Temporal stability and directional change in a color cline of a marine snail from NW Spain.

The evolution and maintenance of color clines is a classic topic of research in evolutionary ecology. However, studies analyzing the temporal dynamics of such clines are much less frequent, due to the difficulty of obtaining reliable data about past color distributions along environmental gradients. In this article, we describe a case of decades-long temporal stability and directional change in a color cline of the marine snail Littorina saxatilis along the coastal inlet of the Ría de Vigo (NW Spain). L. saxatilis from this area shows a clear color cline with 3 distinct areas from the innermost to the more wave-exposed localities of the Ría: the inner, protected localities show an abundance of fawn-like individuals; the intermediate localities show a high diversity of colors; and the outer, wave-exposed localities show populations with a high frequency of a black and lineated morph. We compare data from the 1970s and 2022 in the same localities, showing that the cline has kept relatively stable for at least over half a century, except for some directional change and local variability in the frequency of certain morphs. Multiple regression analyses and biodiversity measures are presented to provide clues into the selective pressures that might be involved in the maintenance of this color cline. Future research avenues to properly test the explanatory power of these selective agents as well as the possible origins of the cline are discussed.

Shell color polymorphism in marine gastropods.

Marine gastropods are characterized by an incredible variation in shell color. In this review, we aim to introduce researchers to previous studies of shell color polymorphism in this group of animals, trying to provide an overview of the topic and highlighting some potential avenues for future research. For this, we tackle the different aspects of shell color polymorphism in marine gastropods: its biochemical and genetic basis, its patterns of spatial and temporal distribution, as well as its potential evolutionary causes. In particular, we put special emphasis on the evolutionary studies that have been conducted so far to reveal the evolutionary mechanisms responsible for the maintenance of shell color polymorphism in this group of animals, as it constitutes the least addressed aspect in existing literature reviews. Several general conclusions can be drawn from our review: First, natural selection is commonly involved in the maintenance of gastropod color polymorphism; second, although the contribution of neutral forces (gene flow-genetic drift equilibrium) to shell color polymorphism maintenance do not seem to be particularly important, it has rarely been studied systematically; third, a relationship between shell color polymorphism and mode of larval development (related to dispersal capability) may exist. As for future studies, we suggest that a combination of both classical laboratory crossing experiments and -Omics approaches may yield interesting results on the molecular basis of color polymorphism. We believe that understanding the various causes of shell color polymorphism in marine gastropods is of great importance not only to understand how biodiversity works, but also for protecting such biodiversity, as knowledge of its evolutionary causes may help implement conservation measures in those species or ecosystems that are threatened.

Thermodynamic effects drive countergradient responses in the thermal performance of Littorina saxatilis across latitude.

Thermal performance curves (TPCs) provide a powerful framework to assess the evolution of thermal sensitivity in populations exposed to divergent selection regimes across latitude. However, there is a lack of consensus regarding the extent to which physiological adjustments that compensate for latitudinal temperature variation (metabolic cold adaptation; MCA) may alter the shape of TPCs, including potential repercussion on upper thermal limits. To address this, we compared TPCs for cardiac activity in latitudinally-separated populations of the intertidal periwinkle Littorina saxatilis. We applied a non-linear TPC modelling approach to explore how different metrics governing the shape of TPCs varied systematically in response to local adaptation and thermal acclimation. Both critical upper limits, and the temperatures at which cardiac performance was maximised, were higher in the northernmost (cold-adapted) population and displayed a countergradient latitudinal trend which was most pronounced following acclimation to low temperatures. We interpret this response as a knock-on consequence of increased standard metabolic rate in high latitude populations, indicating that physiological compensation associated with MCA may indirectly influence variation in upper thermal limits across latitude. Our study highlights the danger of assuming that variation in any one aspect of the TPC is adaptive without appropriate mechanistic and ecological context.

Proteomic analysis of F1 hybrids and intermediate variants in a Littorina saxatilis hybrid zone.

Proteomic analysis was carried out on the Crab (upper-shore) and Wave (lower-shore) ecotypes of Littorina saxatilis from a hybrid zone at Silleiro Cape, Spain. Proteome profiles of individual snails were obtained. Protein expression in F1 hybrid snails bred in the laboratory and snails with intermediate shell phenotypes collected from the mid-shore were compared with Crab and Wave ecotypes using analytical approaches used to study dominance. Multivariate analysis over many protein spots showed that the F1 snails are distinct from both ecotypes but closer to the Wave ecotype. The intermediate snails are highly variable, some closer to the Crab and others to the Wave ecotype. Considered on a protein by protein basis, some proteins are significantly closer in expression to the Crab and others to the Wave ecotype for both F1 and intermediate snails. Furthermore, a significant majority of proteins were closer in expression to the Wave ecotype for the F1, consistent with the multivariate analysis. No such significant majority toward either the Crab or Wave ecotype was observed for the intermediate snails. The closer similarity of F1 and Wave ecotype expression patterns could be the result of similar selective pressures in the similar mid-shore and low-shore environments. For a significantly larger number of proteins, intermediate snails were closer in expression to the ecotype having the lower expression, for both Crab and Wave ecotypes. This is somewhat unexpected as lower expression might be expected to be an indication of impairment of function and lower fitness. Proteomic analysis could be important for the identification of candidate proteins useful for gaining improved understanding of adaptation and barriers to gene flow in hybrid zones.

Divergence in Thermal Physiology Could Contribute to Vertical Segregation in Intertidal Ecotypes of Littorina saxatilis.

AbstractThermal stress is a potentially important selective agent in intertidal marine habitats, but the role that thermal tolerance might play in local adaptation across shore height has been underexplored. Northwest Spain is home to two morphologically distinct ecotypes of the periwinkle Littorina saxatilis, separated by shore height and subject to substantial differences in thermal stress exposure. However, despite other biotic and abiotic drivers of ecotype segregation being well studied, their thermal tolerance has not been previously characterized. We investigated thermal tolerance across multiple life history stages by employing the thermal death time (TDT) approach to determine (i) whether the two ecotypes differ in thermal tolerance and (ii) how any differences vary with life history stage. Adults of the two ecotypes differed in their thermal tolerance in line with their shore position: the upper-shore ecotype, which experiences more extreme temperatures, exhibited greater endurance of thermal stress compared with the lower-shore ecotype. This difference was most pronounced at the highest temperatures tested. The proximate physiological basis for these differences is unknown but likely due to a multifarious interaction of traits affecting different parts of the TDT curve. Differences in tolerance between ecotypes were less pronounced in early life history stages but increased with ontogeny, suggesting partial divergence of this trait during development. Thermal tolerance could potentially play an important role in maintaining population divergence and genetic segregation between the two ecotypes, since the increased thermal sensitivity of the lower-shore ecotype may limit its dispersal onto the upper shore and so restrict gene flow.

Negative frequency-dependent selection maintains shell banding polymorphisms in two marine snails (Littorina fabalis and Littorina saxatilis).

The presence of shell bands is common in gastropods. Both the marine snails Littorina fabalis and Lttorina saxatilis are polymorphic for this trait. Such polymorphism would be expected to be lost by the action of genetic drift or directional selection, but it appears to be widespread at relatively constant frequencies. This suggests it is maintained by balancing selection on the trait or on a genetically linked trait. Using long time series of empirical data, we compared potential effects of genetic drift and negative frequency-dependent selection (NFDS) in the two species. The contribution of genetic drift to changes in the frequency of bands in L. fabalis was estimated using the effective population size estimated from microsatellite data, while the effect of genetic drift in L. saxatilis was derived from previously published study. Frequency-dependent selection was assessed by comparing the cross-product estimator of fitness with the frequency of the polymorphism across years using a regression analysis. Both studied species showed patterns of NFDS. In addition, in L. fabalis, contributions from genetic drift could explain some of the changes in banding frequency. Overdominance and heterogeneous selection did not fit well to our data. The possible biological explanations resulting in the maintenance of the banding polymorphism are discussed.

Acceptance and knowledge of evolutionary theory among third-year university students in Spain.

The theory of evolution is one of the greatest scientific achievements in the intellectual history of humankind, yet it is still contentious within certain social groups. Despite being as robust and evidence-based as any other notable scientific theory, some people show a strong reluctance to accept it. In this study, we used the Measure of Acceptance of the Theory of Evolution (MATE) and Knowledge of Evolution Exam (KEE) questionnaires with university students from four academic degree programs (Chemistry, English, History, and Biology) of ten universities from Spain to measure, respectively, acceptance and knowledge of evolutionary theory among third-year undergraduate students (nMATE = 978; nKEE = 981). Results show that acceptance of evolution is relatively high (87.2%), whereas knowledge of the theory is moderate (5.4 out of 10) although there are differences across degrees (Biology>Chemistry>History>English), and even among various universities (ranging from 4.71 to 5.81). Statistical analysis reveals that knowledge of evolutionary theory among Biology students is partially explained by the relative weight of evolutionary themes within the curriculum, suggesting that an increase in the number of hours dedicated to this topic could have a direct influence on students' knowledge of it. We also found that religion may have a significant-although relatively small-negative influence on evolutionary theory acceptance. The moderate knowledge of evolution in our undergraduate students, together with the potential problem of acceptance in certain groups, suggests the need for a revision of the evolutionary concepts in the teaching curricula of our students since primary school.

Geographic and Temporal Variation of Distinct Intracellular Endosymbiont Strains of Wolbachia sp. in the Grasshopper Chorthippus parallelus: a Frequency-Dependent Mechanism?

Wolbachia is an intracellular endosymbiont that can produce a range of effects on host fitness, but the temporal dynamics of Wolbachia strains have rarely been experimentally evaluated. We compare interannual strain frequencies along a geographical region for understanding the forces that shape Wolbachia strain frequency in natural populations of its host, Chorthippus parallelus (Orthoptera, Acrididae). General linear models show that strain frequency changes significantly across geographical and temporal scales. Computer simulation allows to reject the compatibility of the observed patterns with either genetic drift or sampling errors. We use consecutive years to estimate total Wolbachia strain fitness. Our estimation of Wolbachia fitness is significant in most cases, within locality and between consecutive years, following a negatively frequency-dependent trend. Wolbachia spp. B and F strains show a temporal pattern of variation that is compatible with a negative frequency-dependent natural selection mechanism. Our results suggest that such a mechanism should be at least considered in future experimental and theoretical research strategies that attempt to understand Wolbachia biodiversity.

Population genomics of parallel evolution in gene expression and gene sequence during ecological adaptation.

Natural selection often produces parallel phenotypic changes in response to a similar adaptive challenge. However, the extent to which parallel gene expression differences and genomic divergence underlie parallel phenotypic traits and whether they are decoupled or not remains largely unexplored. We performed a population genomic study of parallel ecological adaptation among replicate ecotype pairs of the rough periwinkle (Littorina saxatilis) at a regional geographical scale (NW Spain). We show that genomic changes underlying parallel phenotypic divergence followed a complex pattern of both repeatable differences and of differences unique to specific ecotype pairs, in which parallel changes in expression or sequence are restricted to a limited set of genes. Yet, the majority of divergent genes were divergent either for gene expression or coding sequence, but not for both simultaneously. Overall, our findings suggest that divergent selection significantly contributed to the process of parallel molecular differentiation among ecotype pairs, and that changes in expression and gene sequence underlying phenotypic divergence could, at least to a certain extent, be considered decoupled processes.

Karyotype Characterization of Nine Periwinkle Species (Gastropoda, Littorinidae).

Periwinkles of the family Littorinidae (Children, 1834) are common members of seashore littoral communities worldwide. Although the family is composed of more than 200 species belonging to 18 genera, chromosome numbers have been described in only eleven of them. A molecular cytogenetic analysis of nine periwinkle species, the rough periwinkles Littorina arcana, L. saxatilis, and L. compressa, the flat periwinkles L. obtusata and L. fabalis, the common periwinkle L. littorea, the mangrove periwinkle Littoraria angulifera, the beaded periwinkle Cenchritis muricatus, and the small periwinkle Melarhaphe neritoides was performed. All species showed diploid chromosome numbers of 2n = 34, and karyotypes were mostly composed of metacentric and submetacentric chromosome pairs. None of the periwinkle species showed chromosomal differences between male and female specimens. The chromosomal mapping of major and minor rDNA and H3 histone gene clusters by fluorescent in situ hybridization demonstrated that the patterns of distribution of these DNA sequences were conserved among closely related species and differed among less related ones. All signals occupied separated loci on different chromosome pairs without any evidence of co-localization in any of the species.

A novel method for estimating the strength of positive mating preference by similarity in the wild.

Mating preference can be a driver of sexual selection and assortative mating and is, therefore, a key element in evolutionary dynamics. Positive mating preference by similarity is the tendency for the choosy individual to select a mate which possesses a similar variant of a trait. Such preference can be modelled using Gaussian-like mathematical functions that describe the strength of preference, but such functions cannot be applied to empirical data collected from the field. As a result, traditionally, mating preference is indirectly estimated by the degree of assortative mating (using Pearson's correlation coefficient, r) in wild captured mating pairs. Unfortunately, r and similar coefficients are often biased due to the fact that different variants of a given trait are nonrandomly distributed in the wild, and pooling of mating pairs from such heterogeneous samples may lead to "false-positive" results, termed "the scale-of-choice effect" (SCE). Here we provide two new estimators of mating preference (Crough and Cscaled) derived from Gaussian-like functions which can be applied to empirical data. Computer simulations demonstrated that r coefficient showed robust estimations properties of mating preference but it was severely affected by SCE, Crough showed reasonable estimation properties and it was little affected by SCE, while Cscaled showed the best properties at infinite sample sizes and it was not affected by SCE but failed at biological sample sizes. We recommend using Crough combined with the r coefficient to infer mating preference in future empirical studies.

Size selection by a gape-limited predator of a marine snail: Insights into magic traits for speciation.

The intertidal snail Littorina saxatilis has repeatedly evolved two parallel ecotypes assumed to be wave adapted and predatory shore crab adapted, but the magnitude and targets of predator-driven selection are unknown. In Spain, a small, wave ecotype with a large aperture from the lower shore and a large, thick-shelled crab ecotype from the upper shore meet in the mid-shore and show partial size-assortative mating. We performed complementary field tethering and laboratory predation experiments; the first set compared the survival of two different size-classes of the crab ecotype while the second compared the same size-class of the two ecotypes. In the first set, the large size-class of the crab ecotype survived significantly better than the small size-class both on the upper shore and in the laboratory. In the second set, the small size-class of the crab ecotype survived substantially better than that of the wave ecotype both on the upper shore and in the laboratory. Shell-breaking predation on tethered snails was almost absent within the lower shore. In the laboratory shore crabs (Pachygrapsus marmoratus) with larger claw heights selected most strongly against the small size-class of the crab ecotype, whereas those with medium claw heights selected most strongly against the thin-shelled wave ecotype. Sexual maturity occurred at a much larger size in the crab ecotype than in the wave ecotype. Our results showed that selection on the upper shore for rapid attainment of a size refuge from this gape-limited predator favors large size, thick shells, and late maturity. Model parameterization showed that size-selective predation restricted to the upper shore resulted in the evolution of the crab ecotype despite gene flow from the wave ecotype snails living on the lower shore. These results on gape-limited predation and previous ones showing size-assortative mating between ecotypes suggest that size may represent a magic trait for the thick-shelled ecotype.

Limited proteomic response in the marine snail Melarhaphe neritoides after long-term emersion.

Rocky intertidal organisms are commonly exposed to environmental gradients, promoting adaptations to these conditions. Emersion time varies along the intertidal range and in the supralittoral zone is frequently larger than a single tidal cycle, even lasting for weeks. The planktonic-dispersing gastropod Melarhaphe neritoides is a common species of the high shore, adapted to reduce water loss in order to survive during long-term emersion. In this study, we investigated the molecular response, at the proteome level, of M. neritoides collected in high-shore tide pools to a series of emersion periods, from 8 to 24 days, in laboratory conditions. We compared this response to individuals maintained submerged during this period, because this was their original habitat. We also included a reversion treatment in the study, in which emersed individuals were returned to the submerged conditions. Although we detected an increase in overall protein concentration with longer emersion periods, contrary to general expectation, the two dimensional electrophoresis (2DE)-based proteomic analysis did not show significant differences between the treatments at the level of individual protein spots, even after an emersion period of 24 days. Our results suggest that the metabolism remains unaltered independent of the treatment carried out or the changes are very subtle and therefore difficult to detect with our experimental design. We conclude that M. neritoides could be equally adapted to emersion and submersion without drastic physiological changes.

Historical Biogeography of the Marine Snail Littorina saxatilis Inferred from Haplotype and Shell Morphology Evolution in NW Spain.

The marine snail Littorina saxatilis exhibits extreme morphological variation between and within geographical regions and represents an excellent model for assessing local adaptation. Previous studies support the hypothesis of parallel evolution in sympatry of two morphologically different ecotypes (named as RB and SU) that co-inhabit different habitats from Galician rocky shores (NW Spain), and which are interrupted by sheltered areas inhabited by a different morph never studied before (named as SRB). Here, we use morphological and mitochondrial DNA (mtDNA) sequence data to test hypotheses on the origin and diversification of SRB snails and to assess their evolutionary relationships with RB and SU ecotypes. Our results show that the SRB morph displays the largest size and shell elongation and the smallest relative shell aperture, representing an extreme type of the RB vs. SU polymorphism, which has been linked to adaptation to sheltered ecological factors. Phylogenetic analysis shows that the SRB morph shares ancestry with RB and SU ecotypes, rejecting the hypothesis that the SRB morph marks relict populations from which these ecotypes evolved in Galician coasts. Our data support that genetic differentiation among SRB, RB and SU morphs results from a general pattern of restricted gene flow and isolation by distance linked to the colonization of Galician coasts by two independent mtDNA lineages, rather than from a random fragmentation of the initial distributional range. Therefore, the confinement of distinct lineages to specific geographical areas denote evident limits to the distances these snails can disperse. Morphological analysis indicates no association between mtDNA lineage and a specific morphotype, and suggests the independent gain of convergent morphological patterns within each mtDNA lineage in populations occupying contrasting habitats following the colonization of Galician coasts.

The scale-of-choice effect and how estimates of assortative mating in the wild can be biased due to heterogeneous samples.

The mode in which sexual organisms choose mates is a key evolutionary process, as it can have a profound impact on fitness and speciation. One way to study mate choice in the wild is by measuring trait correlation between mates. Positive assortative mating is inferred when individuals of a mating pair display traits that are more similar than those expected under random mating while negative assortative mating is the opposite. A recent review of 1134 trait correlations found that positive estimates of assortative mating were more frequent and larger in magnitude than negative estimates. Here, we describe the scale-of-choice effect (SCE), which occurs when mate choice exists at a smaller scale than that of the investigator's sampling, while simultaneously the trait is heterogeneously distributed at the true scale-of-choice. We demonstrate the SCE by Monte Carlo simulations and estimate it in two organisms showing positive (Littorina saxatilis) and negative (L. fabalis) assortative mating. Our results show that both positive and negative estimates are biased by the SCE by different magnitudes, typically toward positive values. Therefore, the low frequency of negative assortative mating observed in the literature may be due to the SCE's impact on correlation estimates, which demands new experimental evaluation.

Parallel evolution of local adaptation and reproductive isolation in the face of gene flow.

Parallel evolution of similar phenotypes provides strong evidence for the operation of natural selection. Where these phenotypes contribute to reproductive isolation, they further support a role for divergent, habitat-associated selection in speciation. However, the observation of pairs of divergent ecotypes currently occupying contrasting habitats in distinct geographical regions is not sufficient to infer parallel origins. Here we show striking parallel phenotypic divergence between populations of the rocky-shore gastropod, Littorina saxatilis, occupying contrasting habitats exposed to either wave action or crab predation. This divergence is associated with barriers to gene exchange but, nevertheless, genetic variation is more strongly structured by geography than by ecotype. Using approximate Bayesian analysis of sequence data and amplified fragment length polymorphism markers, we show that the ecotypes are likely to have arisen in the face of continuous gene flow and that the demographic separation of ecotypes has occurred in parallel at both regional and local scales. Parameter estimates suggest a long delay between colonization of a locality and ecotype formation, perhaps because the postglacial spread of crab populations was slower than the spread of snails. Adaptive differentiation may not be fully genetically independent despite being demographically parallel. These results provide new insight into a major model of ecologically driven speciation.

Proteomics in evolutionary ecology: linking the genotype with the phenotype.

The study of the proteome (proteomics), which includes the dynamics of protein expression, regulation, interactions and its function, has played a less prominent role in evolutionary and ecological investigations in comparison with the study of the genome and transcriptome. There are, however, a number of arguments suggesting that this situation should change. First, the proteome is closer to the phenotype than the genome or the transcriptome, and as such may be more directly responsive to natural selection, and thus closely linked to adaptation. Second, there is evidence of a low correlation between protein and transcript expression levels across genes in many different organisms. Finally, there have been some recent important technological improvements in proteomics methods that make them feasible, practical and useful to address a wide range of evolutionary questions even in nonmodel organisms. The different proteomic methods, their limitations and problems when interpreting empirical data are described and discussed. In addition, the proteomic literature pertaining to evolutionary ecology is reviewed with examples, and potential applications of proteomics in a variety of evolutionary contexts are outlined. New proteomic research trends such as the study of posttranslational modifications and protein-protein interactions, as well as the combined use of the different -omics approaches, are discussed in relation to the development of a more functional and integrated perspective, needed for achieving a more comprehensive knowledge of evolutionary change.

Insights into the role of differential gene expression on the ecological adaptation of the snail Littorina saxatilis.

BACKGROUND: In the past 40 years, there has been increasing acceptance that variation in levels of gene expression represents a major source of evolutionary novelty. Gene expression divergence is therefore likely to be involved in the emergence of incipient species, namely, in a context of adaptive radiation. In this study, a genome-wide expression profiling approach (cDNA-AFLP), validated by quantitative real-time polymerase chain reaction (qPCR) were used to get insights into the role of differential gene expression on the ecological adaptation of the marine snail Littorina saxatilis. This gastropod displays two sympatric ecotypes (RB and SU) which are becoming one of the best studied systems for ecological speciation. RESULTS: Among the 99 transcripts shared between ecotypes, 12.12% showed significant differential expression. At least 4% of these transcripts still displayed significant differences after correction for multiple tests, highlighting that gene expression can differ considerably between subpopulations adapted to alternative habitats in the face of gene flow. One of the transcripts identified was Cytochrome c Oxidase subunit I (COI). In addition, 6 possible reference genes were validated to normalize and confirm this result using qPCR. α-Tubulin and histone H3.3 showed the more stable expression levels, being therefore chosen as the best option for normalization. The qPCR analysis confirmed a higher COI expression in SU individuals. CONCLUSIONS: At least 4% of the transcriptome studied is being differentially expressed between ecotypes living in alternative habitats, even when gene flow is still substantial between ecotypes. We could identify a candidate transcript of such ecotype differentiation: Cytochrome c Oxidase Subunit I (COI), a mitochondrial gene involved in energy metabolism. Quantitative PCR was used to confirm the differences found in COI and its over-expression in the SU ecotype. Interestingly, COI is involved in the oxidative phosphorylation, suggesting an enhanced mitochondrial gene expression (or increased number of mitochondria) to improve energy supply in the ecotype subjected to the strongest wave action.

Repeated evolution of reproductive isolation in a marine snail: unveiling mechanisms of speciation.

Distinct ecotypes of the snail Littorina saxatilis, each linked to a specific shore microhabitat, form a mosaic-like pattern with narrow hybrid zones in between, over which gene flow is 10-30% of within-ecotype gene flow. Multi-locus comparisons cluster populations by geographic affinity independent of ecotype, while loci under selection group populations by ecotype. The repeated occurrence of partially reproductively isolated ecotypes and the conflicting patterns in neutral and selected genes can either be explained by separation in allopatry followed by secondary overlap and extensive introgression that homogenizes neutral differences evolved under allopatry, or by repeated evolution in parapatry, or in sympatry, with the same ecotypes appearing in each local site. Data from Spain, the UK and Sweden give stronger support for a non-allopatric model of ecotype formation than for an allopatric model. Several different non-allopatric mechanisms can, however, explain the repeated evolution of the ecotypes: (i) parallel evolution by new mutations in different populations; (ii) evolution from standing genetic variation; and (iii) evolution in concert with rapid spread of new positive mutations among populations inhabiting similar environments. These models make different predictions that can be tested using comprehensive phylogenetic information combined with candidate loci sequencing.

The role of phenotypic plasticity on the proteome differences between two sympatric marine snail ecotypes adapted to distinct micro-habitats.

BACKGROUND: The role of phenotypic plasticity is increasingly being recognized in the field of evolutionary studies. In this paper we look at the role of genetic determination versus plastic response by comparing the protein expression profiles between two sympatric ecotypes adapted to different shore levels and habitats using two-dimensional protein maps. RESULTS: We compared qualitative and quantitative differences in protein expression between pools of both ecotypes from different environments (field and laboratory conditions). The results suggested that ecotype differences may affect about 7% of the proteome in agreement with previous studies, and moreover these differences are basically insensitive to environmental changes. Thus, observed differences between wild ecotypes can be mainly attributed to genetic factors rather than phenotypic plasticity. CONCLUSIONS: These results confirm the mechanism of adaptation already proposed in this species and a minor role of phenotypic plasticity in this ecological speciation process. In addition, this study provides a number of interesting protein spots potentially involved in adaptation, and therefore candidates for a future identification.