Does the order of copulation matter? Experimental paternity analyses in the earthworm Hormogaster elisae (Annelida: Hormogastridae)

Multiple mating and sperm storage organs allow postcopulatory sexual selection to occur. This is the first time that a paternity test has been performed in an earthworm species. Microsatellite markers are used in Hormogaster elisae to trace paternity for multiple sires. Multiple paternity was detected in some cases, showing sperm mixture even within one cocoon, suggesting the eventual mixture of the allosperm within the spermathecae. Order of copulation influences paternity, the first (P1) and third partner (P3) being the most successful. Interestingly the second partner (P2) almost lacks paternity. The most plausible hypothesis suggests the existence of sperm displacement by flushing out older sperm once the spermathecae are full, which seems to occur when they contain sperm from two partners and P3 donates. At that moment sperm is still stratified thus removing sperm from P2. Afterwards sperm from P1 and P3 would be mixed provoking an equivalent paternity. Given the endogeic nature of the earthworm, copulation was not observed, making the lack of copulation with P2 (due to the need of a recovery time) or copulation without sperm transfer (due to low sperm production rate) possible alternative hypotheses. The weight of the earthworms is related to the number of cocoons they produce but not to their viability, which in all the cases was low, probably due to laboratory conditions. The sperm was kept viable inside the spermathecae for a maximum of two years and three months, suggesting a very effective nourishment system in the epithelium of the storage structures.