexperienced observers. The results suggest that intra‐observer error can potentially be reduced by including a training period prior to the actual landmark acquisition task and by acquiring landmarks in as few sessions as possible. Additionally, the application of moderate and careful surface simplification and, potentially, also the use of case‐specific optimal combinations of automatic local thresholding algorithms and parameters for segmentation can help reduce intra‐observer error. If landmark data are to be acquired by several observers, it is important to ensure that all observers are consistent in landmark identification. Despite the significant amount of artefactual variance, we have shown that landmark data acquired from microCT‐derived surfaces are precise enough to study the shape of anuran pectoral girdles. Yet, a systematic assessment of measurement error is advisable for all geometric morphometric studies.

Previous myological studies show inconsistencies with regard to the identification and naming of the shoulder joint muscles in frogs and toads (Amphibia: Anura). Those inconsistencies were revealed and resolved by assessing the ontogenetic development, innervation, and adult morphology of selected anuran species representing ancient lineages and two major neobatrachian groups. To do so, digital dissections of volumes acquired by histological serial sectioning, episcopic microtomy, and contrast-enhanced micro-computed tomography scanning were performed and three-dimensional reconstructions were derived. Muscle units crossing the shoulder joint were defined, their ontogenetic development was described, their homology across species was established, and a consistent nomenclature was suggested. The mm. anconaeus, dorsalis scapulae, latissimus dorsi, and the group of scapulohumeralis muscles were ontogenetically derived from the dorsal pre-muscle mass present in all tetrapods. The ventral pre-muscle mass gave rise to the mm. cleidohumeralis, episternohumeralis, supracoracoideus, coracoradialis, subcoracoscapularis, coracobrachialis, and pectoralis. The results indicate that the mm. anconaeus, dorsalis scapulae, latissimus dorsi, coracoradialis, and the portionis sternalis and abdominalis of the m. pectoralis have consistently been recognized and denoted in previous studies, whereas the names for the muscle units commonly denoted as m. coraco-brachialis longus and as parts of the m. deltoideus are misleading with regard to the ontogenetic origin of these muscles. The mm. scapulohumeralis profundus anterior and posterior, although present, have been overlooked in some studies. The mm. cleidohumeralis, supracoracoideus, and coracobrachialis are present with two parts or portions in some species, these portions have previously not always been recognized and assigned correctly.

Frogs and toads (Lissamphibia: Anura) show a diversity of locomotor modes that allow them to inhabit a wide range of habitats. The different locomotor modes are likely to be linked to anatomical specializations of the skeleton within the typical frog Bauplan. While such anatomical adaptations of the hind limbs and the pelvic girdle are comparably well understood, the pectoral girdle received much less attention in the past. We tested for locomotor‐mode‐related shape differences in the pectoral girdle bones of 64 anuran species by means of micro‐computed‐tomography‐based geometric morphometrics. The pectoral girdles of selected species were analyzed with regard to the effects of shape differences on muscle moment arms across the shoulder joint and stress dissipation within the coracoid. Phylogenetic relationships, size, and locomotor behavior have an effect on the shape of the pectoral girdle in anurans, but there are differences in the relative impact of these factors between the bones of this skeletal unit. Remarkable shape diversity has been observed within locomotor groups indicating many‐to‐one mapping of form onto function. Significant shape differences have mainly been related to the overall pectoral girdle geometry and the shape of the coracoid. Most prominent shape differences have been found between burrowing and nonburrowing species with headfirst and backward burrowing species significantly differing from one another and from the other locomotor groups. The pectoral girdle shapes of burrowing species have generally larger moment arms for (simulated) humerus retractor muscles across the shoulder joint, which might be an adaptation to the burrowing behavior. The mechanisms of how the moment arms were enlarged differed between species and were associated with differences in the reaction of the coracoid to simulated loading by physiologically relevant forces.

No abstract

Different kinds of bone measurements like coordinates of landmarks, linear distances, or variables describing trabecular bone are routinely used in biological studies and related fields to answer a multitude of questions. Such measurements are acquired from, among others, computed tomography (CT) volumes or derived polygon surfaces (e.g.

&lt;p&gt;Marine biogenic carbonate deposits are important climate archives as environmental conditions during mineral genesis are recorded chemically in the crystal lattice. When exposed to diagenetic alteration metastable carbonate phases as aragonite are prone to transform into more stable calcite, Mg-calcite or dolomite resulting in the loss of the original geochemical information. As diagenetic alteration is often heterogeneous, numerous carbonate archives are characterized by multi-phase compositions, including potentially unaltered remains of primary formation. Consequently, high-spatial-resolution identification of adjacent carbonate mineral phases is of key interest to constrain diagenetic fronts, as well as the degree and pattern of a carbonate archive alteration, and the deciphering of relevant geochemical proxy information.&lt;/p&gt;&lt;p&gt;The aim of the present study is to contribute to the localization of potentially unaltered localities within heterogeneous carbonate samples, enhancing the retrieval of original geochronological and proxy information. Using natural tropical fossil coral samples as an example, we present an approach for the 3D-identification of multiple carbonate phases using &amp;#181;-computed tomography scans with a resolution on a &amp;#181;m scale. The straightforward discrimination of aragonite, calcite, and dolomite from CT-imaging is principally hampered by the similar X-ray densities of these carbonate phases. To overcome this problem, reference material blocks for each of the three carbonate minerals were scanned together with the carbonate sample. Using the AVIZO 2019 software package in combination with a self-developed Tool command language (Tcl) script each of the carbonate reference blocks within the scanned volume was systematically subsampled for its voxel grey values. The obtained data set was statistically analysed and a robust mean voxel grey-value was calculated for each reference mineral. Subsequently, these mean grey values were used for the automated selection of seed points for subsequent image segmentation throughout the entire scanned volume based on a self-developed Tcl script. After seed point definition mineral identification was carried out throughout the volume using the watershed algorithm as a region-based image segmentation method. The final result yielded an approximation of the 3D-distribution of identified carbonate phases throughout the sample on a &amp;#181;m scale, which represents an excellent starting point for subsampling strategy development.&amp;#160;&lt;/p&gt;

No abstract