Towards a Diagnostic Tool for Turtle Ootaxonomy: Investigation of Microstructural Differences in the Eggshells of Australian Freshwater Turtles

Researchers frequently encounter turtle nests after the nest has been raided, finding only eggshell fragments that are difficult to identify. Valuable information on the breeding biology and needs of different species might still be collected from these nests if remains could be confidently assigned to a particular species. However, this can be difficult if there are multiple turtle species nesting within an area at a given time, and gross-scale characteristics such as egg size and shape are no longer available due to damage. This study examined eggshell microstructure, by scanning electron microscopy (SEM), among multiple species of freshwater turtle eggshell fragments, with the aim of potentially developing a diagnostic tool for species identification. Eggshell fragments were collected from known sources of four species of turtle native to eastern Australia: Eastern Long-necked Turtle (Chelodina longicollis), Murray River Turtle (Emydura macquarii), Bell's Turtle (Myuchelys bellii), and Bellinger River Turtle (M. georgesi). Fragments were scanned and analyzed for microstructural and ultrastructural features, and measured for differences in feature size across species. Central plaque diameter emerged as a potential diagnostic feature for separating species, with longneck turtle eggshells having no visible plaques, Murray River Turtle eggshells having relatively small mean plaque size (19.8 μm ± 10.28 SD), while Bell's Turtle (43.5 μm ± 14.16 SD) and Bellinger River Turtle eggshells (41.9 μm ± 15.41 SD) had relatively large plaques with little overlap between genera. Other measured features (shell unit size, basal knob size, and shell unit density) significantly differed in means among species but overlapped in range, or showed a lack of statistical difference, likely preventing the technique being definitive with a single measure. Some of this variance may be due to inherent confounding factors, such as the replicability of fragment preparation, condition of eggshells, and stage of embryonic development at the time of collection. These results suggest that use of measurements of microstructural features has promise as a means for distinguishing the provenance of eggshell fragments, and further investigation is warranted to develop site-specific protocols for use as a diagnostic tool for turtle ootaxonomy.