Prezygotic Barriers to Hybridization in Marine Broadcast Spawners: Reproductive Timing and Mating System Variation Carla A. Monteiro, Ester A. Serra˜ o, Gareth A. Pearson* CCMAR - CIMAR, University of Algarve, Gambelas, Faro, Portugal Abstract 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, Serra˜o EA, Pearson GA (2012) Prezygotic Barriers to Hybridization in Marine Broadcast Spawners: Reproductive Timing and Mating System Variation. PLoS ONE 7(4): e35978. doi: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. * E-mail: gpearson@ualg.pt Introduction Speciation occurs by the evolution of reproductive barriers that ultimately prevent genetic exchange between previously inter- breeding populations [1]. 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 [2]). In the sea, where reproduction by external fertilization is common, it may be more difficult for extrinsic ecological barriers to arise, resulting in more opportuni- ties for hybridization [3]. Asynchronous gamete release between related taxa, and/or gametic incompatibility are considered the major prezygotic barriers to hybridization between externally fertilizing sympatric animal species [4]. 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 [5], outcrossing, albeit at varying rates, appears to dominate in hermaphroditic broadcast spawners [6]. 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 [7,8]. Despite this, broadcast spawning is a common reproductive mode in diverse marine intertidal taxa, from invertebrates to macroalgae [9–15]. 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 mech- anisms to sense the environment and restrict gamete release, particularly to periods of low environmental water motion (e.g., in PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35978