Geometric vs. traditional morphometric methods for exploring morphological variation of tadpoles at early developmental stages; supplementary material

We conducted a comparative (2D landmark-based geometric and traditional) morphometric analysis on tadpoles at early developmental stages. Two species of brown frog (Rana dalmatina and R. temporaria) and the common toad (Bufo bufo) were involved, all raised in the laboratory from fertilized eggs collected in their natural habitat. Taxonomic identification was confirmed by the DNA barcoding method with the 16S rRNA sequence as the gene marker. Interested to compare the methodologies for quantification and description of morphological differences among tadpoles of mentioned species, we aimed to: 1) calculate interspecies genetic distances as the most relevant measurement for species differentiation, 2) determine and describe size and shape variation, 3) identify relationships among the analyzed species at the morphological level and 4) assess their classification accuracy. Within the framework of the specified aims, both methodologies produced very similar results, i.e., the smallest divergence was between R. dalmatina and R. temporaria, while the most discriminative were B. bufo and R. temporaria. However, we observed subtle shape variation of the distal region of the tail that was detected only by the geometric morphometrics. Our findings support the following. Geometric morphometric method captures more subtle shape differences that were unable to be recovered from linear measurements. It performs slightly better in classification rate. Although it was not quantified, it stands to reason that there is no difference in time investment between the two approaches. Geometric morphometrics provides more information that can be leveraged to answer further questions and it has a clear advantage in visualizing.