1 Department of Environmental Sciences, Section of Conservation Biology (NLU), University of Basel, Basel, Switzerland; 2 Laboratory for Molecular Oncology, University Hospital Zuerich, Zuerich, Switzerland; 3 Department of Environmental Sciences, Section of Plant Ecology, University of Basel, Basel, Switzerland; 4 Anatomical Institute, University of Basel, Basel, Switzerland Evolution of female sperm-storage organs in the carrefour of stylommatophoran gastropods K. Beese 1,2 , G. F. J. Armbruster 1,3 , K. Beier 4 and B. Baur 1 Abstract The presence of specialized female sperm-storage organs has been recognized as an important factor influencing postcopulatory sexual selection via sperm competition and cryptic female choice in internally fertilizing species. We morphologically examined the complexity of sperm-storage organs in the carrefour (spermatheca and fertilization pouch) in 47 species of stylommatophoran gastropods. We used partial 28S rDNA sequences to construct a molecular phylogeny, and applied maximum likelihood (ML) and Bayesian methods to investigate the history of spermatheca diversification and to test different hypotheses of sperm-storage organ evolution. The phylogenetic reconstruction supported several gains and losses of spermathecae. Moreover, a complex spermatheca was associated with the occurrence of love darts or other kinds of auxiliary copulatory organs, the presence of a long penial flagellum, and cross-fertilization as the predominant mating system. However, our results also suggest associations of carrefour complexity with body size, reproductive strategy (semelparity versus iteroparity), reproductive mode (oviparity versus ovoviviparity), and habitat type. Carrefour length in 17 snail species possessing a spermatheca was positively correlated with sperm length. Our results indicate that postcopulatory sexual selection as well as life history and habitat specificity may have influenced the evolution of female sperm-storage organs in hermaphroditic gastropods. Key words: Comparative phylogenetic analysis – dart shooting – habitat – hermaphrodite – life history – mating system – postcopulatory sexual selection Introduction In the majority of internally fertilizing animals the processes of insemination and fertilization are uncoupled in space and time. In these species, sperm are preserved in the reproductive tract of the female for weeks, months or even years before being used to fertilize eggs (Birkhead and Møller 1998). The ability to store sperm is an integral part of the speciesÕ reproductive strategy and can provide important advantages (Neubaum and Wolfner 1999). However, despite recent studies demonstrating that females of many taxa possess highly complex organs for storage of sperm (e.g. insects: Pitnick et al. 1999; crustaceans: Bauer and Martin 1991; reptiles: Olsson and Madsen 1998; and birds: Shugart 1988), the causes of interspecific divergence in the presence and morphology of these organs are still not well understood, particularly in hermaphroditic animal species. A variety of adaptive explanations have been proposed to explain the diversity of female sperm-storage organs. One widely supported hypothesis is that postcopulatory sexual selection has played an important role in the evolution of sperm-storage organs, due to the potential influence of female sperm stores on the extent of non-random paternity (Eberhard 1996; Pitnick et al. 1999). A prerequisite for sexual selection via sperm competition is that the sperm of two or more males coexist within the reproductive tract of the female at the time of fertilization (Parker 1970). In the past few years, increasing attention has been paid to the possibility that females of many species are active not only in precopulatory choice but also in controlling the processes of sperm storage and use (Eberhard 1996; Birkhead and Pizzari 2002). The presence of storage organs may allow females to maintain viable sperm from multiple mates and thus selectively bias the fertilization success of sperm in relation to male behaviour (Siva-Jothy and Hooper 1995) or male genotype (Ward 1998a). Males always try to monopolize females (Chapman et al. 2003). Females, however, may benefit from increased between- male variance in sperm traits in their reproductive tract (Jennions and Petrie 2000). The resulting male–female conflict over sperm use could have favoured the evolution of adapta- tions in the female that control the events after copula and, vice versa, counter-adaptations by the male that manipulate sperm storage processes (Parker 1979; Rice and Holland 1997). These adaptations often involve harmful behaviour and might lead to perpetual antagonistic coevolution between certain traits (Rice and Holland 1997) or the evolution of new traits (Lessells 2006), resulting in increased inter-sexual specializa- tions. The presence of female sperm-storage organs should therefore be linked with the presence of other complex or peculiar reproductive traits. Moreover, diverse mating systems that impose different levels of selection pressure on postcop- ulatory processes are expected to covary with the presence of sperm-storage organs and their complexity. The differentiation of female sperm-storage organs could also be dictated by demands of sperm storage capacity arising from differences in animal longevity and⁄or egg productivity, or by selection for functional design to match sperm mor- phology in order to efficiently store and utilize sperm (Pitnick et al. 1999). Females with a high longevity or producing multiple egg clutches in consecutive years may require more specialized organs to provide nourishment or protection (e.g. through anchoring the sperm inside the storage organ) to maintain the viability of sperm (Tingari and Lake 1973; Smith and Yanagimachi 1990). Consequently, the evolution of sperm-storage organs should be coupled with life history. Moreover, female reproductive morphology is presumably associated with habitat specificity, because of adaptations of the life-history traits to local conditions. For example, small body size and ovoviviparity could be adaptations to habitats Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Verlag, Berlin Accepted on 23 May 2008 J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2008.00491.x J Zool Syst Evol Res (2009) 47(1), 49–60