Effect of ethanol preservation on cephalopod beak geometric morphometrics over time: Sepia esculenta as a case

Recent studies increasingly recognize that preservation of biological specimens can influence the morphometry of animals and their tissues. Although cephalopod beaks are often preserved in ethanol reagents, there is a lack of quantitative studies that investigate the effect of ethanol preservation on beak morphology. In this study, we used geometric morphometric (GM) methods to characterize and quantify the morphology of Sepia esculenta beaks across five preservation periods: original (unpreserved), 50 days, 150 days, 360 days, and 720 days, and we tested the effect of preservation on downstream statistical analysis. Our results indicate that ethanol preservation causes significant changes in both the shape and size of cephalopod beaks, with the degree of change increasing as preservation time extends. Additionally, preservation can negatively impact the accuracy of downstream statistical analyses by inducing shape changes that disrupt sex-specific traits and reduce classification reliability. Therefore, beak samples from different preservation periods should not be combined when conducting downstream statistical analyses to avoid compromising accuracy. Further research is needed to assess the generalizability of these findings across different cephalopod species and preservation methods.