Fine scale diversity in the lava: genetic and phenotypic diversity in small populations of Arctic charr Salvelinus alpinus.

BACKGROUND: A major goal in evolutionary biology is to understand the processes underlying phenotypic variation in nature. Commonly, studies have focused on large interconnected populations or populations found along strong environmental gradients. However, studies on small fragmented populations can give strong insight into evolutionary processes in relation to discrete ecological factors. Evolution in small populations is believed to be dominated by stochastic processes, but recent work shows that small populations can also display adaptive phenotypic variation, through for example plasticity and rapid adaptive evolution. Such evolution takes place even though there are strong signs of historical bottlenecks and genetic drift. Here we studied 24 small populations of the freshwater fish Arctic charr (Salvelinus alpinus) found in groundwater filled lava caves. Those populations were found within a few km2-area with no apparent water connections between them. We studied the relative contribution of neutral versus non-neutral evolutionary processes in shaping phenotypic divergence, by contrasting patterns of phenotypic and neutral genetic divergence across populations in relation to environmental measurements. This allowed us to model the proportion of phenotypic variance explained by the environment, taking in to account the observed neutral genetic structure. RESULTS: These populations originated from the nearby Lake Mývatn, and showed small population sizes with low genetic diversity. Phenotypic variation was mostly correlated with neutral genetic diversity with only a small environmental effect. CONCLUSIONS: Phenotypic diversity in these cave populations appears to be largely the product of neutral processes, fitting the classical evolutionary expectations. However, the fact that neutral processes did not explain fully the phenotypic patterns suggests that further studies can increase our understanding on how neutral evolutionary processes can interact with other forces of selection at early stages of divergence. The accessibility of these populations has provided the opportunity for long-term monitoring of individual fish, allowing tracking how the environment can influence phenotypic and genetic divergence for shaping and maintaining diversity in small populations. Such studies are important, especially in freshwater, as habitat alteration is commonly breaking populations into smaller units, which may or may not be viable.

Effects of anthropogenic stress on hosts and their microbiomes: Treated wastewater alters performance and gut microbiome of a key detritivore (Asellus aquaticus).

Human activity is a major driver of ecological and evolutionary change in wild populations and can have diverse effects on eukaryotic organisms as well as on environmental and host-associated microbial communities. Although host-microbiome interactions can be a major determinant of host fitness, few studies consider the joint responses of hosts and their microbiomes to anthropogenic changes. In freshwater ecosystems, wastewater is a widespread anthropogenic stressor that represents a multifarious environmental perturbation. Here, we experimentally tested the impact of treated wastewater on a keystone host (the freshwater isopod Asellus aquaticus) and its gut microbiome. We used a semi-natural flume experiment, in combination with 16S rRNA amplicon sequencing, to assess how different concentrations (0%, 30%, and 80%) of nonfiltered wastewater (i.e. with chemical toxicants, nutrients, organic particles, and microbes) versus ultrafiltered wastewater (i.e. only dissolved pollutants and nutrients) affected host survival, growth, and food consumption as well as mid- and hindgut bacterial community composition and diversity. Our results show that while host survival was not affected by the treatments, host growth increased and host feeding rate decreased with nonfiltered wastewater - potentially indicating that A. aquaticus fed on organic matter and microbes available in nonfiltered wastewater. Furthermore, even though the midgut microbiome (diversity and composition) was not affected by any of our treatments, nonfiltered wastewater influenced bacterial composition (but not diversity) in the hindgut. Ultrafiltered wastewater, on the other hand, affected both community composition and bacterial diversity in the hindgut, an effect that in our system differed between sexes. While the functional consequences of microbiome changes and their sex specificity are yet to be tested, our results indicate that different components of multifactorial stressors (i.e. different constituents of wastewater) can affect hosts and their microbiome in distinct (even opposing) manners and have a substantial impact on eco-evolutionary responses to anthropogenic stressors.

Demographic Basis of Spatially Structured Fluctuations in a Threespine Stickleback Metapopulation.

AbstractUncovering the demographic basis of population fluctuations is a central goal of population biology. This is particularly challenging for spatially structured populations, which require disentangling synchrony in demographic rates from coupling via movement between locations. In this study, we fit a stage-structured metapopulation model to a 29-year time series of threespine stickleback abundance in the heterogeneous and productive Lake Mývatn, Iceland. The lake comprises two basins (North and South) connected by a channel through which the stickleback disperse. The model includes time-varying demographic rates, allowing us to assess the potential contributions of recruitment and survival, spatial coupling via movement, and demographic transience to the population's large fluctuations in abundance. Our analyses indicate that recruitment was only modestly synchronized between the two basins, whereas survival probabilities of adults were more strongly synchronized, contributing to cyclic fluctuations in the lake-wide population size with a period of approximately 6 years. The analyses further show that the two basins were coupled through movement, with the North Basin subsidizing the South Basin and playing a dominant role in driving the lake-wide dynamics. Our results show that cyclic fluctuations of a metapopulation can be explained in terms of the combined effects of synchronized demographic rates and spatial coupling.

Spatial variation in the evolutionary potential and constraints of basal metabolic rate and body mass in a wild bird.

An organism's energy budget is strongly related to resource consumption, performance, and fitness. Hence, understanding the evolution of key energetic traits, such as basal metabolic rate (BMR), in natural populations is central for understanding life-history evolution and ecological processes. Here we used quantitative genetic analyses to study evolutionary potential of BMR in two insular populations of the house sparrow (Passer domesticus). We obtained measurements of BMR and body mass (Mb ) from 911 house sparrows on the islands of Leka and Vega along the coast of Norway. These two populations were the source populations for translocations to create an additional third, admixed 'common garden' population in 2012. With the use of a novel genetic group animal model concomitant with a genetically determined pedigree, we differentiate genetic and environmental sources of variation, thereby providing insight into the effects of spatial population structure on evolutionary potential. We found that the evolutionary potential of BMR was similar in the two source populations, whereas the Vega population had a somewhat higher evolutionary potential of Mb than the Leka population. BMR was genetically correlated with Mb in both populations, and the conditional evolutionary potential of BMR (independent of body mass) was 41% (Leka) and 53% (Vega) lower than unconditional estimates. Overall, our results show that there is potential for BMR to evolve independently of Mb , but that selection on BMR and/or Mb may have different evolutionary consequences in different populations of the same species.

Genome-phenotype-environment associations identify signatures of selection in a panmictic population of threespine stickleback.

Adaptive genetic divergence occurs when selection imposed by the environment causes the genomic component of the phenotype to differentiate. However, genomic signatures of natural selection are usually identified without information on which trait is responding to selection by which selective agent(s). Here, we integrate whole-genome sequencing with phenomics and measures of putative selective agents to assess the extent of adaptive divergence in threespine stickleback occupying the highly heterogeneous lake Mývatn, NE Iceland. We find negligible genome wide divergence, yet multiple traits (body size, gill raker structure and defence traits) were divergent along known ecological gradients (temperature, predatory bird densities and water depth). SNP based heritability of all measured traits was high (h2  = 0.42-0.65), indicating adaptive potential for all traits. Environment-association analyses further identified thousands of loci putatively involved in selection, related to genes linked to, for instance, neuron development and protein phosphorylation. Finally, we found that loci linked to water depth were concurrently associated with pelvic spine length variation - supporting the conclusion that divergence in pelvic spine length occurred in the face of gene flow. Our results suggest that whilst there is substantial genetic variation in the traits measured, phenotypic divergence of Mývatn stickleback is mostly weakly associated with environmental gradients, potentially as a result of substantial gene flow. Our study illustrates the value of integrative studies that combine genomic assays of multivariate trait variation with landscape genomics.

Variation in egg size and offspring phenotype among and within seven Arctic charr morphs.

Maternal effects have the potential to alter early developmental processes of offspring and contribute to adaptive diversification. Egg size is a major contributor to offspring phenotype, which can influence developmental trajectories and potential resource use. However, to what extent intraspecific variation in egg size facilitates evolution of resource polymorphism is poorly understood. We studied multiple resource morphs of Icelandic Arctic charr, ranging from an anadromous morph-with a phenotype similar to the proposed ancestral phenotype-to sympatric morphs that vary in their degree of phenotypic divergence from the ancestral anadromous morph. We characterized variation in egg size and tested whether egg size influenced offspring phenotype at early life stages (i.e., timing of- and size at- hatching and first feeding [FF]). We predicted that egg size would differ among morphs and be less variable as morphs diverge away from the ancestral anadromous phenotype. We also predicted that egg size would correlate with offspring size and developmental timing. We found morphs had different egg size, developmental timing, and size at hatching and FF. Egg size increased as phenotypic proximity to the ancestral anadromous phenotype decreased, with larger eggs generally giving rise to larger offspring, especially at FF, but egg size had no effect on developmental rate. The interaction between egg size and the environment may have a profound impact on offspring fitness, where the resulting differences in early life-history traits may act to initiate and/or maintain resource morphs diversification.

Short-term responses of Rana arvalis tadpoles to pH and predator stress: adaptive divergence in behavioural and physiological plasticity?

Environmental stress is a major driver of ecological and evolutionary processes in nature. To cope with stress, organisms can adjust through phenotypic plasticity and/or adapt through genetic change. Here, we compared short-term behavioural (activity) and physiological (corticosterone levels, CORT) responses of Rana arvalis tadpoles from two divergent populations (acid origin, AOP, versus neutral origin, NOP) to acid and predator stress. Tadpoles were initially reared in benign conditions at pH 7 and then exposed to a combination of two pH (acid versus neutral) and two predator cue (predator cue versus no predator cue) treatments. We assessed behavioural activity within the first 15 min, and tissue CORT within 8 and 24 h of stress exposure. Both AOP and NOP tadpoles reduced their activity in acidic pH, but the response to the predator cue differed between the populations: AOP tadpoles increased whereas NOP tadpoles decreased their activity. The AOP and NOP tadpoles differed also in their CORT responses, with AOP being more responsive (CORT levels of NOP tadpoles did not differ statistically across treatments). After 8 h exposure, AOP tadpoles had elevated CORT levels in the acid-predator cue treatment and after 24 h exposure they had elevated CORT levels in all three stress treatments (relative to the benign neutral-no-cue treatment). These results suggest that adaptation to environmental acidification in R. arvalis is mediated, in part, via behavioural and hormonal plasticity.

Context dependent variation in corticosterone and phenotypic divergence of Rana arvalis populations along an acidification gradient.

BACKGROUND: Physiological processes, as immediate responses to the environment, are important mechanisms of phenotypic plasticity and can influence evolution at ecological time scales. In stressful environments, physiological stress responses of individuals are initiated and integrated via the release of hormones, such as corticosterone (CORT). In vertebrates, CORT influences energy metabolism and resource allocation to multiple fitness traits (e.g. growth and morphology) and can be an important mediator of rapid adaptation to environmental stress, such as acidification. The moor frog, Rana arvalis, shows adaptive divergence in larval life-histories and predator defense traits along an acidification gradient in Sweden. Here we take a first step to understanding the role of CORT in this adaptive divergence. We conducted a fully factorial laboratory experiment and reared tadpoles from three populations (one acidic, one neutral and one intermediate pH origin) in two pH treatments (Acid versus Neutral pH) from hatching to metamorphosis. We tested how the populations differ in tadpole CORT profiles and how CORT is associated with tadpole life-history and morphological traits. RESULTS: We found clear differences among the populations in CORT profiles across different developmental stages, but only weak effects of pH treatment on CORT. Tadpoles from the acid origin population had, on average, lower CORT levels than tadpoles from the neutral origin population, and the intermediate pH origin population had intermediate CORT levels. Overall, tadpoles with higher CORT levels developed faster and had shorter and shallower tails, as well as shallower tail muscles. CONCLUSIONS: Our common garden results indicate among population divergence in CORT levels, likely reflecting acidification mediated divergent selection on tadpole physiology, concomitant to selection on larval life-histories and morphology. However, CORT levels were highly environmental context dependent. Jointly these results indicate a potential role for CORT as a mediator of multi-trait divergence along environmental stress gradients in natural populations. At the same time, the population level differences and high context dependency in CORT levels suggest that snapshot assessment of CORT in nature may not be reliable bioindicators of stress.

Wastewater constituents impact biofilm microbial community in receiving streams.

Microbial life in natural biofilms is dominated by prokaryotes and microscopic eukaryotes living in dense association. In stream ecosystems, microbial biofilms influence primary production, elemental cycles, food web interactions as well as water quality. Understanding how biofilm communities respond to anthropogenic impacts, such as wastewater treatment plant (WWTP) effluent, is important given the key role of biofilms in stream ecosystem function. Here, we implemented 16S and 18S rRNA gene sequencing of stream biofilms upstream (US) and downstream (DS) of WWTP effluents in four Swiss streams to test how bacterial and eukaryotic communities respond to wastewater constituents. Stream biofilm composition was strongly affected by geographic location - particularly for bacteria. However, the abundance of certain microbial community members was related to micropollutants in the wastewater - among bacteria, micropollutant-associated members were found e.g. in Alphaproteobacteria, and among eukaryotes e.g. in Bacillariophyta (algal diatoms). This study corroborates several previously characterized responses (e.g. as seen in diatoms), but also reveals previously unknown community responses - such as seen in Alphaproteobacteria. This study advances our understanding of the ecological impact of the current wastewater treatment practices and provides information about potential new marker organisms to assess ecological change in stream biofilms.

Differences among families in craniofacial shape at early life-stages of Arctic charr (Salvelinus alpinus).

BACKGROUND: Organismal fitness can be determined at early life-stages, but phenotypic variation at early life-stages is rarely considered in studies on evolutionary diversification. The trophic apparatus has been shown to contribute to sympatric resource-mediated divergence in several taxa. However, processes underlying diversification in trophic traits are poorly understood. Using phenotypically variable Icelandic Arctic charr (Salvelinus alpinus), we reared offspring from multiple families under standardized laboratory conditions and tested to what extent family (i.e. direct genetic and maternal effects) contributes to offspring morphology at hatching (H) and first feeding (FF). To understand the underlying mechanisms behind early life-stage variation in morphology, we examined how craniofacial shape varied according to family, offspring size, egg size and candidate gene expression. RESULTS: Craniofacial shape (i.e. the Meckel's cartilage and hyoid arch) was more variable between families than within families both across and within developmental stages. Differences in craniofacial morphology between developmental stages correlated with offspring size, whilst within developmental stages only shape at FF correlated with offspring size, as well as female mean egg size. Larger offspring and offspring from females with larger eggs consistently had a wider hyoid arch and contracted Meckel's cartilage in comparison to smaller offspring. CONCLUSIONS: This study provides evidence for family-level variation in early life-stage trophic morphology, indicating the potential for parental effects to facilitate resource polymorphism.

Stream microbial communities and ecosystem functioning show complex responses to multiple stressors in wastewater.

Multiple anthropogenic drivers are changing ecosystems globally, with a disproportionate and intensifying impact on freshwater habitats. A major impact of urbanization are inputs from wastewater treatment plants (WWTPs). Initially designed to reduce eutrophication and improve water quality, WWTPs increasingly release a multitude of micropollutants (MPs; i.e., synthetic chemicals) and microbes (including antibiotic-resistant bacteria) to receiving environments. This pollution may have pervasive impacts on biodiversity and ecosystem services. Viewed through multiple lenses of macroecological and ecotoxicological theory, we combined field, flume, and laboratory experiments to determine the effects of wastewater (WW) on microbial communities and organic-matter processing using a standardized decomposition assay. First, we conducted a mensurative experiment sampling 60 locations above and below WWTP discharges in 20 Swiss streams. Microbial respiration and decomposition rates were positively influenced by WW inputs via warming and nutrient enrichment, but with a notable exception: WW decreased the activation energy of decomposition, indicating a "slowing" of this fundamental ecosystem process in response to temperature. Second, next-generation sequencing indicated that microbial community structure below WWTPs was altered, with significant compositional turnover, reduced richness, and evidence of negative MP influences. Third, a series of flume experiments confirmed that although diluted WW generally has positive influences on microbial-mediated processes, the negative effects of MPs are "masked" by nutrient enrichment. Finally, transplant experiments suggested that WW-borne microbes enhance decomposition rates. Taken together, our results affirm the multiple stressor paradigm by showing that different aspects of WW (warming, nutrients, microbes, and MPs) jointly influence ecosystem functioning in complex ways. Increased respiration rates below WWTPs potentially generate ecosystem "disservices" via greater carbon evasion from streams and rivers. However, toxic MP effects may fundamentally alter ecological scaling relationships, indicating the need for a rapprochement between ecotoxicological and macroecological perspectives.

On biological evolution and environmental solutions.

Drawing insights from multiple disciplines is essential for finding integrative solutions that are required to tackle complex environmental problems. Human activities are causing unprecedented influence on global ecosystems, culminating in the loss of species and fundamental changes in the selective environments of organisms across the tree of life. Our collective understanding about biological evolution can help identify and mitigate many of the environmental problems in the Anthropocene. To this end, we propose a stronger integration of environmental sciences with evolutionary biology.

Drift versus selection as drivers of phenotypic divergence at small spatial scales: The case of Belgjarskógur threespine stickleback.

Divergence in phenotypic traits is facilitated by a combination of natural selection, phenotypic plasticity, gene flow, and genetic drift, whereby the role of drift is expected to be particularly important in small and isolated populations. Separating the components of phenotypic divergence is notoriously difficult, particularly for multivariate phenotypes. Here, we assessed phenotypic divergence of threespine stickleback (Gasterosteus aculeatus) across 19 semi-interconnected ponds within a small geographic region (~7.5 km2) using comparisons of multivariate phenotypic divergence (PST), neutral genetic (FST), and environmental (EST) variation. We found phenotypic divergence across the ponds in a suite of functionally relevant phenotypic traits, including feeding, defense, and swimming traits, and body shape (geometric morphometric). Comparisons of PSTs with FSTs suggest that phenotypic divergence is predominantly driven by neutral processes or stabilizing selection, whereas phenotypic divergence in defensive traits is in accordance with divergent selection. Comparisons of population pairwise PSTs with ESTs suggest that phenotypic divergence in swimming traits is correlated with prey availability, whereas there were no clear associations between phenotypic divergence and environmental difference in the other phenotypic groups. Overall, our results suggest that phenotypic divergence of these small populations at small geographic scales is largely driven by neutral processes (gene flow, drift), although environmental determinants (natural selection or phenotypic plasticity) may play a role.

A way forward with eco evo devo: an extended theory of resource polymorphism with postglacial fishes as model systems.

A major goal of evolutionary science is to understand how biological diversity is generated and altered. Despite considerable advances, we still have limited insight into how phenotypic variation arises and is sorted by natural selection. Here we argue that an integrated view, which merges ecology, evolution and developmental biology (eco evo devo) on an equal footing, is needed to understand the multifaceted role of the environment in simultaneously determining the development of the phenotype and the nature of the selective environment, and how organisms in turn affect the environment through eco evo and eco devo feedbacks. To illustrate the usefulness of an integrated eco evo devo perspective, we connect it with the theory of resource polymorphism (i.e. the phenotypic and genetic diversification that occurs in response to variation in available resources). In so doing, we highlight fishes from recently glaciated freshwater systems as exceptionally well-suited model systems for testing predictions of an eco evo devo framework in studies of diversification. Studies on these fishes show that intraspecific diversity can evolve rapidly, and that this process is jointly facilitated by (i) the availability of diverse environments promoting divergent natural selection; (ii) dynamic developmental processes sensitive to environmental and genetic signals; and (iii) eco evo and eco devo feedbacks influencing the selective and developmental environments of the phenotype. We highlight empirical examples and present a conceptual model for the generation of resource polymorphism - emphasizing eco evo devo, and identify current gaps in knowledge.

Gene expression in the phenotypically plastic Arctic charr (Salvelinus alpinus): A focus on growth and ossification at early stages of development.

Gene expression during development shapes the phenotypes of individuals. Although embryonic gene expression can have lasting effects on developmental trajectories, few studies consider the role of maternal effects, such as egg size, on gene expression. Using qPCR, we characterize relative expression of 14 growth and/or skeletal promoting genes across embryonic development in Arctic charr (Salvelinus alpinus). We test to what extent their relative expression is correlated with egg size and size at early life-stages within the study population. We predict smaller individuals to have higher expression of growth and skeletal promoting genes, due to less maternal resources (i.e., yolk) and prioritization of energy toward ossification. We found expression levels to vary across developmental stages and only three genes (Mmp9, Star, and Sgk1) correlated with individual size at a given developmental stage. Contrary to our hypothesis, expression of Mmp9 and Star showed a non-linear relationship with size (at post fertilization and hatching, respectively), whilst Sgk1 was higher in larger embryos at hatching. Interestingly, these genes are also associated with craniofacial divergence of Arctic charr morphs. Our results indicate that early life-stage variation in gene expression, concomitant to maternal effects, can influence developmental plasticity and potentially the evolution of resource polymorphism in fishes.

De novo oviduct transcriptome of the moor frog Rana arvalis: a quest for maternal effect candidate genes.

Maternal effects can substantially affect ecological and evolutionary processes in natural populations. However, as they often are environmentally induced, establishing their genetic basis is challenging. One important, but largely neglected, source of maternal effects are egg coats (i.e., the maternally derived extracellular matrix that surrounds the embryo). In the moor frog, the gelatinous egg coats (i.e., egg jelly) are produced in the mother's oviduct and consist primarily of highly glycosylated mucin type O-glycans. These O-glycans affect jelly water balance and, subsequently, contribute to adaptive divergence in embryonic acid tolerance. To identify candidate genes for maternal effects, we conducted RNAseq transcriptomics on oviduct samples from seven R. arvalis females, representing the full range of within and among population variation in embryonic acid stress tolerance across our study populations. De novo sequencing of these oviduct transcriptomes detected 124,071 unigenes and functional annotation analyses identified a total of 57,839 unigenes, of which several identified genes likely code for variation in egg jelly coats. These belonged to two main groups: mucin type core protein genes and five different types of glycosylation genes. We further predict 26,711 gene-linked microsatellite (simple sequence repeats) and 231,274 single nucleotide polymorphisms. Our study provides the first set of genomic resources for R. arvalis, an emerging model system for the study of ecology and evolution in natural populations, and gives insight into the genetic architecture of egg coat mediated maternal effects.

What genomic data can reveal about eco-evolutionary dynamics.

Recognition that evolution operates on the same timescale as ecological processes has motivated growing interest in eco-evolutionary dynamics. Nonetheless, generating sufficient data to test predictions about eco-evolutionary dynamics has proved challenging, particularly in natural contexts. Here we argue that genomic data can be integrated into the study of eco-evolutionary dynamics in ways that deepen our understanding of the interplay between ecology and evolution. Specifically, we outline five major questions in the study of eco-evolutionary dynamics for which genomic data may provide answers. Although genomic data alone will not be sufficient to resolve these challenges, integrating genomic data can provide a more mechanistic understanding of the causes of phenotypic change, help elucidate the mechanisms driving eco-evolutionary dynamics, and lead to more accurate evolutionary predictions of eco-evolutionary dynamics in nature.

Cryptic lineages of a common alpine mayfly show strong life-history divergence.

Understanding ecological divergence of morphologically similar but genetically distinct species - previously considered as a single morphospecies - is of key importance in evolutionary ecology and conservation biology. Despite their morphological similarity, cryptic species may have evolved distinct adaptations. If such ecological divergence is unaccounted for, any predictions about their responses to environmental change and biodiversity loss may be biased. We used spatio-temporally replicated field surveys of larval cohort structure and population genetic analyses (using nuclear microsatellite markers) to test for life-history divergence between two cryptic lineages of the alpine mayfly Baetis alpinus in the Swiss Alps. We found that the more widespread and abundant cryptic lineage represents a 'generalist' with at least two cohorts per year, whereas the less abundant lineage is restricted to higher elevations and represents a 'specialist' with a single cohort per year. Importantly, our results indicate partial temporal segregation in reproductive periods between these lineages, potentially facilitating local coexistence and reproductive isolation. Taken together, our findings emphasize the need for a taxonomic revision: widespread and apparently generalist morphospecies can hide cryptic lineages with much narrower ecological niches and distribution ranges.

Molecular phenotyping of maternally mediated parallel adaptive divergence within Rana arvalis and Rana temporaria.

When similar selection acts on the same traits in multiple species or populations, parallel evolution can result in similar phenotypic changes, yet the underlying molecular architecture of parallel phenotypic divergence can be variable. Maternal effects can influence evolution at ecological timescales and facilitate local adaptation, but their contribution to parallel adaptive divergence is unclear. In this study, we (i) tested for variation in embryonic acid tolerance in a common garden experiment and (ii) used molecular phenotyping of egg coats to investigate the molecular basis of maternally mediated parallel adaptive divergence in two amphibian species (Rana arvalis and Rana temporaria). Our results on three R. arvalis and two R. temporaria populations show that adaptive divergence in embryonic acid tolerance is mediated via maternally derived egg coats in both species. We find extensive polymorphism in egg jelly coat glycoproteins within both species and that acid-tolerant clutches have more negatively charged egg jelly - indicating that the glycosylation status of the jelly coat proteins is under divergent selection in acidified environments, likely due to its impact on jelly water balance. Overall, these data provide evidence for parallel mechanisms of adaptive divergence in two species. Our study highlights the importance of studying intraspecific molecular variation in egg coats and, specifically, their glycoproteins, to increase understanding of underlying forces maintaining variation in jelly coats.