Reproductive Synchrony as Hybridization Barrier Figure 1. Map showing the location of the study site at Viana do Castelo, Portugal, and detailed view of the shore indicating the positions of the sampling sites A and B. doi:10.1371/journal.pone.0035978.g001 Periodicity of receptacle maturation In order to compare the temporal variability of gamete maturation in both species, total and mature oogonia were quantified every two days during 2002 and once a week during 2003. In five haphazardly chosen individuals within each site and species, one receptacle was collected at low tide. The total number of oogonia and the mature oogonia were quantified under a microscope in 3 conceptacles of each receptacle. Mature oogonia were defined as those in which cleavage furrows were evident (as in [17]). Sampling at hourly – tidal timescales The timing of egg release during the day was studied in June to July 2009, in natural populations of F. vesiculosus, F. spiralis and F. guiryi. Along the vertical direction, the distance between species/ entities was approximately 10 m, and at each height female gamete release by 5 individuals (females only for F. vesiculosus) was monitored in each of two replicate sites separated by approxi- mately 5 m. Nylon mesh bags (40 mm) were used to enclose 2–3 receptacles per individual. A mesh size of 40 mm was chosen in order to retain eggs, while allowing water to circulate as freely as possible. The bags were attached to individuals with plastic clips with neoprene seals to provide a full seal around the thalli. During neap tide days, eggs were sampled every 2 h between 6:00 and 20:00, with a final sample taken at 21:30, prior to darkness. Bags were replaced carefully to prevent any egg loss in the field and were immediately taken to the laboratory where eggs were counted under a dissecting microscope. The bags were collected underwater or out of water, according to the tidal levels at each sampling time. At maximum high tide it was not possible to sample F. vesiculosus and the results presented are for 2 h periods before and after the high tide. Data analysis We tested the hypothesis that relaxed constraint on reproductive assurance due to selfing in F. guiryi has led to a reduction in reproductive synchrony, relative to the obligate outcrossing species F. vesiculosus. To do this, we used two-tailed F-tests (degrees of freedom are the number of observations-1 for each of the distributions) to compare the variance in the distribution of spawning times (defined as the time of the first high tide after sunrise) for spawning events of a particular threshold magnitude (e.g. $10% or $20% of the maximum value). These data were obtained from the complete seasonal – semilunar settlement dataset from Feb 2002–Jul 2003. The cumulative frequency distribution of egg release during neap tide cycles (2 h sampling intervals; see above) was plotted against ranks (i.e., Pareto or rank/frequency plots). For this, egg counts for each bag (n = 10 bags per 2 h sampling interval) were used to generate cumulative ranks, starting with the largest number of eggs released and adding sequentially the next largest observation until all observations have been summed. Linear regression lines were then fitted to such cumulated frequencies plotted in rank order and using logarithmic scales on both axes. Pairwise comparisons of linear regression slopes between species/ entities fitted to double logarithmic plots were performed by standard techniques using dummy (or indicator) variables to compare linear models using the R statistical package and a custom script. In addition, the distributions were also compared using more robust non-parametric Kolmogorov-Smirnoff tests. Results Seasonal periodicity of reproductive output Gamete release assessed by egg settlement in the field over 2 years was strongly seasonal in all study species (Figs. 2a and b). At a monthly temporal scale the patterns were indistinguishable between the three species. Although some egg settlement was detected throughout the year (see Figs. 2a and b insets plotted on Log scale), major settlement was detected only in May to September in 2002, and also only after May in 2003, until at least July 2003, when the survey was completed. Both total and mature oogonia were observed throughout the year, peaking in April–May, immediately prior to the onset of major settlement events (Figs. 3a and b). During the main reproductive season there was a general decline in oogonia, although numbers were similar in periods when settlement was still PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35978