For Littorina saxatilis, ecotypes-pairs have been described for several areas across Atlantic...
1
For Littorina saxatilis, ecotypes-pairs have been described for several areas across Atlantic shores, which evolved and are maintained in the face of gene flow, due to strong disruptive evolution (Rolán-Alvarez et al., 2007). 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 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). Model system Figure 1. Sympatric ecotypes adapted to different shore levels or ecological conditions can be observed in several species of Littorina. Two sympatric ecotypes are found in waved-exposed rocky shores from Galicia (Spain): SU adapted to lower shore levels; and RB adapted to upper shore levels. RB RB SU SU 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 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 DNA’s fragmented sperms were counted. Hierarchical G-tests (Sokal and Rohlf, 1995) were then conducted to test for the existence of significant differences in DNA fragmentation both within and between each of the three morphological classes of individuals. Methodology Hybrids show significantly higher degree of SDF than each of the two pure forms in both sampling locations (Tables 1 and 2), suggesting the existence of an incipient post-zygotic isolation mechanism between the two ecotypes. As females are highly promiscuous, the higher SDF rates observed in hybrids 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 addition, it could be a necessary step for reinforcement to occur, although evidence for reinforcement has not yet been detected in this model system. 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 with different degrees of genome introgression in all morphological classes and/or to environmental factors causing SDF in all ecotypes under certain conditions. To test the former hypothesis, genetics markers diagnostic between two forms will be screened to test for the existence of a correlation between hybrid index and SDF. To test for the second hypothesis analyses of SDF rates in lab reared individuals will be necessary. Results and discussion 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. Acknowledgements 5033 3119 6660 NS 17 11 22 N 0 0 0 p 10 21 16 DF 1236.98 313.23 1080.91 G 638.89 25.83 283.31 Variance 14.99 5.17 9.71 Mean Senín 1194 1316 1297 NS 12 13 13 N 5.96E-19 3.03E-07 0.10 p 12 12 11 DF 115.09 53.75 17.25 G 79.08 15.85 2.91 Variance 7.95 3.23 2.00 Mean Silleiro Hy RB SU Table 1. Results of Sperm DNA Fragmentation (SDF) analysis. Mean: mean SDF value observed in each morphological class; Variance: variance observed in SDF; G: G-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 unbanded ecotype; RB: ridged and banded ecotype; Hy: hybrids. 7.55E-19 4.63E-49 3.91E-11 p 1 1 1 Df 78.61 216.75 43.66 G Senín 0.07 8.01E-08 3.05E-12 p 1 1 1 Df 3.35 28.80 48.65 G Silleiro RB / SU RB / Hy SU / Hy Table 2. Results of G-tests conducted for testing for the existence of significant differences in Sperm DNA fragmentation between pairs of morphological classes. 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. Reference s 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. Mónica Martínez-Fernández 1 , Cármen López-Fernández 2 , Alexandra Sá-Pinto 3* , Zita Ferreira 3 , Rute Pereira 3 , Emílio Rolán-Alvarez 4 * [email protected] 1 Unidad 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; 3 CIBIO - Centro de Investigação em Biodiversidade e Recursos Genéticos, Vairão, Portugal; 4 Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidade de Vigo, 36310 Vigo, Spain Evolution of post-zygotic barriers in a scenario of non- allopatric ecological speciation 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. State of the art & major goals Figure 2. Sperm treated with Halomax Proto-Littorina, observed under Fluorescence microscope (a) and after informatic image correction (b). Red arrows point to sperm with DNA fragmentation and yellow arrows point to normal sperms. b a
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Transcript of For Littorina saxatilis, ecotypes-pairs have been described for several areas across Atlantic...
For Littorina saxatilis, ecotypes-pairs have been described for several areas across Atlantic shores, which
evolved and are maintained in the face of gene flow, due to strong disruptive evolution (Rolán-Alvarez et
al., 2007).
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
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).
Model systemFigure 1. Sympatric ecotypes adapted to
different shore levels or ecological
conditions can be observed in several
species of Littorina. Two sympatric
ecotypes are found in waved-exposed
rocky shores from Galicia (Spain): SU
adapted to lower shore levels; and RB
adapted to upper shore levels.
RB
RBSU
SU
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
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
DNA’s fragmented sperms were counted. Hierarchical G-tests (Sokal and Rohlf, 1995) were then conducted
to test for the existence of significant differences in DNA fragmentation both within and between each of
the three morphological classes of individuals.
Methodology
Hybrids show significantly higher degree of SDF than each of the two pure forms in both sampling
locations (Tables 1 and 2), suggesting the existence of an incipient post-zygotic isolation mechanism
between the two ecotypes. As females are highly promiscuous, the higher SDF rates observed in hybrids
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
addition, it could be a necessary step for reinforcement to occur, although evidence for reinforcement has
not yet been detected in this model system.
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
with different degrees of genome introgression in all morphological classes and/or to environmental
factors causing SDF in all ecotypes under certain conditions. To test the former hypothesis, genetics
markers diagnostic between two forms will be screened to test for the existence of a correlation between
hybrid index and SDF. To test for the second hypothesis analyses of SDF rates in lab reared individuals will
be necessary.
Results and discussion
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.
Acknowledgements
503331196660NS171122N000p
102116DF1236.98313.231080.91G638.8925.83283.31Variance14.995.179.71MeanSenín119413161297NS
121313N5.96E-193.03E-070.10p
121211DF115.0953.7517.25G79.0815.852.91Variance7.953.232.00MeanSilleiroHyRBSU
Table 1. Results of Sperm DNA Fragmentation (SDF) analysis.
Mean: mean SDF value observed in each morphological class; Variance: variance observed in SDF; G: G-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 unbanded ecotype; RB: ridged and banded ecotype; Hy: hybrids.
7.55E-194.63E-493.91E-11p
111Df
78.61216.7543.66GSenín
0.078.01E-083.05E-12p
111Df
3.3528.8048.65GSilleiro
RB / SURB / HySU / Hy
Table 2. Results of G-tests conducted for testing for the existence of significant differences in Sperm DNA fragmentation between pairs of morphological classes.
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.
ReferencesCoyne 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/eyl023Rolán-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.
Mónica Martínez-Fernández 1, Cármen López-Fernández 2, Alexandra Sá-Pinto 3*, Zita Ferreira 3, Rute Pereira 3, Emílio Rolán-Alvarez 4
* [email protected] 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; 3CIBIO - 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
Evolution of post-zygotic barriers in a scenario of non-allopatric ecological speciation
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.
State of the art & major goals
Figure 2. Sperm treated with Halomax Proto-Littorina, observed under Fluorescence microscope (a) and after informatic image
correction (b). Red arrows point to sperm with DNA fragmentation and yellow arrows point to normal sperms.
b
a
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