Quantitative Analysis of Fish Morphology Through Landmark and Outline-based Geometric Morphometrics with Free Software.

Morphology underpins key biological and evolutionary processes that remain elusive. This is in part due to the limitations in robustly and quantitatively analyzing shapes within and between groups in an unbiased and high-throughput manner. Geometric morphometrics (GM) has emerged as a widely employed technique for studying shape variation in biology and evolution. This study presents a comprehensive workflow for conducting geometric morphometric analysis of fish morphology. The step-by-step manual provides detailed instructions for using popular free software, such as the TPS series, MorphoJ, ImageJ, and R, to carry out generalized Procrustes analysis (GPA), principal component analysis (PCA), discriminant function analysis (DFA), canonical variate analysis (CVA), mean shape analysis, and thin plate spline analysis (TPS). The Momocs package in R is specifically utilized for in-depth analysis of fish outlines. In addition, selected functions from the dplyr package are used to assist in the analysis. The full process of fish outline analysis is covered, including extracting outline coordinates, converting and scaling data, defining landmarks, creating data objects, analyzing outline differences, and visualizing results. In conclusion, the current protocol compiles a detailed method for evaluating fish shape variation based on landmarks and outlines. As the field of GM continues to evolve and related software develops rapidly, the limitations associated with morphological analysis of fish are expected to decrease. Interoperable data formats and analytical methods may facilitate the sharing of morphological data and help resolve related scientific problems. The convenience of this protocol allows for fast and effective morphological analysis. Furthermore, this detailed protocol could be adapted to assess image-based differences across a broader range of species or to analyze morphological data of the same species from different origins. Key features • This protocol provides a comprehensive set of commonly used GM-analyzing methods and visualizing skills plus supporting information to help assess the appropriate analysis method • By incorporating both landmarks and outlines, this protocol facilitates a thorough analysis of two-dimensional shape variation in fish, covering a wide range of morphological features • The simplified workflow and detailed procedures make it accessible for non-experienced users to successfully complete the analysis while also providing valuable insights for experienced users Graphical overview The steps include image acquisition as data sources, digitization of fish morphology using landmark-based methods, analysis of shape variation characteristics, and visualization of the results in relation to biological interpretation. Largemouth bass () is used as an example in the schematic representation.