ORIGINAL ARTICLE doi:10.1111/j.1558-5646.2007.00150.x THE RISK OF POLYSPERMY IN THREE CONGENERIC SEA URCHINS AND ITS IMPLICATIONS FOR GAMETIC INCOMPATIBILITY AND REPRODUCTIVE ISOLATION Don R. Levitan,1,2 Casey P. terHorst,1 and Nicole D. Fogarty1 1Department of Biological Science, Florida State University, Tallahassee, Florida 32306-1100 2E-mail: [email protected] Received October 25, 2006 Accepted March 19, 2007 Developmental failure caused by excess sperm (polyspermy) is thought to be an important mechanism driving the evolution of gamete-recognition proteins, reproductive isolation, and speciation in marine organisms. However, these theories assume that there is heritable variation in the susceptibility to polyspermy and that this variation is related to the overall affinity between sperm and eggs. These assumptions have not been critically examined. We investigated the relationship between ease of fertilization and susceptibility to polyspermy within and among three congeneric sea urchins. The results from laboratory studies indicate that, both within and among species, individuals and species that produce eggs capable of fertilization at relatively low sperm concentrations are more susceptible to polyspermy, whereas individuals and species producing eggs that require higher concentrations of sperm to be fertilized are more resistant to polyspermy. This relationship sets the stage for selection on gamete traits that depend on sperm availability and for sexual conflict that can influence the evolution of gamete-recognition proteins and eventually lead to reproductive isolation. KEY WORDS: Fertilization, gamete, polyspermy, reproductive isolation, sexual conflict, sperm availability, Strongylocentrotus. The evolution of gametic incompatibility and its effects on re- fusion of more than one spermatozoan with an egg). Under con- productive isolation are based on the affinity between eggs and ditions of sexual conflict, novel egg-recognition proteins may be sperm. Although complete gametic incompatibility has been selected that slightly mismatch with sperm, lowering the affinity noted among distantly related taxa, incompatibility is generally between sperm and eggs and decreasing the risk of polyspermy a continuum (Hagstrom and Lonning 1967; Strathmann 1981; (Palumbi 1999; Gavrilets 2000; Haygood 2004). This conflict is Minor et al. 1991; Willis et al. 1997; Pernet 1999; Levitan 2002a; thought to be one mechanism that can result in the rapid evolution McCartney and Lessios 2002) that, in part, is related to the genetic of gamete-recognition proteins noted in a variety of taxa (reviewed similarity of a mating pair’s gamete-recognition proteins (Zigler in Swanson and Vacquier 2002). However, this premise hinges on et al. 2005). Theory suggests that gametic incompatibility and the assumption that mating pairs with matched recognition alleles reproductive isolation can evolve through a sexual conflict over not only have a higher probability of fertilization but are also more fertilization rate. Males under intense sperm competition are se- likely to face the risk of polyspermy (see, e.g., Palumbi 1999). lected to have high rates of fertilization to outcompete other males, Although recent data from intraspecific field experiments sug- whereas females under these conditions may be under selection gest that matched alleles are favored under conditions of sperm for lower fertilization rates that reduce the risk of polyspermy (the limitation but perform poorly under conditions of high levels of C 2007 The Author(s). Journal compilation C 2007 The Society for the Study of Evolution. 2007 Evolution 61-8: 2007–2014 DON R. LEVITAN ET AL. polyspermy (Levitan and Ferrell 2006), no comparative data are Table 1. Gamete traits of Strongylocentrotus droebachiensis (Sd), available within and across species on how the likelihood of fer- S. franciscanus (Sf), and S. purpuratus (Sp). Egg diameter in mm tilization influences the likelihood of polyspermy. (Egg size), estimated proportion of sperm collisions that results in a fertilization event (Fertilizability), average log amount of con- Variation within and among species exists for the influence generic sperm needed to fertilize 50% of eggs in the laboratory of sperm availability on fertilization success of externally fertil- (Hybrid resistance), velocity of sperm in mm/s (Sperm velocity), izing taxa. Some species produce eggs that can be fertilized at slope of the relationship between sperm half-life as a function low sperm concentrations, whereas others require much higher of log sperm concentration-—higher slopes are sperm that have a concentrations (reviewed in Levitan 2006). The existing evidence more pronounced reduction in half-life with sperm dilution (Sperm indicates that the ability to achieve fertilization at low levels of longevity), relative performance of gametes to achieve fertiliza- sperm availability is related to the likelihood of conspecific sperm tion under sperm-limited conditions in the sea (Field performance), availability. For example, species that spawn at lower population and relative degree of sperm availability based on population density and nearest neighbor distances in Barkley Sound, British densities are more likely to experience conditions of sperm limi- Columbia, Canada (Sperm availability). tation and may be selected to achieve fertilization with relatively few sperm encounters (Levitan 1993, 1998, 2002b). Trait Sd Sf Sp Reference At the other end of the continuum of sperm availability, Egg size 0.145 0.135 0.084 1 eggs exposed to very high sperm concentrations run the risk of Fertilizability 0.1667 0.0512 0.0559 1 polyspermy. Polyspermy has been noted in a variety of externally Hybrid resistance 2.47 6.39 7.34 4 fertilizing taxa in the laboratory (Rothschild and Swann 1951; Sperm velocity 0.088 0.130 0.145 1 Oliver and Babcock 1992; Styan and Butler 2000; Huchette et al. Sperm longevity 0.308 0.391 0.457 1 2004; Levitan et al. 2004) and field (Brawley 1992; Franke et al. Field performance High Medium Low 2,3 2002; Levitan 2004). Eggs are protected from multiple sperm fu- Sperm availability Low Medium High 2,3 sions by blocks to polyspermy. These blocks include the fast elec- References: 1. Levitan 1993, 2. Levitan 1998, 3. Levitan 2002b, 4. Levitan trical block and the slower cortical reaction (Tyler et al. 1956a,b). 2002a. Polyspermy occurs when the rates of sperm collision and the rates of fertilization exceed the ability of the egg to block excess sperm. lationship among these species in susceptibility to polyspermy. What is not clear is whether the ease of fertilization and the However, sperm velocity, which influences rates of fertilization susceptibility to polyspermy are related, within and across species. (Levitan 2002b), is inversely related to egg size and ease of Although the mechanisms that might increase the ease with which fertilization in these species. This reciprocal pattern might re- eggs are fertilized differ from those that prevent polyspermy, these flect a sexual conflict (females selected to produce eggs with traits may be correlated because any factor that increases the rate low affinity for sperm to avoid polyspermy, whereas males are of sperm–egg fusion might allow the rapid fusion of several sperm selected to have fast sperm and rapid fertilization to compete before a block to polyspermy can be established. Alternatively, with other males) and confound the relationship between ease greater ease of fertilization might lead to adaptations for a very of fertilization and polyspermy across these species. We there- efficient block to polyspermy. All other things being equal, eggs fore examined the relationship between ease of fertilization and should be selected for both fast fusion under sperm limitation polyspermy in a laboratory study of these three congeneric sea and efficient blocks to polyspermy when sperm are abundant. urchins. However, trade-offs may exist because of the inherent constraints that fast fusion might place on rapid blocks to polyspermy. Species that are subject to different levels of sperm availability may be Methods selected either for fast fusion, which makes eggs easier to fertilize Evidence of polyspermy was quantified in three species of cooc- but more susceptible to polyspermy, or for slow fusion, which curring sea urchins: S. purpuratus (purple sea urchin), S. francis- makes them more resistant to polyspermy. canus (red sea urchin), and S. droebachiensis (green sea urchin). The sea urchins Strongylocentrotus purpuratus, S. francis- Individuals of these species were collected from the Deer Island canus,andS. droebachiensis vary in their egg traits and suscep- Group in Barkley Sound, British Columbia, Canada, in March tibility to fertilization under sperm limitation (Table 1). Strongy- 2005 for an initial study and then in May and June 2006 for a locentrotus purpuratus has the smallest eggs, which lowers the follow-up study. Individuals were kept in an open seawater sys- sperm collision rate, and an egg surface that reduces the chance tem and fed macroalgae for no more than two weeks before use in of fertilization given a spermatozoan collision; S. droebachiensis laboratory crosses. The initial 2005 study was conducted during has the largest and easiest-to-fertilize eggs (Levitan 1993, 1998, spawning season for all three species. Conspecific crosses as well 2002b). This pattern of egg traits also predicts a reciprocal re- as hybrid crosses using S. droebachiensis eggs were examined (all 2008 EVOLUTION AUGUST 2007