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Evolution of post-zygotic barriers in a scenario of non-allopatric ecological speciation Mónica Martínez-Fernández 1, Cármen López-Fernández 2, Alexandra Sá-Pinto 3*, Zita Ferreira 3, Rute Pereira 3, Emílio Rolán-Alvarez 4 * [email protected] 1Unidad 3CIBIO de Oncología Molecular. Dpto. Investigación Básica. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). 28040 Madrid (Spain); 2 Departamento de Biología, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain; - Centro de Investigação em Biodiversidade e Recursos Genéticos, Vairão, Portugal; 4Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidade de Vigo, 36310 Vigo, Spain State of the art & major goals One of the major goals of Evolutionary Biology is to understand how two distinct species evolve from an initial single population. This question prompted research on the timing of emergence of reproductive isolation and on the nature of barriers preventing gene flow (Coyne and Orr, 2004). As an example, pre-zygotic barriers have evolved significantly faster than post-zygotic barriers between species of Drosophila with a current sympatric distribution but not between those that are presently allopatrically distributed (reviewed in Coyne and Orr, 2004). Unfortunately for most of these studies, the geographic context of speciation was not completely clear and/or understood, thus preventing a clear interpretation of such results. In fact, given the few widely accepted examples of non-allopatric speciation, knowledge on the emergence of reproductive barriers in such context and their importance for the speciation process stills scarce. With the present work we will use the snail Littorina saxatilis as a model species to study the evolution of post-zygotic barriers during sympatric speciation. Model system Figure 1. Sympatric ecotypes adapted to different shore levels or conditions can be observed in several evolved and are maintained in the face of gene flow, due to strong disruptive evolution (Rolán-Alvarez et species al., 2007). ecotypes are found in waved-exposed In some exposed Galician shores this species present two ecotypes adapted to distinct shore levels (Fig1). The ridged and banded ecotype (RB) occurs at the upper shore and has a large and robust shell to protect Littorina. SU RB ecological For Littorina saxatilis, ecotypes-pairs have been described for several areas across Atlantic shores, which of RB SU Two sympatric rocky shores from Galicia (Spain): SU adapted to lower shore levels; and RB adapted to upper shore levels. against crab predation and a small aperture to minimize desiccation; the smooth and unbanded ecotype (SU) occurs at lower shore and has a smaller shell with a relatively large aperture, that enclose the strongly muscular foot that avoids dislodgement by the heavy wave action (Rolán-Alvarez, 2007). These two ecotypes meet at mid-shore where they sometimes hybridise forming apparently fertile hybrids (Rolán-Alvarez et al., 2004). Until now, mainly pre-zygotic barriers have been found to contribute to restrict gene flow between both ecotypes (Rolán-Alvarez, 2007), although divergent sexual selection in morphological traits was shown to reduce the fecundity of intermediate morphological hybrids in nature (Cruz et al., 2001). a Methodology Individuals morphologically identified as RB, SU and hybrids were collected at mid-shore, in two Galician localities (Cabo Silleiro and Senín). Individuals were sexed and kept alive in aquariums until dissection. All the males were anesthetised (emerged in a solution of Mg2Cl 7,5 % during 30 minutes) and seminal vesicles were dissected, immediately emerged in a extender solution (Hepes 10 mM, KCl 80 mM, NaCl 45 mM, C2H3NaO2 45 mM, CaCl2 0.4 mM and MgCl2 0.2 mM) and preserved at -80 ºC. The degree of sperm DNA fragmentation (SDF) was determined for each individual as, in other species, SDF was shown to be correlated with individuals’ fertility (Giwercman et al., 2010). For each seminal vesicle collected, a b minimum of 100 sperms were randomly taken, treated according to the protocol of a specific prototype assay for Littorina (Halomax Proto-Littorina) and observed under fluorescence microscope. Normal and Figure 2. Sperm treated with Halomax Proto-Littorina, observed under Fluorescence microscope (a) and after informatic image DNA’s fragmented sperms were counted. Hierarchical G-tests (Sokal and Rohlf, 1995) were then conducted correction (b). Red arrows point to sperm with DNA fragmentation and yellow arrows point to normal sperms. to test for the existence of significant differences in DNA fragmentation both within and between each of Table 1. Results of Sperm DNA Fragmentation (SDF) analysis. the three morphological classes of individuals. Silleiro Results and discussion Hybrids show significantly higher degree of SDF than each of the two pure forms in both sampling Senín SU RB Hy Mean Variance 2.00 3.23 7.95 2.91 79.08 G DF p 17.25 11 15.85 53.75 12 115.09 12 0.10 3.03E-07 5.96E-19 N NS Mean 13 1297 13 1316 12 1194 9.71 5.17 14.99 locations (Tables 1 and 2), suggesting the existence of an incipient post-zygotic isolation mechanism Variance 283.31 25.83 638.89 between the two ecotypes. As females are highly promiscuous, the higher SDF rates observed in hybrids G 1080.91 313.23 1236.98 DF 16 21 10 p 0 0 0 N 22 11 17 NS 6660 3119 5033 may reduce the fertility of these individuals in nature as their lower quality sperm may be out-competed by sperm of pure forms. The existence of this incipient post-zygotic barrier between the ecotypes may be contributing to the speciation process as it could strength the genetic barrier between these ecotypes. In Mean: mean SDF value observed in each morphological class; Variance: variance observed in SDF; G: G- addition, it could be a necessary step for reinforcement to occur, although evidence for reinforcement has Value of G test, Df: degrees of freedom; P: probability of homogeneity within groups (significant results indicated in bold); N: number of males analysed; NS: total number of sperms analysed; SU: smooth and not yet been detected in this model system. unbanded ecotype; RB: ridged and banded ecotype; Hy: hybrids. With the exception of SU in Silleiro, significant heterogeneity in SDF rates was detected in all morphological classes in both sampling locations (Table 1). This may be due to the presence of individuals Table 2. Results of G-tests conducted for testing for the existence of significant differences in Sperm DNA fragmentation between pairs of morphological classes. with different degrees of genome introgression in all morphological classes and/or to environmental SU / Hy RB / Hy RB / SU G 48.65 28.80 3.35 markers diagnostic between two forms will be screened to test for the existence of a correlation between Df 1 1 1 hybrid index and SDF. To test for the second hypothesis analyses of SDF rates in lab reared individuals will p 3.05E-12 8.01E-08 0.07 G 43.66 216.75 78.61 Df 1 1 1 p 3.91E-11 4.63E-49 7.55E-19 factors causing SDF in all ecotypes under certain conditions. To test the former hypothesis, genetics Silleiro Senín be necessary. G: value of G test; Df: degrees of freedom; P: probability of homogeneity between groups (significant results indicated in bold); SU: smooth and unbanded ecotype; RB: ridged and banded ecotype; Hy: hybrids. Acknowledgements This work is funded by Fundação para a Ciência e Tecnologia (FCT), through the project PTDC/BIA-BEC/105221/2008. Alexandra Sá Pinto is supported by an FCT grant (SFRH/BPD/48750/2008). M. Martínez-Fernández is currently funded by a “Juan de la Cierva” research fellowship (JCI-2010-06167) from MICINN. References Coyne JA, Orr HA (2004) Speciation. Sinauer Associates, Sunderland. Cruz, R. Rolán-Álvarez,E., Garcia, C. (2001). Sexual selection on phenotypic traits in a hybrid zone of Littorina saxatilis. J. Evol. Biol. 14: 773-785. Giwercman A, Lindstedt L, Larsson M, et al. (2010) Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study. Int. J. Androl. 33(1): e221-7. Rolán-Alvarez, E. (2007). Sympatric speciation as a by-product of ecological adaptation in the Galician Littorina saxatilis hybrid zone. Journal of Molluscan Studies doi:10.1093/mollus/eyl023 Rolán-Alvarez, E. et al. (2004). Nonallopatric and parallel origin oflocal reproductive barriers between two snails ecotypes. Molecular Ecology 13: 3415-3424. Sokal, R.R., and F.J. Rohlf. (1995). Biometry: The principles and practice of statistics in biological research. 3rd edition. W.H. Freeman, New York.