Microsatellite markers reveal multiple paternity and sperm storage in the Mediterranean spur-thighed tortoise, Testudo graeca

Abstract

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 alleles. 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.

Sperm storage and low incidence of multiple paternity in the European pond turtle, Emys orbicularis: A secure but costly strategy?

“…These results may be due to a mutation of one of the maternal alleles, as these offspring were from the same nest. A similar mutation at the same locus was previously described in Testudo graeca (Roques et al, 2004).…”

“…However, such observations do not identify offspring from different males, so that the quantitative and qualitative contributions of stored sperm to successive clutches could not be evaluated. Identification of paternal alleles in successive clutches has confirmed both multiple paternity and sperm storage over both short and long periods in turtles Crim et al, 2002;Moore and Ball, 2002;Roques et al, 2004). For species that lay multiple 9 clutches per season, the time for effective matings is the interval between oviposition and the subsequent ovulation (Gist and Congdon, 1998).…”

“…Therefore, starting egg maturation early would enable a female to lay a higher number of clutches within a season. Sperm storage within the same breeding season is common in most turtle species and has generally been viewed as a mechanism that increases the number of clutches per year Roques et al, 2004). Alternatively, the percent of reproductive E. orbicularis females was very small in years of drought (Keller, 1997).…”

“…Mating systems have important implications for effective population size, level of inbreeding, and genetic diversity within and among populations. Multiple paternity is rare in E. orbicularis in south-western Spain, while it was more frequent in other turtles Ireland et al, 2003;Lee and Hays, 2004;Roques et al, 2004). This means that this population may also lack the advantages associated with multiple paternity, such as an increased effective population size and higher genetic variability (Sugg and Chesser, 1994).…”

“…For species that lay multiple 9 clutches per season, the time for effective matings is the interval between oviposition and the subsequent ovulation (Gist and Congdon, 1998). Given the short length of this interval, sperm storage was considered to be a strategy that enabled females to fertilize consecutive clutches in a single reproductive season, without the necessity of additional matings (Fitzsimmons, 1998;Kichler et al, 1999;Roques et al, 2004). On the other hand, the frequency and the benefits of long term sperm storage (across years) are not well known in turtles (Palmer et al, 1998;.…”