Evolution of male copulatory organs in box crabs (Decapoda: Eubrachyura: Calappidae de Haan, 1833)
Male genitalia are extraordinarily diverse, and this diversity makes them valuable taxonomic and phylogenetic markers. Genitalia of true crabs (Brachyura) consist of two pairs of modified appendages, or gonopods. The first pair is tubular, and holds the second pair during copulation. Whether the second pair is shorter or longer than the first pair has important implications for copulation. Its length is often similar in closely related species, e.g. within genera and families, but is highly variable in the Brachyura at large. The genus Calappa Weber, 1795 contains species with long, short, and intermediate-length second gonopods, representing an unusually variable pattern. We investigated the evolution of the second gonopods in Calappa and other members of Calappidae. We assembled a molecular multi-locus phylogeny (mitochondrial marker: COI and 16S, nuclear marker: Enolase, H3 and 28S). Based on this phylogeny, long second gonopods are the ancestral state for Calappa as well as Calappidae in general. Short second gonopods evolved once, while intermediate-length second gonopods evolved independently at least four times. Scanning electron microscopic images of second gonopods of 22 species of Calappidae reveal that shortening of the second gonopod is always achieved by a shortening of the terminal segment. Congruently, taxon-specific structures at the tip of the terminal segment of the second gonopod, such as spines and pistil-like protrusions, are limited to long second gonopods. Based on current knowledge, a shortening of the second gonopod should be accompanied by extensive changes in the copulatory mechanism of males and females, including changes to the copulatory mechanism, mechanism of species recognition, targets of sexual selection, and the potential for sperm removal. Calappa therefore provides a vital example for the study of changes in copulatory mechanisms.