Sympatric assemblages of congeners with incomplete reproductive barriers offer the opportunity to study the roles that ecological and non-ecological factors play in reproductive isolation. While interspecific asynchrony in gamete release and gametic incompatibility are known prezygotic barriers to hybridization, the role of mating system variation has been emphasized in plants. Reproductive isolation between the sibling brown algal species Fucus spiralis, Fucus guiryi (selfing hermaphrodite) and Fucus vesiculosus (dioecious) was studied because they form hybrids in parapatry in the rocky intertidal zone, maintain species integrity over a broad geographic range, and have contrasting mating systems. We compared reproductive synchrony (spawning overlap) between the three species at several temporal scales (yearly/seasonal, semilunar/tidal, and hourly during single tides). Interspecific patterns of egg release were coincident at seasonal (single peak in spring to early summer) to semilunar timescales. Synthesis of available data indicated that spawning is controlled by semidiurnal tidal and daily light-dark cues, and not directly by semilunar cycles. Importantly, interspecific shifts in timing detected at the hourly scale during single tides were consistent with a partial ecological prezygotic hybridization barrier. The species displayed patterns of gamete release consistent with a power law distribution, indicating a high degree of reproductive synchrony, while the hypothesis of weaker selective constraints for synchrony in selfing versus outcrossing species was supported by observed spawning in hermaphrodites over a broader range of tidal phase than in outcrossers. Synchronous gamete release is critical to the success of external fertilization, while high-energy intertidal environments may offer only limited windows of reproductive opportunity. Within these windows, however, subtle variations in reproductive timing have evolved with the potential to form ecological barriers to hybridization.
Citation: Monteiro CA, Serrão EA, Pearson GA (2012) Prezygotic Barriers to Hybridization in Marine Broadcast Spawners: Reproductive Timing and Mating System Variation. PLoS ONE7(4): e35978. https://doi.org/10.1371/journal.pone.0035978
Editor: William J. Etges, University of Arkansas, United States of America
Received: December 12, 2011; Accepted: March 24, 2012; Published: April 26, 2012
Copyright: © 2012 Monteiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by projects from the Portuguese Science Foundation (http://www.fct.pt) co-sponsored by FEDER (ERDF, European Regional Development Fund), and an FCT and ESF (European Social Fund) PhD fellowship to CAM. The European Union Network of Excellence MARBEF-Marine Biodiversity and Ecosystem Function and the NSF (USA) CORONA network provided opportunities for interesting insights and discussions on the topic of this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Speciation occurs by the evolution of reproductive barriers that ultimately prevent genetic exchange between previously interbreeding populations . Prezygotic isolating mechanisms range from ecological barriers to gametic incompatibility, while postzygotic isolation may arise through hybrid inviability, sterility, or reduced hybrid fitness. Ecological barriers are commonly the first to arise, and are more important than intrinsic postzygotic factors (reviewed for plants by ). In the sea, where reproduction by external fertilization is common, it may be more difficult for extrinsic ecological barriers to arise, resulting in more opportunities for hybridization . Asynchronous gamete release between related taxa, and/or gametic incompatibility are considered the major prezygotic barriers to hybridization between externally fertilizing sympatric animal species . In contrast to terrestrial plants, where mating system (particularly selfing) can maintain almost complete reproductive isolation between sympatric species in the presence of gene flow , outcrossing, albeit at varying rates, appears to dominate in hermaphroditic broadcast spawners .
The intertidal zone imposes unique constraints on external fertilization synchrony because during tidal emersion efficient gamete mixing is prevented and abiotic stresses can be intense, while during immersion hydrodynamic conditions may result in rapid gamete dilution and/or shear stresses on gametes that may severely reduce fertilization success , . Despite this, broadcast spawning is a common reproductive mode in diverse marine intertidal taxa, from invertebrates to macroalgae –.
Synchronous gamete release is critical to reproductive success in broadcast spawners, but the cyclical fluctuation of environmental conditions arising from interacting cycles at diurnal, tidal, semilunar and seasonal temporal scales suggests that external fertilization in intertidal species may be constrained within narrow windows of opportunity. We predict that selective pressures to restrict gamete release within these temporal windows will be strong. Indeed, some intertidal taxa possess sophisticated mechanisms to sense the environment and restrict gamete release, particularly to periods of low environmental water motion (e.g., in fucoid algae, –), maximising the probability of gamete encounters. However, some species seen to be stimulated to spawn by storms (e.g. Patella sp.; ).
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  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 , 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 , 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 , , and dioecious, outcrossing Fucus vesiculosus. These species coexist as distinct entities , ,  in the mid to high shore throughout most of their range in the eastern Atlantic . Analyses of both multilocus genotypes  and variation in sexual phenotype  indicate that hybridization with introgression 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 . 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 reproduction to narrow temporal windows of opportunity in the intertidal zone.
Materials and Methods
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.
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. . In all species, sperm and eggs develop inside gametangia called antheridia and oogonia, respectively, and both are released unfertilized (see review  for details). Fertilization is external and Fucus eggs are large (ca. 70 µm diameter), negatively buoyant and settle immediately as they are released. Sperm are negatively phototactic. Empirical studies –, as well as local patterns of genetic structure , , ,  all indicate that gamete and zygote dispersal is highly restricted. Eggs tend to fall immediately below the releasing alga  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 , ; 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 ; they are fertile and appear as vigorous as their parental species , . 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 , 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 cm−2) with a complex surface to promote egg retention (substrate preparation, fixation and sampling were as described in ). 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  and at each site the nearest individuals of any other Fucus species were more than 5 m away.
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 ).
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 approximately 5 m. Nylon mesh bags (40 µm) were used to enclose 2–3 receptacles per individual. A mesh size of 40 µm 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.
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.
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.
Periods when sampling was not carried out are represented by n.d. (no data). Insets show the data plotted on a Logarithmic scale.
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 observed (in August) and when settlement was near zero (October–November). Abundance of mature receptacles declined following the main reproductive period, coinciding with declining reproductive output (pers. obs.). However, the continued presence of mature oogonia following the end of the main reproductive period in October suggests that gamete release may not be directly related to the availability of gametangia.
Consistently lower settlement occurred in F. spiralis (site A), which had low density of reproductive individuals in the immediate vicinity of settlement disks. We included these data in Fig. 2a to illustrate that settled eggs originated from reproductive individuals immediately adjacent to sampling disks. Egg settlement quantification at the study site at various distances from the nearest females also consistently demonstrated low dispersal (Monteiro, Pearson, Serrão, unpubl. data). In addition to supporting the absence of interspecific contamination between sites monitored for each species, the ca. 2-fold lower settlement observed in F. vesiculosus compared with F. guiryi (site B, Fig. 2) may also be related to the low egg dispersal, since all individuals of hermaphroditic species produce eggs, compared with only half of dioecious F. vesiculosus (assuming equal sex-ratios); randomly-placed settlement substrates closer to males than females may therefore account for the reduced overall egg count for F. vesiculosus.
Periodicity of gamete release on a daily scale within the semilunar cycle
On a finer daily scale during the semilunar cycle, gamete release was highly discrete, with peaks of release coinciding with neap (minimum amplitude) tides in both 2002 and 2003 (Fig. 4). In 2002, 9 major settlement events, and in 2003 a further 6 were recorded over a total of 311 days. In most cases, release occurred over 1–2 days, more rarely with an additional 1–2 days of low release before or after the main peak. In 2003, release events were somewhat broader in time (5–6 days, compared with 1–2 days in 2002) and peak settlement was higher than in 2002. Greater gamete release in 2003 may have contributed to the temporal broadening of release events as a result of sampling efficiency (i.e., increased detection efficiency of larger release events may have contributed to apparent peak broadening). Overall, the data show that the large majority of days on which release occurred were shared between species, and reveal no temporal barriers to interspecific gamete encounters in the water column at a semilunar time scale.
Values are means ± SE from n = 10 (Site A) or n = 15 (site B) sampling disks. Tidal amplitude (black line) and lunar phase for the sampling period are shown in a) and b); grey shading indicates the timing and duration of major settlement events from both species/sites.
We compared the daily tidal phase on days of major gamete release in this study with those of Berndt et al.  for F. vesiculosus on western Atlantic shores in Maine, USA, because these authors observed release during spring tides, while we observed release exclusively during neap tides. However, the tidal phase (i.e., the timing of the low and high tides during the day) when gamete release occurred was very similar in both studies (Fig. 5a). Gamete release in F. vesiculosus, both in our study and in Berndt et al. , happens mainly on days when the first diurnal high tide occurs in the late morning. In Figure 5b we added tidal data from Brawley  for days with maximum gamete release in the estuarine dioecious fucoid, F. ceranoides. In this case peak gamete release appears shifted forwards by about 2 h relative to F. vesiculosus.
Data are from Pemaquid, Maine, USA (Oct 8 and Nov 8, 1999; from Berndt et al. ), Viana do Castelo, Portugal (Jun 23, Jul 22, 2003; this study), and Port Erin, Isle of Man (Jul 17, Aug 1–3, 1989, and Jul 22, Aug 4, 1990; from Brawley ).
Interspecific comparison of synchrony on a daily scale within the semilunar cycle
To compare interspecific reproductive synchrony within semilunar cycles, we used the daily egg settlement data from 2002–2003 to compare the variance in the timing of settlement events above certain threshold values. The data were normalized as a percentage of the maximum value observed, and plotted against the time of the first daylight high tide (i.e., phase in the semilunar cycle) on the day of release (Fig. 6). Gamete release from hermaphroditic F. guiryi had a higher variance (i.e., was less synchronous) with respect to semilunar phase than that from F. vesiculosus. Mean settlement values ≥10% of the maximum, occurred on days when the maximum level of the day-time high tide (defined as the first high tide after sunrise) fell within the time interval 07:41–14:27 h in F. guiryi (a range >6 h), but were restricted to days with peak high tides within the time interval 08:27–13:13 h in F. vesiculosus (ranging ca. 4:46 h) (two-tailed F-test; F(32, 41) = 2.083, P = 0.027). For settlement events of ≥20% of the maximum the values were 07:41–13:49 h in F. guiryi (06:08 h) and 09:01–12:31 h in F. vesiculosus (03:30 h) (two-tailed F-test; F(26, 30) = 2.772, P = 0.008).
Intraspecific timing and synchrony of gamete release: hourly scale during the tidal cycle
During the 4 neap tide periods studied, major egg release events (defined as >1000 eggs per bag in a 2 h sampling period) were observed on 8 days in F. vesiculosus (June 2–3, June 15–17, and July 17–19); 4 days in F. guiryi (June 3, 14, 17 and 18); and 6 days in F. spiralis (June 3, 5, 14, 17, 18 and July 3). While the three species showed co-occurring gamete release on several days, the largest events were shared mainly by the two hermaphrodites, F. spiralis and F. guiryi. (Figs. 7a and b), to the exclusion of F. vesiculosus (Fig. 7c). Moreover, the timing of gamete release within the tidal cycle was divergent between F. vesiculosus and the two hermaphrodite species. While F. vesiculosus released gametes almost exclusively immediately prior to, and especially following, the high tide (mid-morning to early afternoon), F. spiralis and F. guiryi released gametes consistently at the earliest sampling time of 06:00. Although we were unable to measure gamete release during peak high tide in F. vesiculosus due to the difficulty of sampling immersed individuals in situ, our data are consistent with this being the case, as reported by Berndt et al.  who observed the onset of release occurring prior to immersion. The precise timing of release in F. spiralis and F. guiryi remains uncertain, with both the dark to dawn transition, or release in the dark after the last sampling bags were attached (i.e., after 21:30 on the previous day) being possible.
Grey lines are tidal amplitude and grey bars the dark periods during the daily cycle.
Double logarithmic plots of cumulative frequency against ranked egg release magnitude for 2 hourly intervals (Pareto plots) revealed power-law distributions for all three entities (F. spiralis, F. guiryi and F. vesiculosus; Fig. 8). The relationship deviated from linearity only at values of egg release >ca. 105 eggs per sampling bag (2–3 receptacles), which probably approaches the upper limit of eggs contained in the sample receptacles. The regression fit to the F. vesiculosus data was improved (r2 = 0.987 versus 0.952) by removing values ≥105, so this was chosen as a cut-off value for number of eggs sampled. The fit of the regressions were also high for hermaphrodite species (r2 = 0.9813 and 0.9701 for F. guiryi and F. spiralis, respectively), and were highly significant in all cases (P<0.0001). Pairwise comparisons of the regression slopes showed no significant difference between the two hermaphrodite species (P = 0.208), but that both were significantly different from F. vesiculosus (P<0.0001 in both cases). The same was found using Kolmogorov-Smirnoff tests; the distributions of egg release data did not differ between the hermaphroditic species (F. spiralis versus F. guiryi, p = 0.800) but differed between these and the dioecious F. vesiculosus (F. guiryi versus F. vesiculosus, p = 0.000; F. spiralis versus F. vesiculosus, p = 0.000). It follows that the exponent or scaling factor, α, for the power law relationships calculated from the regression slopes varied little between the two hermaphrodite species (0.4760 and 0.4883 for the F. spiralis and F. guiryi, respectively), but was lower for F. vesiculosus (0.3454).
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) = 2.853−0.476*Log(x), R2 = 0.981; 2) Fucus guiryi: Log(y) = 2.767−0.488*Log(x), R2 = 0.970, and 3) Fucus vesiculosus: Log(y) = 2.812−0.345*Log(x), R2 = 0.987.
This study provides one of the most comprehensive descriptions to date of reproductive timing (egg release) at annual to hourly scales in the marine environment. We had two main goals; firstly to investigate the potential for interspecific variation in spawning time to act as a prezygotic ecological barrier to hybridization between sympatric, externally-fertilizing congeners. Our data show that, while coincident at seasonal and semilunar scales, differences in the timing of gamete release during single tides are consistent with a partial ecological barrier to hybridization. Secondly, we addressed the hypothesis that selfing species are under reduced selective constraint for spawning synchrony relative to obligately outcrossing species. We discovered that while reproductive synchrony remains a feature in selfing hermaphroditic species, it is reduced relative to sister species with obligate outcrossing. Reduced synchrony was identified on daily time scales as increased variance of major spawning periods during the semilunar reproductive cycle, and from a relative excess of small spawning events during single favourable tides. Finally, we provide evidence from these data and previously published studies on both sides of the Atlantic ,  that gamete release in natural fucoid populations is controlled by environmental cues arising from the interaction of tidal and diurnal cycles, rather than semilunar cycles.
Spawning patterns at seasonal to daily temporal scales were highly coincident for F. vesiculosus, F. spiralis and F. guiryi. Reproductive output, estimated as daily egg settlement, a close proxy for gamete release , , , 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., , , see also review by ).
Gamete release in all study species followed a≈14 day period, synchronized with neap tides during semilunar cycles. Synchronous spawning with the same periodicity has been reported for both dioecious and hermaphrodite Fucus species –, , . 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) , , as well as those for an estuarine fucoid, F. ceranoides in the Irish Sea, UK , 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 environmental cycles of differing periodicity was shown to underlie the apparent semilunar spawning patterns of a puffer fish . While 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  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 , although empirical data from conspecific broadcast spawning algae and corals , , , as well as theoretical models  indicate that it does occur, with important evolutionary consequences 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 . Our data for F. vesiculosus broadly agree with those of Berndt et al.  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  for details). In contrast, major spawning events in hermaphrodite 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 . 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 (, reviewed by ), and can release gametes at low tide in the field . 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 .
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  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  and that hybrids are found mainly in the contact zones along the vertical zonation . 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 , . 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 . entities, or that other pre- or postzygotic barriers exist. Mating system variation can reinforce speciation processes, e.g. a shift from outcrossing to selfing resulted in almost complete reproductive isolation between potentially hybridizing plant species . However, while flowering plants rely on pollen vectors (e.g., insects) for cross-fertilization, or have developed effective selfing mechanisms like cleistogamous (non-opening) flowers, it is less clear how effective mating system variation can be in broadcast spawning marine external fertilizers. Temporal differences in spawning times occur also in sympatric and inter-fertile marine invertebrates, and may play a role in prezygotic reproductive isolation, reinforcing some degree of gametic incompatibility (e.g., ). Since syngamy occurs shortly after spawning, selfing rates presumably depend on the spatial proximity of the eggs and sperm released from the same individual. Eggs from different fucoid species share the same pheromonal sperm attractant , and dioecious male conceptacles produce an order of magnitude more sperm than hermaphrodites . Therefore, as a hybridization barrier in mixed stands of broadcast spawning congeneric species, mating system alone is expected to be an imperfect isolating mechanism.
Theoretically, the selection pressure to maintain spawning synchrony in dioecious (i.e., obligately outcrossing) broadcast spawning species should be stronger than for self-fertilizing hermaphrodites. Separate male and female individuals are selectively constrained to ensure reproductive success by spawning into the water column at the same time, while individuals of selfing hermaphrodites can assure reproductive success by releasing male and female gametangia from the same reproductive tissue (conceptacles), and are not therefore constrained in the same way. Our finding of larger temporal variance in spawning time during semilunar cycles in F. spiralis and F. guiryi than in F. vesiculosus supports the hypothesis of a reduced constraint on synchrony in hermaphroditic species. Despite this, at a smaller temporal scale, power law-like distributions of spawning intensity during reproductive periods (neap tide cycles) were observed in all three taxa/genetic entities, indicating very few large release events and many small events. This indicates that, for both dioecious and hermaphroditic species, reproduction (and therefore recruitment, as the two processes are closely linked in fucoids) depend for their success on very limited temporal “windows of opportunity". During these rare events of synchronous spawning, the predominant form of crossing may still be between neighbouring individuals due to the limited gamete dispersal , which may explain high levels of biparental inbreeding observed in several F. vesiculosus populations . Although our data support a reduced constraint on synchrony in hermaphroditic species at semilunar scales, at smaller, hourly, scales within peak release days, the opposite trend is observed, possibly resulting from their use of different environmental triggers for circadian release timing. The shallower slope (significantly lower α) of the power law relationship in dioecious F. vesiculosus (i.e., greater “evenness"), indicates less synchrony at hourly scales during the neap tide release periods. A possible reason for this contrast could be use of different cues for gamete release timing within circadian scales. Spawning in F. vesiculosus was observed to coincide with immersion at high tide, a more extended interval including the immediately prior and following emersion periods. In contrast, the shift to low tide (emersed) release in F. spiralis and F. guiryi, possibly in response to putative dark/light shift signals, appears to result in more synchronous release at this smaller temporal scale.
On a semi-lunar scale, our data support the prediction that constraints on reproductive synchrony are reduced in selfing species in the intertidal. However, the broadly coincident semilunar spawning patterns, as well the power-law relationships for egg release, showing high synchrony for all entities independent of mating system, both indicate that any synchrony differences are quite small. Why is reproductive synchrony maintained in hermaphrodite species at all? Possible reasons include evolutionary contingency, given the very recent divergence of the two species , , maintenance of adaptive potential by some degree of continued outcrossing , , and/or ecological factors unrelated to reproduction directly. If F. spiralis and F. guiryi arose recently from a dioecious ancestral lineage , then synchrony may simply be a retained ancestral trait. Populations of F. spiralis and F. guiryi show evidence of high levels of inbreeding ,, which may be favoured in marginal habitats (such as the upper intertidal shoreline where F. spiralis and F. guiryi occur), and where maintaining locally adaptive gene combinations and/or purging of deleterious alleles may be particularly advantageous (reviewed by ).. Nevertheless, the benefits of occasional outcrossing may be maintained via continued selection for reproductive synchrony. A third possible reason may relate to other life-history traits such as limiting dispersal to favourable potential habitats. Since in fucoids zygotes settle and develop immediately post-fertilization, the timing of spawning may also be selected for optimal recruit survival in the steep vertical abiotic gradients of the intertidal zone, as suggested earlier . Releasing gametes at low tide by these hermaphroditic taxa might favour retention of propagules at their higher intertidal range as the upwards moving tide washes pre-released gametes onto local safe micro-sites. Thus gamete release at slack high tide , or at low tide in selfing species, besides maximising fertilization success, might simultaneously limit zygote wastage from dispersal out of their narrow favourable intertidal range.
Fucoid algae are a fascinating system in which to address, from an ecological viewpoint, some of the problems associated with maintaining reproductive barriers and more broadly with speciation/hybridization processes, in broadcast spawning marine species. The timing of reproduction in the intertidal zone may be particularly constrained for broadcast spawners, with very limited temporal windows for reproductive assurance imposed by immersion-emersion cycles and the dilution effects of surf zone hydrodynamism . Therefore species with incomplete reproductive isolation must balance reproductive assurance (e.g., fertilization success under optimal hydrodynamic conditions) while minimizing hybridization with sympatric congeners. The species studied here have incomplete reproductive isolation, and low proportions of hybrids are commonly observed, mainly at contact zones , , , , but are nevertheless maintained as genetically distinct entities in sympatry.
While the seasonal, tidal and diurnal cues entraining gamete release are shared by sister taxa, a temporal shift in reproductive timing within single tides constitutes an ecological barrier to gene flow between the selfing hermaphrodite, F. spiralis and F. guiryi and dioecious F. vesiculosus. Mating system variation plays two important roles; while dioecious species require high tide immersion to mix gametes and a high degree of reproductive synchrony, selfing hermaphrodites are less constrained, possibly including reproduction during emersion at low tide . The mechanism(s) preventing gene flow between the two hermaphroditic species F. spiralis and F. guiryi remains to be seen, but subtle differences in reproductive timing , gamete release during low tide emersion, and selfing  may, separately or in concert, be sufficient prezygotic barriers to maintain them as distinct genetic entities in sympatry.
We would like to thank “Associação dos Amigos do Mar" (Viana do Castelo) and the staff for providing facilities for laboratory work, and Fernando Cánovas for help in using R.
Conceived and designed the experiments: CAM EAS GAP. Performed the experiments: CAM. Analyzed the data: CAM GAP. Contributed reagents/materials/analysis tools: EAS GAP. Wrote the paper: CAM EAS GAP.
- 1. Mayr E (1963) Animal species and evolution. 797 p.E. Mayr1963Animal species and evolution.Harvard University Press, Cambridge. 797 Harvard University Press, Cambridge.
- 2. Rieseberg LH, Willis JH (2007) Plant speciation. Science 317: 910–4.LH RiesebergJH Willis2007Plant speciation.Science3179104
- 3. Palumbi SR (1994) Genetic divergence, reproductive isolation, and marine speciation. Ann Rev Ecol Syst 25: 547–572.SR Palumbi1994Genetic divergence, reproductive isolation, and marine speciation.Ann Rev Ecol Syst25547572
- 4. Levitan DR, Fukami H, Jara J, Kline D, McGovern TM, et al. (2004) Mechanisms of reproductive isolation among sympatric broadcast-spawning corals of the Montastraea annularis species complex. Evolution 58: 308–23.DR LevitanH. FukamiJ. JaraD. KlineTM McGovern2004Mechanisms of reproductive isolation among sympatric broadcast-spawning corals of the Montastraea annularis species complex.Evolution5830823
- 5. Martin NH, Willis JH (2007) Ecological divergence associated with mating system causes nearly complete reproductive isolation between sympatric Mimulus species. Evolution 61: 68–82.NH MartinJH Willis2007Ecological divergence associated with mating system causes nearly complete reproductive isolation between sympatric Mimulus species.Evolution616882
- 6. Heyward AJ, Babcock RC (1986) Self- and cross-fertilization in scleractinian corals. Mar Biol 90: 191–195.AJ HeywardRC Babcock1986Self- and cross-fertilization in scleractinian corals.Mar Biol90191195
- 7. Denny M, Dairiki J, Distefano S (1992) Biological consequences of topography on wave-swept rocky shores: I. Enhancement of external fertilization. Biol Bull 183: 220–232.M. DennyJ. DairikiS. Distefano1992Biological consequences of topography on wave-swept rocky shores: I. Enhancement of external fertilization.Biol Bull183220232
- 8. Mead KS, Denny MW (1995) The effects of hydrodynamic shear stress on fertilization and early development of the purple sea urchin Strongylocentrotus purpuratus. Biol Bull 188: 46–56.KS MeadMW Denny1995The effects of hydrodynamic shear stress on fertilization and early development of the purple sea urchin Strongylocentrotus purpuratus.Biol Bull1884656
- 9. Berndt M, Callow J, Brawley S (2002) Gamete concentrations and timing and success of fertilization in a rocky shore seaweed. Mar Ecol Prog Ser 226: 273–285.M. BerndtJ. CallowS. Brawley2002Gamete concentrations and timing and success of fertilization in a rocky shore seaweed.Mar Ecol Prog Ser226273285
- 10. Brawley SH (1992) Fertilization in natural populations of the dioecious brown alga Fucus ceranoides and the importance of the polyspermy block. Mar Biol 113: 145–157.SH Brawley1992Fertilization in natural populations of the dioecious brown alga Fucus ceranoides and the importance of the polyspermy block.Mar Biol113145157
- 11. Ladah L, Bermudez R, Pearson G, Serrão E (2003) Fertilization success and recruitment of dioecious and hermaphroditic fucoid seaweeds with contrasting distributions near their southern limit. Mar Ecol Prog Ser 262: 173–183.L. LadahR. BermudezG. PearsonE. Serrão2003Fertilization success and recruitment of dioecious and hermaphroditic fucoid seaweeds with contrasting distributions near their southern limit.Mar Ecol Prog Ser262173183
- 12. Ladah LB, Feddersen F, Pearson GA, Serrão EA (2008) Egg release and settlement patterns of dioecious and hermaphroditic fucoid algae during the tidal cycle. Mar Biol 155: 583–591.LB LadahF. FeddersenGA PearsonEA Serrão2008Egg release and settlement patterns of dioecious and hermaphroditic fucoid algae during the tidal cycle.Mar Biol155583591
- 13. Marshall DJ (2002) In situ measures of spawning synchrony and fertilization success in an intertidal, free-spawning invertebrate. Mar Ecol Prog Ser 236: 113–119.DJ Marshall2002In situ measures of spawning synchrony and fertilization success in an intertidal, free-spawning invertebrate.Mar Ecol Prog Ser236113119
- 14. Marshall DJ, Semmens D, Cook C (2004) Consequences of spawning at low tide: limited gamete dispersal for a rockpool anemone. Mar Ecol Prog Ser 266: 135–142.DJ MarshallD. SemmensC. Cook2004Consequences of spawning at low tide: limited gamete dispersal for a rockpool anemone.Mar Ecol Prog Ser266135142
- 15. Pearson GA, Brawley SH (1996) Reproductive ecology of Fucus distichus (Phaeophyceae): an intertidal alga with successful external fertilization. Mar Ecol Progr Ser 143: 211–223.GA PearsonSH Brawley1996Reproductive ecology of Fucus distichus (Phaeophyceae): an intertidal alga with successful external fertilization.Mar Ecol Progr Ser143211223
- 16. Pearson GA, Brawley SH (1998) A model for signal transduction during gamete release in the fucoid alga Pelvetia compressa. Plant Physiol 118: 305–313.GA PearsonSH Brawley1998A model for signal transduction during gamete release in the fucoid alga Pelvetia compressa.Plant Physiol118305313
- 17. Pearson GA, Serrão EA, Brawley SH (1998) Control of gamete release in fucoid algae: Sensing hydrodynamic conditions via carbon acquisition. Ecology 79: 1725–1739.GA PearsonEA SerrãoSH Brawley1998Control of gamete release in fucoid algae: Sensing hydrodynamic conditions via carbon acquisition.Ecology7917251739
- 18. Pearson GA, Serrão EA (2006) Revisiting synchronous gamete release by fucoid algae in the intertidal zone: fertilization success and beyond? Integr Comp Biol 46: 587–597.GA PearsonEA Serrão2006Revisiting synchronous gamete release by fucoid algae in the intertidal zone: fertilization success and beyond?Integr Comp Biol46587597
- 19. Serrão EA, Pearson G, Kautsky L, Brawley SH (1996) Successful external fertilization in turbulent environments. Proc Natl Acad Sci USA 93: 5286–5290.EA SerrãoG. PearsonL. KautskySH Brawley1996Successful external fertilization in turbulent environments.Proc Natl Acad Sci USA9352865290
- 20. Orton JH, Southward AJ, Dodd JM (1956) Studies on the biology of limpets. II. The breeding of Patella vulgate L. in Britain. J Mar Biol Ass UK 35: 149–176.JH OrtonAJ SouthwardJM Dodd1956Studies on the biology of limpets. II. The breeding of Patella vulgate L. in Britain.J Mar Biol Ass UK35149176
- 21. Serrão EA, Havenhand J (2009) Fertilization strategies. In: Wahl M, editor. Marine Hard Bottom Communities. Springer-Verlag, Berlin, Heidelberg. pp. 149–164.EA SerrãoJ. Havenhand2009Fertilization strategies.M. WahlMarine Hard Bottom CommunitiesSpringer-Verlag, Berlin, Heidelberg149164
- 22. Rundle HD, Whitlock MC (2001) A genetic interpretation of ecologically dependent isolation. Evolution 55: 198–201.HD RundleMC Whitlock2001A genetic interpretation of ecologically dependent isolation.Evolution55198201
- 23. Engel CR, Daguin C, Serrão EA (2005) Genetic entities and mating system in hermaphroditic Fucus spiralis and its close dioecious relative F. vesiculosus (Fucaceae, Phaeophyceae). Mol Ecol 14: 2033–46.CR EngelC. DaguinEA Serrão2005Genetic entities and mating system in hermaphroditic Fucus spiralis and its close dioecious relative F. vesiculosus (Fucaceae, Phaeophyceae).Mol Ecol14203346
- 24. Perrin C, Daguin C, Van De Vliet M, Engel CR, Pearson GA, et al. (2007) Implications of mating system for genetic diversity of sister algal species: Fucus spiralis and Fucus vesiculosus (Heterokontophyta, Phaeophyceae). Eur J Phycol 42: 219–230.C. PerrinC. DaguinM. Van De VlietCR EngelGA Pearson2007Implications of mating system for genetic diversity of sister algal species: Fucus spiralis and Fucus vesiculosus (Heterokontophyta, Phaeophyceae).Eur J Phycol42219230
- 25. Billard E, Daguin C, Pearson G, Serrão E, Engel C, et al. (2005a) Genetic isolation between three closely related taxa: Fucus vesiculosus, F. spiralis, and F. ceranoides (Phaeophyceae). J Phycol 41: 900–905.E. BillardC. DaguinG. PearsonE. SerrãoC. Engel2005aGenetic isolation between three closely related taxa: Fucus vesiculosus, F. spiralis, and F. ceranoides (Phaeophyceae).J Phycol41900905
- 26. Zardi GI, Nicastro KR, Canovas F, Costa JF, Serrão EA, et al. (2011) Adaptive traits are maintained on steep selective gradients despite gene flow and hybridization in the intertidal zone. PloS ONE 6(6): e19402.GI ZardiKR NicastroF. CanovasJF CostaEA Serrão2011Adaptive traits are maintained on steep selective gradients despite gene flow and hybridization in the intertidal zone.PloS ONE66e19402
- 27. Lüning K (1990) Seaweeds. K. Lüning1990Seaweeds.Their environment, biogeography, and ecophysiology. Wiley, New York. Their environment, biogeography, and ecophysiology. Wiley, New York.
- 28. Billard E, Serrão EA, Pearson GA, Engel CR, Destombe C, et al. (2005b) Analysis of sexual phenotype and prezygotic fertility in natural populations of Fucus spiralis, F. vesiculosus (Fucaceae, Phaeophyceae) and their putative hybrids. Eur J Phycol 40: 397–407.E. BillardEA SerrãoGA PearsonCR EngelC. Destombe2005bAnalysis of sexual phenotype and prezygotic fertility in natural populations of Fucus spiralis, F. vesiculosus (Fucaceae, Phaeophyceae) and their putative hybrids.Eur J Phycol40397407
- 29. Dudgeon S, Kubler JE, Wright WA, Vadas RL, Petraitis PS (2001) Natural variability in zygote dispersal of Ascophyllum nodosum at small spatial scales. Funct Ecol 15: 595–604.S. DudgeonJE KublerWA WrightRL VadasPS Petraitis2001Natural variability in zygote dispersal of Ascophyllum nodosum at small spatial scales.Funct Ecol15595604
- 30. Dudgeon S, Petraitis PS (2001) Scale-dependent recruitment and divergence of intertidal communities. Ecology 82: 991–1006.S. DudgeonPS Petraitis2001Scale-dependent recruitment and divergence of intertidal communities.Ecology829911006
- 31. Serrão EA, Kautsky L, Lifvergren T, Brawley SH (1997) Gamete dispersal and pre-recruitment mortality in Baltic Fucus vesiculosus. Phycologia 36: 101–102.EA SerrãoL. KautskyT. LifvergrenSH Brawley1997Gamete dispersal and pre-recruitment mortality in Baltic Fucus vesiculosus.Phycologia36101102
- 32. Coleman MA, Brawley SH (2005a) Spatial and temporal variability in dispersal and population genetic structure of a rockpool alga. Mar Ecol Prog Ser 300: 63–77.MA ColemanSH Brawley2005aSpatial and temporal variability in dispersal and population genetic structure of a rockpool alga.Mar Ecol Prog Ser3006377
- 33. Coleman MA, Brawley SH (2005b) Are life history characteristics good predictors of genetic diversity and structure? A case study of the intertidal alga Fucus spiralis (Heterikontophyta; Phaeophyceae). J Phycol 41: 753–762.MA ColemanSH Brawley2005bAre life history characteristics good predictors of genetic diversity and structure? A case study of the intertidal alga Fucus spiralis (Heterikontophyta; Phaeophyceae).J Phycol41753762
- 34. Billard E, Serrão E, Pearson G, Destombe C, Valero M (2010) Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level. Mar Ecol Prog Ser 405: 163–174.E. BillardE. SerrãoG. PearsonC. DestombeM. Valero2010Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level.Mar Ecol Prog Ser405163174
- 35. Moalic Y, Arnaud-Haond S, Perrin C, Gareth GA, Ester EA (2011) Travelling in time with networks: revealing present day hybridization versus ancestralpolymorphism between two species of brown algae, Fucus vesiculosus and F. spiralis. BMC Evol Biol 11: 33.Y. MoalicS. Arnaud-HaondC. PerrinGA GarethEA Ester2011Travelling in time with networks: revealing present day hybridization versus ancestralpolymorphism between two species of brown algae, Fucus vesiculosus and F. spiralis.BMC Evol Biol1133
- 36. Harrison PL, Babcock RC, Bull GD, Oliver JK, Wallace CC, et al. (1984) Mass spawning in tropical reef corals. Science 223: 1186–1189.PL HarrisonRC BabcockGD BullJK OliverCC Wallace1984Mass spawning in tropical reef corals.Science22311861189
- 37. Clifton KE (1997) Mass spawning by green algae on coral reefs. Science 275: 1116–1118.KE Clifton1997Mass spawning by green algae on coral reefs.Science27511161118
- 38. Serrão EA (1996) Reproductive biology of Fucus vesiculosus L. in the Baltic Sea. EA Serrão1996Reproductive biology of Fucus vesiculosus L. in the Baltic Sea.Dissertation, University of Maine. Dissertation, University of Maine.
- 39. Yamahira K (2004) How do multiple environmental cycles in combination determine reproductive timing in marine organisms? A model and test. Funct Ecology 18: 4–15.K. Yamahira2004How do multiple environmental cycles in combination determine reproductive timing in marine organisms? A model and test.Funct Ecology18415
- 40. Schmidt PS, Serrão EA, Pearson GA, Riginos C, Rawson PD, et al. (2008) Ecological genetics in the north atlantic: environmental gradients and adaptation at specific loci. Ecology 89: S91–S107.PS SchmidtEA SerrãoGA PearsonC. RiginosPD Rawson2008Ecological genetics in the north atlantic: environmental gradients and adaptation at specific loci.Ecology89S91S107
- 41. Coyne JA, Orr HA (2004) Speciation. 545 p.JA CoyneHA Orr2004Speciation.Sinauer Associates, Sunderland. 545 Sinauer Associates, Sunderland.
- 42. Knowlton N, Maté JL, Guzmán HM, Rowan R, Jara J (1997) Direct evidence for reproductive isolation among the three species of the Montastraea annularis complex in Central America (Panamá and Honduras). Mar Biol 127: 705–711.N. KnowltonJL MatéHM GuzmánR. RowanJ. Jara1997Direct evidence for reproductive isolation among the three species of the Montastraea annularis complex in Central America (Panamá and Honduras).Mar Biol127705711
- 43. Tomaiuolo M, Hansen TF, Levitan DR (2007) A theoretical investigation of sympatric evolution of temporal reproductive isolation as illustrated by marine broadcast spawners. Evolution 61: 2584–95.M. TomaiuoloTF HansenDR Levitan2007A theoretical investigation of sympatric evolution of temporal reproductive isolation as illustrated by marine broadcast spawners.Evolution61258495
- 44. Cánovas F, Mota CF, Serrão EA, Pearson GA (2011) Driving south: A multi-gene phylogeny of the brown algal family Fucaceae reveals relationships and recent drivers of a marine radiation. BMC Evolutionary Biology 11: 371.F. CánovasCF MotaEA SerrãoGA Pearson2011Driving south: A multi-gene phylogeny of the brown algal family Fucaceae reveals relationships and recent drivers of a marine radiation.BMC Evolutionary Biology11371
- 45. Johnson LE, Brawley SH (1998) Dispersal and recruitment of a canopy-forming intertidal alga: the relative roles of propagule availability and post-settlement processes. Oecologia 117: 517–526.LE JohnsonSH Brawley1998Dispersal and recruitment of a canopy-forming intertidal alga: the relative roles of propagule availability and post-settlement processes.Oecologia117517526
- 46. Forgarty ND, Steven VV, Levitan DR (2012) Weak prezygotic isolating mechanisms in threatened Caribbean Acropora Corals. PloS ONE 7(2): e30486.ND ForgartyVV StevenDR Levitan2012Weak prezygotic isolating mechanisms in threatened Caribbean Acropora Corals.PloS ONE72e30486
- 47. Müller DG, Seferiadis K (1977) Specificity of sexual chemotaxis in Fucus serratus and Fucus vesiculosus (Phaeophyceae). Z Pflanzenphysiol 84: 85–94.DG MüllerK. Seferiadis1977Specificity of sexual chemotaxis in Fucus serratus and Fucus vesiculosus (Phaeophyceae).Z Pflanzenphysiol848594
- 48. Serrão EA, Alice LA, Brawley SH (1999) Evolution of the Fucaceae (Phaeophyceae) inferred from nrDNA-ITS. J Phycol 35: 382–394.EA SerrãoLA AliceSH Brawley1999Evolution of the Fucaceae (Phaeophyceae) inferred from nrDNA-ITS.J Phycol35382394
- 49. Stebbins GL (1957) Self fertilization and population variability in the higher plants. Am Nat 91: 337–354.GL Stebbins1957Self fertilization and population variability in the higher plants.Am Nat91337354
- 50. Takebayashi N, Morrell PL (2001) Is self-fertilization an evolutionary dead end? Revisiting an old hypothesis with genetic theories and a macroevolutionary approach. Am J Bot 88: 1143–1150.N. TakebayashiPL Morrell2001Is self-fertilization an evolutionary dead end? Revisiting an old hypothesis with genetic theories and a macroevolutionary approach.Am J Bot8811431150
- 51. Denny MW, Shibata MF (1989) Consequences of surf-zone turbulence for settlement and external fertilization. Am Nat 134: 859–889.MW DennyMF Shibata1989Consequences of surf-zone turbulence for settlement and external fertilization.Am Nat134859889
- 52. Brawley SH, Johnson LE, Pearson GA, Speransky V, Li R, et al. (1999) Gamete release at low tide in Fucoid algae: maladaptive or advantageous? Am Zool 39: 218–229.SH BrawleyLE JohnsonGA PearsonV. SperanskyR. Li1999Gamete release at low tide in Fucoid algae: maladaptive or advantageous?Am Zool39218229