Microsatellite markers reveal multiple paternity and sperm storage in the Mediterranean spurthighed tortoise, Testudo graeca

The spur-thighed tortoise (Testudo graeca) is a terrestrial species in which multiple mating is frequently observed. We assessed the probability of multiple paternity in clutches (n = 15) laid by eight females, including successive clutches. Paternity was determined by microsatellite analyses at three loci. A large number of alleles per locus (n = 15-22) provided high probabilities of detecting multiple paternity, particularly at all loci combined (P = 0.989). Multiple paternity was found in 20% of the clutches in which offspring displayed more than two paternal alletes. However, this frequency may have been underestimated, given the small clutch sizes and the few loci used. Also, T graeca is able to store sperm from single or multiple matings and can use it to fertilize subsequent clutches of eggs, as indicated by the fact that the second clutch of a captive female was sired by a different male and that clutches of another female were multiply sired by the same males. These results confirm that multiple paternity exists in T graeca and that sperm storage in this species may be an important reproductive strategy to fertilize multiple clutches per year.