Figure 8. Pareto double Log10 plot of cumulative rank distribution of egg release. Data were collected during 2 h sampling intervals over four neap tide periods in June and July 2009 (see text and Fig. 7). Summary results of the regressions are 1) Fucus spiralis: Log(y) = 2.85320.476*Log(x), R2 = 0.981; 2) Fucus guiryi: Lo- g(y) = 2.76720.488*Log(x), R2 = 0.970, and 3) Fucus vesiculosus: Lo- g(y) = 2.81220.345*Log(x), R2 = 0.987. doi:10.1371/journal.pone.0035978.g008 proxy for gamete release [10,15,19], or oogonial maturation, followed essentially the same temporal pattern for the two hermaphrodite species F. spiralis and F. guiryi, and for dioecious F. vesiculosus. Maximum reproductive output occurred from late spring to summer (May–Sep). We are not aware of other comparative long term spawning datasets for Fucus spp. or other intertidal organisms. The restriction of most gamete release to massive spawning events over a few days during the reproductive season is typical, and parallels reports of mass reproductive events in broadcast spawning corals or green algae in tropical reef systems (e.g., [36,37], see also review by [21]). Gamete release in all study species followed a<14 day period, synchronized with neap tides during semilunar cycles. Synchro- nous spawning with the same periodicity has been reported for both dioecious and hermaphrodite Fucus species [10–12,15,38]. However, the timing within the semilunar cycle we observed (i.e., neap tide periods) is out of phase with data for F. vesiculosus from the eastern Atlantic (Maine coast, USA) [9,38], as well as those for an estuarine fucoid, F. ceranoides in the Irish Sea, UK [10], both of which show peak gamete release during spring tides around full and new moon. This apparent conflict is resolved by considering tidal, rather than semilunar cycles; tidal and diurnal cycles are similar between Maine and the Irish Sea, where mid – late morning high tides occur near full and new moons, whereas in northern Portugal, similar timing of high tides occurs during the neap period (i.e., around quarter moons). These results strongly suggest that gamete release in intertidal fucoids at the daily scale is entrained by the interaction between semidiurnal cycles of high and low tides and daily light-dark cues, rather than by cues arising from semilunar (spring – neap tide) cycles (Fig. 5). Similarly, a model combining fitness components associated with environmen- tal cycles of differing periodicity was shown to underlie the apparent semilunar spawning patterns of a puffer fish [39]. While Reproductive Synchrony as Hybridization Barrier our data are compelling, independent confirmation could be obtained by comparisons of spawning times in natural populations in the eastern Atlantic, subject to a north – south gradient in the timing of tides [40] or in areas with striking tidal phase shifts along short distances, versus the western Atlantic, where there are no such tidal gradients. At hourly time scales, spawning was largely synchronous between hermaphroditic F. spiralis and F. guiryi, but asynchronous between hermaphroditic and dioecious species, at daily and particularly hourly time scales (Fig. 7). Major spawning events in hermaphrodites occurred mainly on days when spawning by F. vesiculosus was very low or did not occur. Temporal reproductive isolation is a potentially effective means of reinforcing reproductive isolation, and a key ecological factor in sympatric speciation. Temporal isolation in sympatry has been considered unlikely [41], although empirical data from conspecific broadcast spawning algae and corals [4,37,42], as well as theoretical models [43] indicate that it does occur, with important evolutionary conse- quences for reproductive isolation and sympatric speciation in natural communities. These changes in synchrony and timing of reproduction in Fucus have evolved recently, since we now know that F. spiralis and F. guiryi most likely arose during the Pleistocene glacial cycles, and that their evolution coincided with a switch in reproductive mode from the ancestral dioecious state shared with F. vesiculosus [44]. Our data for F. vesiculosus broadly agree with those of Berndt et al. [9] in suggesting that major spawning events occur during daytime high tide immersion, since we observed that release began prior to, and continued after, the high tide (see also [12] for details). In contrast, major spawning events in hermaph- rodite species were observed by the first sampling interval (06:00, around dawn), implying either that spawning occurred during the light to dark transition, during the night (the last sampling bags were placed at 21:30), or at the dark to light transition at dawn [37]. In this respect it is interesting to consider that a more distantly related member of the family Fucaceae (Silvetia compressa) releases gametes in the laboratory on a light to dark transition after a period under favourable conditions in the light ([16], reviewed by [18]), and can release gametes at low tide in the field [45]. Whatever the exact timing in hermaphrodite species, the interspecific shift in spawning of congeneric hermaphroditic and dioecious species observed here is consistent with selection favouring ecological reproductive isolation, since in congeneric corals a shift in spawning of only 2 h was inferred to reinforce reproductive isolation [4]. Two hypothetical selective mechanisms could have originated the temporal reproductive isolation between the dioecious (F. vesiculosus) and hermaphroditic (F. spiralis and F. guiryi) lineages, sympatric speciation or reinforcement against hybridization. Before the split of the hermaphroditic and dioecious lineages [44] mutations leading to spawning timing difference could have created the reproductive isolation that originated, or contributed to, speciation. Alternatively, after lineage split, reinforcement might have occurred if hybrids were less fit than their parental species along each specific vertical zone, in agreement with the observations that these species vertical distribution is correlated with different stress resilience [26] and that hybrids are found mainly in the contact zones along the vertical zonation [34]. In such a scenario, populations that remained non-introgressed due to different spawning times would have been positively selected. The species F. spiralis and F. guiryi were only recently detected as distinct genetic entities with limited gene flow [26,44]. We found no evidence for ecological prezygotic barriers to gene flow between these species, suggesting that either mating system is a sufficient hybridization barrier in these compatible and selfing [24]. entities, PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35978