Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell

•We analyze multiple paternity in T. hermanni boettgeri from amniotic fluid from the eggshell.•In addition to STR analysis we evaluate SNP variability to improve informativity of monomorphic markers.•We find multiple paternity in 52.9% of the clutches analyzed.

We identified multiple paternity in 52.9% of the clutches of Hermann's tortoise Testudo hermanni boettgeri using polymorphic microsatellite markers. In addition we demonstrated sperm storage across seasons. DNA was extracted from the amniotic fluid adhering to the eggshell's inner surface, a procedure suitable for easy, non-invasive DNA sampling in conservation and breeding programs. To improve the informative value of monomorphic single tandem repeat (STR) markers we additionally analyzed single nucleotide polymorphism (SNP) variability.