Automated landmarking via multiple templates.

Manually collecting landmarks for quantifying complex morphological phenotypes can be laborious and subject to intra and interobserver errors. However, most automated landmarking methods for efficiency and consistency fall short of landmarking highly variable samples due to the bias introduced by the use of a single template. We introduce a fast and open source automated landmarking pipeline (MALPACA) that utilizes multiple templates for accommodating large-scale variations. We also introduce a K-means method of choosing the templates that can be used in conjunction with MALPACA, when no prior information for selecting templates is available. Our results confirm that MALPACA significantly outperforms single-template methods in landmarking both single and multi-species samples. K-means based template selection can also avoid choosing the worst set of templates when compared to random template selection. We further offer an example of post-hoc quality check for each individual template for further refinement. In summary, MALPACA is an efficient and reproducible method that can accommodate large morphological variability, such as those commonly found in evolutionary studies. To support the research community, we have developed open-source and user-friendly software tools for performing K-means multi-templates selection and MALPACA.