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As part of a study into paternity patterns in hawksbill turtles (Eretmochelys imbricata), we sampled tissue from nesting females and emerging hatchlings on Cousine Island, Republic of Seychelles, in the 2007/08 and 2008/09 nesting seasons. We generated DNA profiles of these samples using an extremely powerful array of 32 variable microsatellite loci (probability of two randomly-chosen individuals having identical genotypes = 9.95 ×10^-31), and used mother and offspring data to reconstruct the paternal genotypes in the paternity analysis software COLONY 2.0 (Wang & Santure 2009). For a full description of the molecular methodology and paternal genotype reconstruction, see Phillips et al. (2013). Over the following two seasons (2009/10 and 2010/11), 12 of these females were observed returning to nest on Cousine (re-migration intervals of 2-3 years), and we again sampled their offspring and reconstructed the paternal genotypes. The fathers of the offspring of 11 of these returning females were new males. However, the offspring of the twelfth female were all sired by the exact same male as in her previous visit two years earlier. We genotyped 79 of this female’s offspring in her first year and 32 in her second.

However one interprets this finding, it is remarkable. A chance re-encounter with the same male is possible but seems unlikely, given the rarity of male re-sightings in our study (three other males in this data set were seen in two separate years of the study) and the conclusion from this is that the number of available male mates is likely very large and/or highly mobile (see Phillips et al. 2013). A repeat encounter may be more likely if individuals use the same, idiosyncratic migration routes across years, but testing this hypothesis would require tracking individuals of both sexes over several remigration periods (e.g., Broderick et al. 2007). Another possibility is that the encounter has resulted from some form of pre- copulatory mate choice, but this seems even less likely – studies on sexual selection in marine turtles have yet to demonstrate biases in paternity patterns (e.g., Phillips et al. 2013) or benefits to females from given mating strategies (e.g., Wright et al. 2013).

A third explanation is that this single female stored viable sperm over two years. This raises the possibility that all females store unused sperm from one season as a means of ensuring fertility, utilizing it if they don’t manage to mate successfully during their next fertile period. Should they re-mate, females would presumably eject their store from the previous season at some point during the courtship/copulation process (selective control of sperm stores by females is well known in other taxa, e.g., Bretman et al. 2009; Løvlie et al. 2013), as otherwise we might expect to see a higher rate of multiple paternity resulting from stored sperm mixing with new sperm. We saw relatively few cases of multiple paternity (< 10% of females), and in the one case of multiple paternity in our 12 re-migrant females, neither male was the father of that female’s offspring in the previous season. Interestingly, if our focal female has stored sperm for two years, her reproductive output has not significantly changed between seasons (number of hatchlings per nest (mean±SE) = 163.5±13.8 vs.146.4±24.4, n_nests = 4 and 5; t = 0.61, df = 6.12, P = 0.56), suggesting that the viability of the sperm has remained high between seasons. However, 66% of eggs in her final observed nest in her second season failed to develop, compared with an average of 1-9% over her previous eight nests, which may indicate an eventual depletion of sperm number or quality.

Our inferences are necessarily speculative, but the basic finding should be of interest to marine (and non-marine) turtle biologists however they choose to interpret it. We urge other researchers to keep a look out for such patterns that may indicate long-term sperm storage. However, if one does choose to interpret this case as an incidence of inter-season sperm storage, we should not get too carried away: in social insects, such as ants, a single mating will often supply a queen with sufficient sperm to last decades, fertilizing literally millions of offspring.

Acknowledgements. We thank the staff and management of Cousine Island, especially J. Henwood, S.-M. Jolliffe, and the island’s owner F. Keeley, for initiating this turtle paternity project, for logistic support in the field, and for collecting so many of the samples; J. Mortimer for feedback on the initial research proposal; A. Krupa, D. Dawson, G. Horsburgh, A. Frantz and T. Burke for help with molecular and statistical work; and the University of East Anglia (UEA) Research Computing Service for supporting data analysis software. Genotyping was performed at the Natural Environment Research Council (NERC) Biomolecular Analysis Facility at Sheffield, UK. The project was funded by a UEA Dean of Science Studentship, NERC, and considerable in-kind support from Cousine Island. Turtle tissue samples were collected under a Seychelles Bureau of Standards permit (NRDC/0266 to D.S. Richardson; last updated in 2011) and exported in accordance with the Convention on International Trade in Endangered Species (permits SC9117736A602 to K.G. Jolliffe and 475521/01 to D.S. Richardson, 2011).

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