Marine Snail Littorina Saxatilis Research Articles

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.

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Evolutionary ApplicationsVolume 16, Issue 2 p. i-i COVER IMAGEOpen Access Cover Image First published: 13 February 2023 https://doi.org/10.1111/eva.13395AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Photo caption and photo credit Caption (upper left): View of the Tjärnö Marine Laboratory, host site for the Linnaeus Centre for Marine Evolutionary Biology. Photo credits: Anna-Lena Lundqvist, Göteborgs Universitet Caption (upper right): Tenellia gotlandica sp. nov. holotype (a species of nudibranch Molluscs new to science), described in this issue. Photo credit: Klas Malmberg Caption (middle left): Polymorphism of shell color in a population of the marine snail Littorina saxatilis (rough periwinkle). Photo credit: Juan Gefaell Caption (middle right): The Maroon Anemonefish (Amphiprion biaculeatus), on an Indo-Pacific reef. Photo credit: Leon Green Caption (lower left): Acropora tenuis (kenti). Photo credit: Dr. Tom Bridge, Queensland Museum/James Cook University Caption (lower right): Underwater picture showing a mixture of native and invasive species in on a port seawall. Photo credit: Wilfried Thomas, French National Center for Scientific Research, Biological Station of Roscoff Volume16, Issue2Special Issue: Marine Evolutionary BiologyFebruary 2023Pages i-i RelatedInformation

Genetic architecture of repeated phenotypic divergence in Littorina saxatilis ecotype evolution.

Chromosomal inversions have been shown to play a major role in a local adaptation by suppressing recombination between alternative arrangements and maintaining beneficial allele combinations. However, so far, their importance relative to the remaining genome remains largely unknown. Understanding the genetic architecture of adaptation requires better estimates of how loci of different effect sizes contribute to phenotypic variation. Here, we used three Swedish islands where the marine snail Littorina saxatilis has repeatedly evolved into two distinct ecotypes along a habitat transition. We estimated the contribution of inversion polymorphisms to phenotypic divergence while controlling for polygenic effects in the remaining genome using a quantitative genetics framework. We confirmed the importance of inversions but showed that contributions of loci outside inversions are of similar magnitude, with variable proportions dependent on the trait and the population. Some inversions showed consistent effects across all sites, whereas others exhibited site-specific effects, indicating that the genomic basis for replicated phenotypic divergence is only partly shared. The contributions of sexual dimorphism as well as environmental factors to phenotypic variation were significant but minor compared to inversions and polygenic background. Overall, this integrated approach provides insight into the multiple mechanisms contributing to parallel phenotypic divergence.

A Simulation Study of the Ecological Speciation Conditions in the Galician Marine Snail Littorina saxatilis.

In the last years, the interest in evolutionary divergence at small spatial scales has increased and so did the study of speciation caused by ecologically based divergent natural selection. The evolutionary interplay between gene flow and local adaptation can lead to low-dispersal locally adapted specialists. When this occurs, the evolutionary interplay between gene flow and local adaptation could eventually lead to speciation. The L. saxatilis system consists of two ecotypes displaying a microhabitat-associated intraspecific dimorphism along the wave-exposed rocky shores of Galicia. Despite being a well-known system, the dynamics of the ecotype formation remain unclear and cannot be studied from empirical evidence alone. In this study, individual-based simulations were used to incorporate relevant ecological, spatial, and genetic information, to check different evolutionary scenarios that could evolve non-random mating preferences and finally may facilitate speciation. As main results, we observed the evolution of intermediate values of choice which matches the estimates from empirical data of L. saxatilis in Galician shores and coincides with previous theoretical outcomes. Also, the use of the mating correlation as a proxy for assortative mating led to spuriously inferring greater reproductive isolation in the middle habitat than in the others, which does not happen when directly considering the choice values from the simulations. We also corroborate the well-known fact that the occurrence of speciation is influenced by the strength of selection. Taken together, this means, also according to other L. saxatilis systems, that speciation is not an immediate consequence of local divergent selection and mating preferences, but a fine tuning among several factors including the ecological conditions in the shore levels, the selection strength, the mate choice stringency, and cost to choosiness. The L. saxatilis system could correspond to a case of incomplete reproductive isolation, where the choice intensity is intermediate and local adaptation within the habitat is strong. These results support previous interpretations of the L. saxatilis model system and indicate that further empirical studies would be interesting to test whether the mate choice mechanism functions as a similarity-like mechanism as has been shown in other littorinids.

Using replicate hybrid zones to understand the genomic basis of adaptive divergence.

Combining hybrid zone analysis with genomic data is a promising approach to understanding the genomic basis of adaptive divergence. It allows for the identification of genomic regions underlying barriers to gene flow. It also provides insights into spatial patterns of allele frequency change, informing about the interplay between environmental factors, dispersal and selection. However, when only a single hybrid zone is analysed, it is difficult to separate patterns generated by selection from those resulting from chance. Therefore, it is beneficial to look for repeatable patterns across replicate hybrid zones in the same system. We applied this approach to the marine snail Littorina saxatilis, which contains two ecotypes, adapted to wave-exposed rocks vs. high-predation boulder fields. The existence of numerous hybrid zones between ecotypes offered the opportunity to test for the repeatability of genomic architectures and spatial patterns of divergence. We sampled and phenotyped snails from seven replicate hybrid zones on the Swedish west coast and genotyped them for thousands of single nucleotide polymorphisms. Shell shape and size showed parallel clines across all zones. Many genomic regions showing steep clines and/or high differentiation were shared among hybrid zones, consistent with a common evolutionary history and extensive gene flow between zones, and supporting the importance of these regions for divergence. In particular, we found that several large putative inversions contribute to divergence in all locations. Additionally, we found evidence for consistent displacement of clines from the boulder-rock transition. Our results demonstrate patterns of spatial variation that would not be accessible without continuous spatial sampling, a large genomic data set and replicate hybrid zones.

Genetic variation for adaptive traits is associated with polymorphic inversions in Littorina saxatilis.

Chromosomal inversions have long been recognized for their role in local adaptation. By suppressing recombination in heterozygous individuals, they can maintain coadapted gene complexes and protect them from homogenizing effects of gene flow. However, to fully understand their importance for local adaptation we need to know their influence on phenotypes under divergent selection. For this, the marine snail Littorina saxatilis provides an ideal study system. Divergent ecotypes adapted to wave action and crab predation occur in close proximity on intertidal shores with gene flow between them. Here, we used F2 individuals obtained from crosses between the ecotypes to test for associations between genomic regions and traits distinguishing the Crab‐/Wave‐adapted ecotypes including size, shape, shell thickness, and behavior. We show that most of these traits are influenced by two previously detected inversion regions that are divergent between ecotypes. We thus gain a better understanding of one important underlying mechanism responsible for the rapid and repeated formation of ecotypes: divergent selection acting on inversions. We also found that some inversions contributed to more than one trait suggesting that they may contain several loci involved in adaptation, consistent with the hypothesis that suppression of recombination within inversions facilitates differentiation in the presence of gene flow.

Genomic architecture of parallel ecological divergence: Beyond a single environmental contrast.

The study of parallel ecological divergence provides important clues to the operation of natural selection. Parallel divergence often occurs in heterogeneous environments with different kinds of environmental gradients in different locations, but the genomic basis underlying this process is unknown. We investigated the genomics of rapid parallel adaptation in the marine snail Littorina saxatilis in response to two independent environmental axes (crab-predation versus wave-action and low-shore versus high-shore). Using pooled whole-genome resequencing, we show that sharing of genomic regions of high differentiation between environments is generally low but increases at smaller spatial scales. We identify different shared genomic regions of divergence for each environmental axis and show that most of these regions overlap with candidate chromosomal inversions. Several inversion regions are divergent and polymorphic across many localities. We argue that chromosomal inversions could store shared variation that fuels rapid parallel adaptation to heterogeneous environments, possibly as balanced polymorphism shared by adaptive gene flow.

Genome-wide association analyses reveal polygenic genomic architecture underlying divergent shell morphology in Spanish Littorina saxatilis ecotypes.

Gene flow between diverging populations experiencing dissimilar ecological conditions can theoretically constrain adaptive evolution. To minimize the effect of gene flow, alleles underlying traits essential for local adaptation are predicted to be located in linked genome regions with reduced recombination. Local reduction in gene flow caused by selection is expected to produce elevated divergence in these regions. The highly divergent crab‐adapted and wave‐adapted ecotypes of the marine snail Littorina saxatilis present a model system to test these predictions. We used genome‐wide association (GWA) analysis of geometric morphometric shell traits associated with microgeographic divergence between the two L. saxatilis ecotypes within three separate sampling sites. A total of 477 snails that had individual geometric morphometric data and individual genotypes at 4,066 single nucleotide polymorphisms (SNPs) were analyzed using GWA methods that corrected for population structure among the three sites. This approach allowed dissection of the genomic architecture of shell shape divergence between ecotypes across a wide geographic range, spanning two glacial lineages. GWA revealed 216 quantitative trait loci (QTL) with shell size or shape differences between ecotypes, with most loci explaining a small proportion of phenotypic variation. We found that QTL were evenly distributed across 17 linkage groups, and exhibited elevated interchromosomal linkage, suggesting a genome‐wide response to divergent selection on shell shape between the two ecotypes. Shell shape trait‐associated loci showed partial overlap with previously identified outlier loci under divergent selection between the two ecotypes, supporting the hypothesis of diversifying selection on these genomic regions. These results suggest that divergence in shell shape between the crab‐adapted and wave‐adapted ecotypes is produced predominantly by a polygenic genomic architecture with positive linkage disequilibrium among loci of small effect.

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.

Targeted resequencing reveals geographical patterns of differentiation for loci implicated in parallel evolution.

Parallel divergence and speciation provide evidence for the role of divergent selection in generating biological diversity. Recent studies indicate that parallel phenotypic divergence may not have the same genetic basis in different geographical locations - 'outlier loci' (loci potentially affected by divergent selection) are often not shared among parallel instances of phenotypic divergence. However, limited sharing may be due, in part, to technical issues if false-positive outliers occur. Here, we test this idea in the marine snail Littorina saxatilis, which has evolved two partly isolated ecotypes (adapted to crab predation vs. wave action) in multiple locations independently. We argue that if the low extent of sharing observed in earlier studies in this system is due to sampling effects, we expect outliers not to show elevated FST when sequenced in new samples from the original locations and also not to follow predictable geographical patterns of elevated FST . Following a hierarchical sampling design (within vs. between country), we applied capture sequencing, targeting outliers from earlier studies and control loci. We found that outliers again showed elevated levels of FST in their original location, suggesting they were not generated by sampling effects. Outliers were also likely to show increased FST in geographically close locations, which may be explained by higher levels of gene flow or shared ancestral genetic variation compared with more distant locations. However, in contrast to earlier findings, we also found some outlier types to show elevated FST in geographically distant locations. We discuss possible explanations for this unexpected result.

Non-random paternity of offspring in a highly promiscuous marine snail suggests postcopulatory sexual selection

In the marine snail Littorina saxatilis females are highly polyandrous, resulting in simultaneous broods of offspring sired by ∼20 males. Such high polyandry, in combination with female sperm storage, is likely to promote postcopulatory sexual selection among males. In addition, females may gain direct reproductive benefits from multiple mating. Investigating sexual selection in males and female benefits in relation to the number of sires, we put single virgin females in aquaria with one, two, five or ten males for 80 days, during which each female mated a majority of available males. After removal of the males, females continued to produce offspring during >1 year. Offspring genotypes of 27 families showed multiple paternity with 76 % of males contributing to juveniles, but paternity deviated from random with one or a few males siring the majority of the offspring. Larger males tended to be overrepresented among the sires. Female reproductive output (newborn or juvenile stage) did, however, not increase with number of sires, and females seemed not limited by the number of available males. Because previous studies have shown that females are seemingly indiscriminative in their mate choice, and given that they mate many hundreds of times in their lifespan, this suggests there is comparatively limited scope for premating sexual selection in this species. Hence, we interpret these results mainly in the light of postcopulatory sexual selection. Sexual selection is a main component of evolution. In that context, mating behaviour is central, although it sometimes appears puzzling, even maladaptive. For example, matings are costly; still, in some species, females mate multiple times and with numerous males. Why this behaviour has evolved is intriguing. Extensive numbers of matings suggest less efficient precopulatory mate choice, opening for postcopulatory sexual selection through cryptic female choice and sperm competition. Using a highly promiscuous snail species with internal fertilisation and long-term sperm storage, we identified the fathers of 549 offspring. We found that while females mated most males available, the offspring were non-randomly distributed among mated males and large males were overrepresented among the sires. This suggests that sexual selection predominantly occurs after mating and that males benefit from size-related effects. Yet, we could not find any female benefits from mating multiple males.

What can be learnt from a snail?

The marine snail Littorina saxatilis is a common inhabitant of intertidal shores of the north Atlantic. It is amazingly polymorphic and forms reproductively isolated ecotypes in microhabitats where crabs are either present and wave action is less furious, or where waves are strong and crabs are absent. Decades of research have unveiled much of the ecological and demographic context of the formation of crab- and wave-ecotype snails showing important phenotypic differences being inherited, differential selection being strong over adjacent microhabitats, local dispersal being restricted, and long-distance transports of individuals being rare. In addition, strong assortative mating of ecotypes has been shown to include a component of male mate preference based on female size. Several studies support ecotypes being diverged locally and under gene flow in a parallel and highly replicated fashion. The high level of replication at various levels of independence (from local to pan-European scale) provides excellent opportunities to investigate the detailed mechanisms of microevolution, including the formation of barriers to gene flow. Current investigations benefit from a draft reference genome and an integration of genomic approaches, modelling and experiments to unveil molecular and ecological components of speciation and their interactions.

Do the same genes underlie parallel phenotypic divergence in different Littorina saxatilis populations?

Parallel patterns of adaptive divergence and speciation are cited as powerful evidence for the role of selection driving these processes. However, it is often not clear whether parallel phenotypic divergence is underlain by parallel genetic changes. Here, we asked about the genetic basis of parallel divergence in the marine snail Littorina saxatilis, which has repeatedly evolved coexisting ecotypes adapted to either crab predation or wave action. We sequenced the transcriptome of snails of both ecotypes from three distant geographical locations (Spain, Sweden and United Kingdom) and mapped the reads to the L. saxatilis reference genome. We identified genomic regions potentially under divergent selection between ecotypes within each country, using an outlier approach based on FST values calculated per locus. In line with previous studies indicating that gene reuse is generally common, we expected to find extensive sharing of outlier loci due to recent shared ancestry and gene flow between at least two of the locations in our study system. Contrary to our expectations, we found that most outliers were country specific, suggesting that much of the genetic basis of divergence is not shared among locations. However, we did find that more outliers were shared than expected by chance and that differentiation of shared outliers is often generated by the same SNPs. We discuss two mechanisms potentially explaining the limited amount of sharing we observed. First, a polygenic basis of divergent traits might allow for multiple distinct molecular mechanisms generating the same phenotypic patterns. Second, additional, location-specific axes of selection that we did not focus on in this study may produce distinct patterns of genetic divergence within each site.

The divergence between ecotypes in a Littorina saxatilis hybrid zone is aligned with natural selection, not with intra-ecotype variation

Multivariate covariation matrices occupy a central place in the analysis of evolutionary divergence among populations. They may constrain the multivariate space directions available to the divergence, and at the same time, their structure may be affected by the forces driving the divergence, and may thus provide information about the action of these forces in the past. Unfortunately, the relationship between covariation and divergence is complex and its theoretical analysis has not been completely resolved. To advance in the understanding of this relationship it is therefore necessary to gather information from case studies in which the evolutionary dynamics is as well known as possible. Here I analyse the covariation and divergence in a well-studied hybrid zone of the marine snail Littorina saxatilis. I compare the covariation matrix of seven shell measures with their multivariate divergence both between the two ecotypes in the hybrid zone and among different localities. I also propose an eigenvector-based procedure to predict the multi-character divergence expected under a null model of divergence proportional to within population covariation. The divergence between ecotypes was clearly not proportional to the covariation. Instead, it was in the same direction as the divergent selection pressures estimated in previous studies of this hybrid zone. In addition, the phenotypic divergence between ecotypes, but not between localities, was much larger than expected in terms of Fst estimates already published for this hybrid zone. Finally, I found no evidence of depletion in variance for the traits contributing the most to the divergence. I conclude that the covariation matrix has no direct relationship with the divergence between ecotypes, as it did not seriously constrain the direction of this divergence, nor was influenced in any obvious way by its development in the past.

High proteome variation between ecotypes of Littorina saxatilis cannot be explained by tissue heterogeneity or a common-garden × ecotype effect

In small organisms where gene expression analyses are accomplished on whole specimens rather than individual tissues, the differences observed in gene expression levels between groups of samples are assumed to be caused by regulatory differences in gene expression within tissues. An alternative explanation is the lack of homogeneity distribution of the different tissues across groups of samples. In the case of the marine snail Littorina saxatilis, previous proteomic studies suggested a high differentiation in protein expression between the two (RB and SU) sympatric ecotypes existing on exposed rocky shores of the Galician coast (NW of Spain). As these ecotypes are known to differ in the proportion of muscular section (foot) contributing to the whole body, it remains to be checked which of the above explanations better explains this high proteome differentiation. Results from this new study suggest that tissue heterogeneity cannot explain the high proteomic differences observed between ecotypes of L. saxatilis. On the other hand, different estimates of proteome differentiation between ecotypes were obtained depending on the number of replicates used in each study (ranging from 7 to 30%). A reanalysis of previous published data shows a clear positive relationship between the degree of proteome differentiation observed and the number of biological replicates used in the analysis. This stresses the importance of investigating the effect of sample size on gene expression analyses. Finally, the results from the present study also discard the idea that observed proteome differences between ecotypes under similar laboratory conditions might be due to an interaction effect between common garden and the two different ecotypes. General conclusions drawn from the present study could be useful for setting up future gene expression studies in other species.

Selection on hybrids of ecologically divergent ecotypes of a marine snail: the relative importance of exogenous and endogenous barriers

Unravelling the form of selection acting on hybrids of ecotypes undergoing ecological speciation is essential to understand the mechanisms behind the evolution of reproductive isolation in the face of gene flow. Shell phenotype is known to be affected by natural selection and is involved in the fitness of the marine snail Littorina saxatilis. Here, we studied the association between shell traits and fitness in hybrids in order to determine the relative role of exogenous and endogenous selection in this hybrid zone of L. saxatilis. We show that directional selection is the predominant mode of selection among hybrids. We also show its heterogeneity, affecting different shell traits, within populations at the level of the microhabitat. Therefore, endogenous selection mechanisms are most probably lacking in this hybrid zone and exogenous barriers (pre- and post-zygotic) are possibly one of the main forces behind the evolution of barriers to gene flow between these ecologically divergent ecotypes. This study shows how this barrier might represent an important type of reproductive isolation within ecological speciation, and this should be taken into account in future studies of speciation in hybrid zones. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, , -. ADDITIONAL KEYWORDS: adaptation - introgression - Littorina - mosaic hybrid zone - natural selection - post-zygotic isolation - premating isolation.

Incipient post‐zygotic barrier in a model system of ecological speciation with gene flow

The role of post-zygotic isolation in nonallopatric ecological speciation is still mostly unknown and information on the nature and strength of these barriers in well-known speciation models is essential for a deeper understanding of such processes. The Galician ecotypes of the marine snail Littorina saxatilis represent one of the best studied cases of nonallopatric ecological speciation. Here, we test the existence of incipient post-zygotic isolation by comparing the fertility of male hybrids with that of both pure forms [ridged and banded (RB) and smooth and unbanded (SU) ecotypes]. We analysed the degree of sperm DNA fragmentation (SDF) of individuals morphologically classified as RB, SU and hybrids, sampled from two locations. SDF analyses were chosen to study sperm quality because, in other animal species, SDF rates correlate with important parameters for speciation research, such as fertilization and abortion rates and viability of adult progeny. In the present work, hybrids showed significantly higher SDF rates than RB and SU males in one location and significantly higher variances in both locations. These results suggest the existence of an incipient post-zygotic barrier, the strength of which may vary across the Galician shore, and highlight the potential of SDF analyses for speciation research.

The Effect of Multiple Paternity on Genetic Diversity of Small Populations during and after Colonisation

Genetic variation within and among populations is influenced by the genetic content of the founders and the migrants following establishment. This is particularly true if populations are small, migration rate low and habitats arranged in a stepping-stone fashion. Under these circumstances the level of multiple paternity is critical since multiply mated females bring more genetic variation into founder groups than single mated females. One such example is the marine snail Littorina saxatilis that during postglacial times has invaded mainland refuge areas and thereafter small islands emerging due to isostatic uplift by occasional rafting of multiply mated females. We modelled effects of varying degrees of multiple paternity on the genetic variation of island populations colonised by the founders spreading from the mainland, by quantifying the population heterozygosity during both the transient colonisation process, and after a steady state (with migration) has been reached. During colonisation, multiple mating by males increased the heterozygosity by in comparison with single paternity, while in the steady state the increase was compared with single paternity. In the steady state the increase of heterozygosity due to multiple paternity is determined by a corresponding increase in effective population size. During colonisation, by contrast, the increase in heterozygosity is larger and it cannot be explained in terms of the effective population size alone. During the steady-state phase bursts of high genetic variation spread through the system, and far from the mainland this led to short periods of high diversity separated by long periods of low diversity. The size of these fluctuations was boosted by multiple paternity. We conclude that following glacial periods of extirpation, recolonization of isolated habitats by this species has been supported by its high level of multiple paternity.

The role of local ecology during hybridization at the initial stages of ecological speciation in a marine snail

Hybrid zones of ecologically divergent populations are ideal systems to study the interaction between natural selection and gene flow during the initial stages of speciation. Here, we perform an amplified fragment length polymorphism (AFLP) genome scan in parallel hybrid zones between divergent ecotypes of the marine snail Littorina saxatilis, which is considered a model case for the study of ecological speciation. Ridged-Banded (RB) and Smooth-Unbanded (SU) ecotypes are adapted to different shore levels and microhabitats, although they present a sympatric distribution at the mid-shore where they meet and mate (partially assortatively). We used shell morphology, outlier and nonoutlier AFLP loci from RB, SU and hybrid specimens captured in sympatry to determine the level of phenotypic and genetic introgression. We found different levels of introgression at parallel hybrid zones and nonoutlier loci showed more gene flow with greater phenotypic introgression. These results were independent from the phylogeography of the studied populations, but not from the local ecological conditions. Genetic variation at outlier loci was highly correlated with phenotypic variation. In addition, we used the relationship between genetic and phenotypic variation to estimate the heritability of morphological traits and to identify potential Quantitative Trait Loci to be confirmed in future crosses. These results suggest that ecology (exogenous selection) plays an important role in this hybrid zone. Thus, ecologically based divergent natural selection is responsible, simultaneously, for both ecotype divergence and hybridization. On the other hand, genetic introgression occurs only at neutral loci (nonoutliers). In the future, genome-wide studies and controlled crosses would give more valuable information about this process of speciation in the face of gene flow.

Proteomic evidence of a paedomorphic evolutionary process within a marine snail species: a strategy for adapting to extreme ecological conditions?

The exposed and sheltered ecotypes of the marine snail Littorina saxatilis from European rocky shores are considered a key model system to study adaptation and ecological speciation. Previous studies showed that two ecotypes (RB and SU) of this species in NW Spain have adapted differently to different shore levels and microhabitats. In order to understand how this divergent adaptive process has been accomplished, we followed a quantitative proteomic approach to investigate the proteome variation in a number of different biological factors, that is, ecotype, ontogeny and their interactions. This approach allowed testing the hypothesis that one of the ecotypes has evolved by paedomorphosis, and also whether or not the molecular mechanisms related to ecotype differentiation are set up in early developmental stages. Additionally, the identification of some candidate proteins using mass spectrometry provides some functional insights into these evolutionary processes. Results from this study provided evidence of higher ontogenetic differentiation at proteome level in the RB (metamorphic) than in SU (paedomorphic) ecotype that point to the possibility of juvenile stage retention in this latter ecotype. The level of protein expression (proteome) differences between ecotypes maintained nearly constant from late embryonic stages to adulthood, although some proteins involved in these changes considerably differed in embryonic compared to other ontogenetic stages. Paedomorphosis may be the evolutionary response of the SU ecotype of solving the trade-off during sexually immaturity that is caused by theevolution of small size arising from adaptation to the wave-exposed habitat. Some potential candidate genes of adaptation related to energetic metabolism have been identified, providing a promising baseline for future functional analyses.