fucoid algae, [16–19]), maximising the probability of gamete encounters. However, some species seen to be stimulated to spawn by storms (e.g. Patella sp.; [20]). A major question in the ecology of broadcast spawning taxa is whether patterns of gamete release synchrony at any particular temporal scale may act as a pre-zygotic barrier, a process identified at very fine scale in corals (see review by [21] for details). When hybrids display reduced fitness relative to parental taxa, selective constraints for maximizing reproductive success should be counterbalanced by selection to reinforce pre-zygotic barriers to hybridization. This may be achieved by interspecific variation in spawning time [4], such that congeneric gamete encounters are minimized. A second pre-zygotic mechanism may be mating system variation, particularly self-fertilization within hermaphroditic species, which is expected to dramatically reduce hybridization where individuals release male and female gametes in close proximity with minimal sperm limitation. An alternative to pre-zygotic barriers maintaining species coherence in the face of hybridization, is that hybrid fitness is conditional on the environment [22], and that hybrids are either favoured, or selectively neutral, under certain conditions or in particular microhabitats along the intense gradients of selection in the intertidal. Species capable of coexistence despite hybridization and introgression remain one of the best and most challenging models to study the nature and role of reproductive barriers. This is the case of three sister species with different reproductive modes and mating systems; hermaphroditic Fucus spiralis and Fucus guiryi, in which selfing is the dominant form of reproduction throughout the studied range [23,24], and dioecious, outcrossing Fucus vesiculosus. These species coexist as distinct entities [23,25,26] in the mid to high shore throughout most of their range in the eastern Atlantic [27]. Analyses of both multilocus genotypes [23] and variation in sexual phenotype [28] indicate that hybridization with introgres- sion occurs, and therefore that hybrids are reproductively successful. Investment into male function is an order of magnitude lower in F. spiralis compared with F. vesiculosus, suggesting that sperm limitation is not an important factor in the hermaphrodite [28]. If synchrony is selectively maintained primarily to ensure gamete encounters, then selfing hermaphrodites should display a relaxed synchrony relative to dioecious, obligately outcrossing species. In this study we examined reproductive periodicity from seasonal to hourly timescales for the hermaphroditic fucoids F. spiralis and F. guiryi, and dioecious F. vesiculosus. This allowed us to test two predictions: 1) Reproductive synchrony is more important and therefore under stronger selective constraint in obligate outcrossers (F. vesiculosus) than in selfing species (F. spiralis and F. guiryi); 2) The timing of gamete release should vary between potentially hybridizing species in order to reinforce prezygotic (ecological) barriers to fertilization. Our data support both predictions, and also emphasize the ecological, developmental, and/or physiological constraints that operate to restrict reproduc- tion to narrow temporal windows of opportunity in the intertidal zone. Materials and Methods Ethics Statement No specific permits were required for the described field studies. No specific permissions were required for these locations and activities. The location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species. Reproductive Synchrony as Hybridization Barrier Model organisms The genus Fucus develops specialized reproductive tissue called receptacles on some apical tips of the thallus, and inside the receptacles are numerous spherical conceptacles. Depending on the species, sperm and egg develop inside the same conceptacle (i.e., hermaphroditic species, such as F. spiralis and F. guiryi); or in separate conceptacles from male and female individuals (i.e., dioecious species, as in F. vesiculosus). The three model species can be distinguished morphologically as described in Zardi et al. [26]. In all species, sperm and eggs develop inside gametangia called antheridia and oogonia, respectively, and both are released unfertilized (see review [18] for details). Fertilization is external and Fucus eggs are large (ca. 70 mm diameter), negatively buoyant and settle immediately as they are released. Sperm are negatively phototactic. Empirical studies [29–31], as well as local patterns of genetic structure [23,24,32,33] all indicate that gamete and zygote dispersal is highly restricted. Eggs tend to fall immediately below the releasing alga [31] allowing, in settlement studies conducted in mono-specific patches, the assignment of egg origin to species with a high degree of confidence. The vertical distribution of these species on the shore is distinct although with overlapping zones [26,34]; F. spiralis occurring mainly above F. guiryi, and these above the dioecious species F. vesiculosus. Hybrids between these species are uncommon and can be found mainly in the intertidal ranges where their parental species overlap [34]; they are fertile and appear as vigorous as their parental species [23,28]. The restriction of hybrids to vertical contact zones is additional support for the low dispersal of gametes. Study site and sampling The study was conducted on a rocky shore at Viana do Castelo (Northern Portugal), at Praia Norte (Fig. 1). This location is protected from severe wave action by offshore rocky bluffs. The region is the southernmost limit for the sympatric occurrence of F. vesiculosus, F. spiralis and F. guiryi in Europe, and hybridization is more frequent in this contact range [35], possibly as a consequence of lack of reinforcement in southern allopatric populations. The distribution of these species continues to the south, but F. vesiculosus is confined to estuaries and sheltered coastal lagoons, while F. spiralis and F. guiryi continues to occur on the open coast where suitable rocky substrate is available. Sampling at seasonal – semilunar timescales Periodicity of egg release The periodicity of eggs settled was monitored daily from February 2002 to July 2003, in two sites for F. vesiculosus and one site for F. spiralis and F. guiryi. The eggs were collected on artificial substrates (each with 5.96 cm22) with a complex surface to promote egg retention (substrate preparation, fixation and sampling were as described in [11]). Ten disks (or fifteen at sites where due to strong wave action disk loss appeared more likely) were fixed under the algal canopy in Site A and B, respectively. Disks were replaced daily during low tide and were returned to the laboratory where egg release was quantified under a dissecting microscope. Eggs of both species are morphologically similar; all eggs, which settled on disks, were assumed to be from the species that cover the disks, since Fucus eggs tend to fall within less than 0.5 m of the releasing alga [31] and at each site the nearest individuals of any other Fucus species were more than 5 m away. PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35978