Geometric morphometrics of microscopic animals as exemplified by model nematodes

While a host of molecular techniques are utilized by evolutionary developmental (evo-devo) biologists, tools for quantitative evaluation of morphology are still largely underappreciated, especially in studies on microscopic animals. Here, we provide a standardized protocol for geometric morphometric analyses of 2D landmark data sets using a combination of the geomorph and Morpho R packages. Furthermore, we integrate clustering approaches to identify group structures within such datasets. We demonstrate our protocol by performing exemplary analyses on stomatal shapes in the model nematodes Caenorhabditis and Pristionchus . Image acquisition for 80 worms takes 3–4 d, while the entire data analysis requires 10–30 min. In theory, this approach is adaptable to all microscopic model organisms to facilitate a thorough quantification of shape differences within and across species, adding to the methodological toolkit of evo-devo studies on morphological evolution and novelty. This protocol describes how to perform geometric morphometrics on microscopic animals, as exemplified by analysis of nematode mouth morphology. The procedure provides guidance for microscopy data acquisition, data analysis and validation.