In species where females mate promiscuously, the reproductive success of males depends both on their ability to acquire mates (pre-copulatory sexual selection) and ability of their ejaculates to outcompete those of other males (post-copulatory sexual selection). Sperm competition theory predicts a negative relationship between investment in body traits favouring mate acquisition (secondary sexual characters, SSCs) and investment in ejaculate size or quality, due to the inherent costs of sperm production. In contrast, the phenotype-linked fertility hypothesis posits that male fertilizing efficiency is reliably reflected by the phenotypic expression of male SSCs, allowing females to obtain direct benefits by selecting more ornamented males as copulation partners. In this study, we investigated the relationships between male SSCs and size and quality (viability and longevity) of ejaculates allocated to females in mating trials of the freshwater crayfish Austropotamobius italicus. We showed that the relative size of male weapons, the chelae, was negatively related to ejaculate size, and that chelae asymmetry, resulting from regeneration of lost chelipeds, negatively covaried with sperm longevity. Moreover, males allocated more viable sperm to mates from their own rather than different stream of origin. Our findings thus suggest that, according to sperm competition theory, pre-copulatory sexual selection for large weapons used in male fighting may counteract post-copulatory sperm competition in this crayfish species, and that investment in cheliped regeneration may impair ejaculate quality.

The protection of freshwater biodiversity has become a priority task for conservation practices, as freshwater ecosystems host high levels of cryptic diversity, while also record similarly high rates of extinction. The Italian white-clawed crayfish Austropotamobius italicus is an endemic freshwater crustacean, threatened by several anthropogenic impacts such as habitat fragmentation, pollution, invasion of exotics, and climate change. Previous phylogenetic studies conducted in Italy pointed out a complex phylogeographic framework for the species, with four different subspecies currently recognized. Conservation efforts, particularly when involving restocking and reintroduction, require a detailed knowledge of their population genetics. In this study we describe the genetic structure of A. italicus populations in northern Italy (Lombardy Alpine foothills and northern Apennines) by using the informative mitochondrial marker cytochrome c oxidase subunit I, in order to assess their current evolutionary diversity and past phylogeographic history from a conservation perspective. Our results contribute to the mapping of the contact area among A. i. carsicus and A. i. carinthiacus in the Orobie Larian Prealps. More interestingly, we highlight the existence of two deeply differentiated evolutionary lineages within A. i. carsicus, showing alternative phylogeographic patterns and past demographic trends. We propose to consider these two clades as distinct molecular operational taxonomic units for the conservation of this endangered crayfish.