Natural history collections are important data repositories, but different chemical treatments of specimens can influence morphological measurements and DNA extraction, complicating taxonomic and conservation decisions dependent upon these data. One such example is the Bay Springs Salamander (Plethodon ainsworthi), the only United States amphibian categorized as Extinct by the IUCN. Recent research has proposed that P. ainsworthi is an invalid taxon, arguing that the 55-year-old type specimens’ morphological distinctiveness from syntopic P. mississippi is a preservation artifact. To address this controversy, we tested for morphological changes across five experimental treatments in proxy P. shermani specimens, and we re-examined the datasets used to support the invalidity of P. ainsworthi. We also tested recently developed DNA extraction techniques on the putatively formalin-fixed P. ainsworthi holotype. We used Bayesian models to demonstrate that preservation method can differentially bias morphological measurements, with most methods causing lower estimates of mass and modestly higher estimates of snout-vent-length:head width ratio. These results are broadly consistent with previous studies of other vertebrates, but inconsistent with the hypothesis that P. ainsworthi type specimens are actually poorly preserved P. mississippi. Attempts to extract DNA from the P. ainsworthi holotype unfortunately proved unsuccessful, preventing conclusive resolution of its status and emphasizing the limitations of promising new methods. Nonetheless, we tentatively recommend continued recognition of P. ainsworthi as a valid but possibly extinct taxon. More generally, we invite all authors who study preserved specimens to recognize and report how certain chemical treatments might impact their results.

These files include: 1) our raw morphological measurements; 2) our R scripts; and 3) our Stan file.