During evolution, the morphology of animal organs can vary drastically, such as the reduction or loss of structures. This phenomenon is recurrent in the male copulatory organs of many spiders, where structures that are usually well-developed, are missing or vestigial in some taxa. One example is the retrolateral tibial apophysis (RTA) of the male pedipalp of the most diverse lineage of spiders, the RTA clade. It frequently performs a key function during genital coupling, which is the first and main mechanical engagement during genital coupling (i.e., primary locking). However, in several lineages the RTA is either lost or reduced, raising questions on how genital coupling is achieved in these taxa, and what are the potential drivers for the regression of the RTA. To address these questions, we used the subfamily Amaurobioidinae (Anyphaenidae) as a model and studied the genital mechanics of nine species with different degrees of RTA size. Genital coupling was reconstructed using microcomputed tomography (micro-CT) data of cryofixed mating pairs, revealing that, except for a species with a prominent RTA, primary locking is solely achieved by inserting the conductor (a male genital sclerite) into one of the female copulatory openings. Our phylogenetic analyses indicate that the RTA has been independently lost or reduced six times in Amaurobioidinae, and that at least one functional replacement of the RTA by the conductor has occurred within the subfamily. We hypothesize that the use of the conductor for primary locking replaced the primary function of the RTA, allowing its repeated regressions.
