A Brief Introductory Guide to Nanoindentation for Comparative and Evolutionary Biologists, with a Case Study of Bone Material Property Diversity across Artiodactyl Skulls.

Quantifying the material properties of hard biological materials can improve understanding of the relationships between form, function, and performance. This study illustrates the use of nanoindentation as a tool for evaluating material properties in a comparative biology framework. We provide a step-by-step guide for comparative and evolutionary biologists illustrating the collection and analysis of nanoindentation data from samples of artiodactyl skull bones. We assess the impact of methodological decisions on the output of nanoindentation tests. We also investigate whether evolutionary variations in skull bone properties are present between artiodactyl species that engage in intraspecific head-to-head combat and those that do not. Elastic modulus exhibited little variation among numbers of indents performed per test and per bone sample. The average elastic modulus was significantly lower when bones were hydrated with deionized water. The skulls of artiodactyls exhibited a gradient of elastic modulus values in which the anterior of the skull is less stiff than more posterior locations. Species involved in head-to-head combat showed little difference in elastic modulus values compared to non-combat species. This suggests that ecological factors influence the evolutionary diversity of bone material properties, rather than strictly phylogenetic constraints. In a phylogenetic context, nanoindentation reveals tetrapod bone heterogeneity and provides insights into the evolution of these traits.