1. Butterflies are frequently used in comparative studies of sexual selection because of their diverse mating systems. In Heliconius, the two major clades in the genus are characterised by contrasting pupal-mating and adult-mating strategies. Adult-mating females are considered to be promiscuous whereas pupal-mating females are thought to be monandrous.
2. Counting spermatophores in female Lepidoptera is a common method for assessing patterns of female remating. However, in pupal-mating Heliconius butterflies spermatophores can become completely degraded, potentially leading to underestimation of female remating rates.
3. We qualitatively characterised the different states of spermatophore degradation, and showed that complete degradation takes approximately 3 weeks in captive-bred H. erato females.
4. We counted spermatophores and/or assayed spermatophore degradation in > 500 Heliconius females across 28 species sampled from natural populations. Among pupal-maters these observations yielded a few rare observations of double mating by recently eclosed females, but generally indicated a lack of rematings. In contrast, approximately 25% of sampled adult-mating females remated at least once.
5. Using a novel statistical analysis, we estimated remating rates from patterns of spermatophore degradation or from counts stratified by age, as indicated by wing-wear. This analysis showed no statistically significant evidence for remating for the pupal-mating H. erato whereas significant remating rates were detected for adult-mating species.
6. The present results support the established view of Heliconius mating systems in which pupal-maters are largely monandrous, whereas adult-maters are polyandrous.