Contrasting patterns of quantitative and neutral genetic variation in locally adapted populations of the natterjack toad, Bufo calamita

The relative importance of natural selection and genetic drift in determining patterns of phenotypic diversity observed in nature is still unclear. The natterjack toad (Bufo calamita) is one of a few amphibian species capable of breeding in saline ponds, even though water salinity represents a considerable stress for them. Results from two common-garden experiments showed a pattern of geographic variation in embryonic salinity tolerance among populations from either fresh or brackish environments, consistent with the hypothesis of local adaptation. Full-sib analysis showed increased variation in survival among sibships within population for all populations as osmotic stress was increased (broad-sense heritability increased as salinity raised). Nevertheless, toads native to the brackish water environment had the highest overall survival under brackish conditions. Levels of population genetic differentiation for salinity tolerance were higher than those of neutral genetic differentiation, the latter obtained through the analysis of eight microsatellite loci. Microsatellite markers also revealed little population differentiation, lack of an isolation-by-distance pattern, and moderate gene flow connecting the populations. Therefore, environmental stress tolerance appears to have evolved in absence of geographic isolation, and consequently we reject the null hypothesis of neutral differentiation.     

Adaptation or exaptation? An experimental test of hypotheses on the origin of salinity tolerance in Bufo calamita

The natterjack toad (Bufo calamita) shows variation in embryonic and larval salinity tolerance across populations in southern Spain. However, its aquatic/terrestrial biphasic life cycle, together with remarkable differences in salinity tolerance between Spanish and UK freshwater populations suggest an alternative hypothesis to local adaptation. Drought resistance during the terrestrial phase and salinity tolerance during the aquatic phase are both related to osmotic stress tolerance, and if there were an association between them, one could have evolved as an exaptation from the other. To test such an association, we reared B. calamita juveniles from three populations known to differ genetically in their salinity tolerance, under either dry or humid conditions. Drought decreased growth rate, enhanced burying behaviour, and decreased foraging activity and efficiency. No significant population x treatment interaction was found for any variable, i.e. populations were equally affected by drought. These results do not support the hypothesis of a genetic association between salinity and drought tolerance.     

Developmental alterations and osmoregulatory physiology of a larval anuran under osmotic stress

Water salinity represents an environmental stress for many species. Amphibians are particularly sensitive because they are generally poor osmoregulators, and most species are completely absent from brackish and saline environments. We experimentally examined the effect of different salinity levels on larvae of the toad Bufo calamita L., a species that occupies freshwater ponds but can also breed in brackish ponds. Two independent experiments are reported here. In both experiments, tadpoles under saline conditions (ranging between 85 and 200 mOsm) showed a slower developmental rate, metamorphosing between 4 and 9 d later than the controls. Bufo calamita tadpoles reared in brackish water increased their osmolality and solute concentration (mainly sodium and chloride), decreased their levels of glucose, and decreased the total protein content, all measured from whole-animal extracts. Although most larval anurans are strictly ammoniotelic until the completion of metamorphosis, a few species exposed to dehydrating environments have evolved the ability to use urea as an osmolyte during the larval phase. The data presented here reveal that although B. calamita seems to be yet another exception to the rule of larval strict ammoniotelism, the tadpoles are not able to use urea as an osmolyte and rely on sodium-chloride balance instead. Preliminary immunoassays of thyroid hormone content suggest a possible decrease in hormone levels induced in water salinity conditions that correlate with a decreased developmental rate.     

Genotype-by-environment interaction for salinity tolerance in the freshwater-invading copepod Eurytemora affinis

This study examined the extent of phenotypic plasticity for salinity tolerance and genetic variation in plasticity in the invasive copepod Eurytemora affinis. Euryemora affinis is a species complex inhabiting brackish to hypersaline environments but has invaded freshwater lakes and reservoirs within the past century. Reaction norm experiments were performed on a relatively euryhaline population collected from a brackish lake with fluctuating salinity. Life history traits (hatching rate, survival, and development time) were measured for 20 full-sib clutches that were split and reared at four salinities (fresh, 5, 10, and 27 practical salinity units [PSU]). On average, higher salinities (10 and 27 PSU) were more favorable for larval growth, yielding greater survival and faster development rate. Clutches differed significantly in their response to salinity, with a significant genotype-by-environment interaction for development time. In addition, genetic (clutch) effects were evident in response to low salinity, given that survival in fresh (lake) water was significantly positively correlated with survival at 5 PSU for individual clutches. Clutches raised in fresh water could not survive beyond metamorphosis, suggesting that acclimation to fresh water could not occur in a single generation. Results suggest the importance of natural selection during freshwater invasion events, given the inability of plasticity to generate a freshwater phenotype, and the presence of genetic variation for plasticity upon which natural selection could act.     

Local adaptation for enhanced salt tolerance reduces non‐adaptive plasticity caused by osmotic stress

Organisms often respond to environmental change via phenotypic plasticity, in which an individual modulates its phenotype according to the environment. Highly variable or changing environments can exceed physiological limits and generate maladapted plastic phenotypes, which is termed nonadaptive plasticity. In some cases, selection may reduce the negative or disruptive impacts of environmental stress and produce locally adapted populations. Salt is an increasingly prevalent contaminant of freshwater systems and can induce nonadaptive plastic phenotypes for freshwater organisms like amphibians. Hyla cinerea is a frog species with populations inhabiting brackish, coastal habitats, so we use this species to test whether coastal populations are locally adapted to tolerate saltwater by determining how salt exposure during the embryonic and larval stages alters mortality and plastic developmental and metamorphic phenotypes of coastal and inland populations. Coastal frogs have higher survival, faster growth rates, and metamorphose sooner than inland frogs across salinities. Coastal frogs also metamorphose smaller (likely a consequence of earlier metamorphosis) yet maintain constant size, while higher salinities reduce metamorphic size for inland frogs. Coastal frogs evolved to minimize nonadaptive and disruptive impacts of saltwater during larval development and accelerate the larval period to reduce time spent in a stressful environment.     

Larval Tolerance to Salinity in Three Species of Australian Anuran: An Indication of Saline Specialisation in Litoria aurea

Recent anthropogenic influences on freshwater habitats are forcing anuran populations to rapidly adapt to high magnitude changes in environmental conditions or face local extinction. We examined the effects of ecologically relevant elevated salinity levels on larval growth, metamorphosis and survival of three species of Australian anuran; the spotted marsh frog (Limnodynastes tasmaniensis), the painted burrowing frog (Neobatrachus sudelli) and the green and golden bell frog (Litoria aurea), in order to better understand the responses of these animals to environmental change. Elevated salinity (16% seawater) negatively impacted on the survival of L. tasmaniensis (35% survival) and N sudelli (0% survival), while reduced salinity had a negative impact on L. aurea. (16% seawater: 85% survival; 0.4% seawater: 35% survival). L. aurea tadpoles survived in salinities much higher than previously reported for this species, indicating the potential for inter-populations differences in salinity tolerance. In L. tasmaniensis and L. aurea, development to metamorphosis was fastest in low and high salinity treatments suggesting it is advantageous for tadpoles to invest energy in development in both highly favourable and developmentally challenging environments. We propose that this response might either maximise potential lifetime fecundity when tadpoles experience favourable environments, or, facilitate a more rapid escape from pond environments where there is a reduced probability of survival.     

Small Changes in Gene Expression of Targeted Osmoregulatory Genes When Exposing Marine and Freshwater Threespine Stickleback (Gasterosteus aculeatus) to Abrupt Salinity Transfers

Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na⁺/K⁺-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.     

A proteomics approach reveals divergent molecular responses to salinity in populations of European whitefish (Coregonus lavaretus)

Osmoregulation is a vital physiological function for fish, as it helps maintain a stable intracellular concentration of ions in environments of variable salinities. We focused on a primarily freshwater species, the European whitefish (Coregonus lavaretus), to investigate the molecular mechanisms underlying salinity tolerance and examine whether these mechanisms differ between genetically similar populations that spawn in freshwater vs. brackishwater environments. A common garden experiment involving 27 families in two populations and five salinity treatments together with a large-scale, high-resolution mass spectrometry experiment that quantified 1500 proteins was conducted to assess phenotypic and proteomic responses during early development, from fertilization until hatching, in the studied populations. The populations displayed drastically different phenotypic and proteomic responses to salinity. Freshwater-spawning whitefish showed a significantly higher mortality rate in higher salinity treatments. Calcium, an ion involved in osmotic stress sensing, had a central role in the observed proteomic responses. Brackishwater-spawning fish were capable of viable osmoregulation, which was modulated by cortisol, an important seawater-adaptation hormone in teleost fish. Several proteins were identified to play key roles in osmoregulation, most importantly a highly conserved cytokine, tumour necrosis factor, whereas calcium receptor activities were associated with salinity adaptation. These results imply that individuals from these populations are most likely adapted to their local environments, even though the baseline level of genetic divergence between them is low (F(ST)=0.049). They also provide clues for choosing candidate loci for studying the molecular basis of salinity adaptation in other species. Further, our approach provides an example of how proteomic methods can be successfully used to obtain novel insights into the molecular mechanisms behind adaptation in non-model organism.     

Effects of reduced salinities on metamorphosis of a freshwater-tolerant sesarmid crab, Armases roberti: Is upstream migration in the megalopa stage constrained by increasing osmotic stress?

Numerous species of estuarine and freshwater-tolerant crabs show an “export strategy”, i.e. an early larval downstream transport towards coastal marine waters, later zoeal development at higher salinities, and a return of the last larval stage, the megalopa, into estuaries or rivers. The speed and extent of the upstream migration of the megalopa through strong salinity gradients may be constrained by increasing hypo-osmotic stress. In an experimental laboratory study with Armases roberti, a freshwater-inhabiting sesarmid crab from the Caribbean region, we studied in the megalopa stage (after zoeal rearing at 25‰) the tolerance of reduced salinities.In the first experiment, the larvae were exposed directly to various constant salinities (1-25‰). For the second experiment, they were transferred stepwise to strongly diluted media (within 6 days from 25‰ to ≤ 3‰), simulating differential scenarios of upstream migration into brackish or freshwater habitats.When postmoult megalopae were exposed directly to salinities ≤ 3‰, they all died within 24 h. A slightly higher salt concentration (5‰), however, allowed for considerable survival (46%) through metamorphosis to the first juvenile crab stage. In treatments with continuous exposure to 10-15‰, as well as in a control group (25‰), survival to metamorphosis was significantly higher (83-96%), and the average duration of development was shorter compared to 5‰ (12-13 vs. 16 days). In the second experiment, with stepwise salinity reductions, gradual acclimation to decreasing osmotic pressures permitted a successful development to metamorphosis at ≤ 3‰ and even in freshwater (< 0.2‰).This strong physiological adaptability enables the megalopa of A. roberti to cross during its upstream migration, within a short time (6 days), strong osmotic gradients, so that metamorphosis is possible also in freshwater habitats where the conspecific adult crabs live. The speed of migration appears to be limited by physiological constraints related to changes in the capability for osmoregulation occurring during the course of the moulting cycle.     

Salinity relationships in a freshwater population of eastern mosquitofish

Tolerance limit to elevated ambient salinity, and plasma osmotic regulatory capabilities were evaluated in a freshwater population of Gambusia holbrooki. The upper tolerance limit of ambient salinity was found to be 25‰. Plasma osmotic concentrations trended sharply upward with increasing salinity, but in a stepwise fashion, unlike patterns in related, but more euryhaline species. These laboratory observations are consistent with field observations that natural populations of G holbrooki are generally restricted to fresh and dilute brackish waters.     

Influence of density and salinity on larval development of salt‐adapted and salt‐naïve frog populations

Environmental change and habitat fragmentation will affect population densities for many species. For those species that have locally adapted to persist in changed or stressful habitats, it is uncertain how density dependence will affect adaptive responses. Anurans (frogs and toads) are typically freshwater organisms, but some coastal populations of green treefrogs (Hyla cinerea) have adapted to brackish, coastal wetlands. Tadpoles from coastal populations metamorphose sooner and demonstrate faster growth rates than inland populations when reared solitarily. Although saltwater exposure has adaptively reduced the duration of the larval period for coastal populations, increases in densities during larval development typically increase time to metamorphosis and reduce rates of growth and survival. We test how combined stressors of density and salinity affect larval development between salt‐adapted (“coastal”) and nonsalt‐adapted (“inland”) populations by measuring various developmental and metamorphic phenotypes. We found that increased tadpole density strongly affected coastal and inland tadpole populations similarly. In high‐density treatments, both coastal and inland populations had reduced growth rates, greater exponential decay of growth, a smaller size at metamorphosis, took longer to reach metamorphosis, and had lower survivorship at metamorphosis. Salinity only exaggerated the effects of density on the time to reach metamorphosis and exponential decay of growth. Location of origin affected length at metamorphosis, with coastal tadpoles metamorphosing slightly longer than inland tadpoles across densities and salinities. These findings confirm that density has a strong and central influence on larval development even across divergent populations and habitat types and may mitigate the expression (and therefore detection) of locally adapted phenotypes.     

Fructans of the saline world

Saline and hypersaline environments make up the largest ecosystem on earth and the organisms living in such water-restricted environments have developed unique ways to cope with high salinity. As such these organisms not only carry significant industrial potential in a world where freshwater supplies are rapidly diminishing, but they also shed light upon the origins and extremes of life. One largely overlooked and potentially important feature of many salt-loving organisms is their ability to produce fructans, fructose polymers widely found in various mesophilic Eubacteria and plants, with potential functions as storage carbohydrates, aiding stress tolerance, and acting as virulence factors or signaling molecules. Intriguingly, within the whole archaeal domain of life, Archaea possessing putative fructan biosynthetic enzymes were found to belong to the extremely halophilic class of Halobacteria only, indicating a strong, yet unexplored link between the fructan syndrome and salinity. In fact, this link may indeed lead to novel strategies in fighting the global salinization problem. Hence this review explores the unknown world of fructanogenic salt-loving organisms, where water scarcity is the main stress factor for life. Within this scope, prokaryotes and plants of the saline world are discussed in detail, with special emphasis on their salt adaptation mechanisms, the potential roles of fructans and fructosyltransferase enzymes in adaptation and survival as well as future aspects for all fructanogenic salt-loving domains of life.     

Survival and behavioral characteristics of amphidromous goby larvae of Sicyopterus japonicus (Tanaka, 1909) during their downstream migration

To understand the ecology and environmental tolerances of newly hatched larvae of the amphidromous fish Sicyopterus japonicus during their downstream migration, the salinity tolerance of eggs, 0-15 day old larvae, and adults, and the temperature tolerance, specific gravity and phototaxis of hatched larvae were examined. Tolerances of adults were measured as survival after a 24 h challenge in freshwater (FW), brackish water (1/3 SW) and seawater (SW). The survival rate of adult S. japonicus was 100% in FW and 1/3 SW, while none survived in SW. Hatching success of eggs (30 eggs each) was significantly higher in FW (mean: 73%) and 1/3 SW (73%) than in SW (19%). Tolerance of newly hatched larvae to salinity and temperature was investigated in different combinations of salinities (FW, 1/3 SW and SW) and temperatures (18, 23 and 28 °C). Larval survival was significantly different in each salinity and temperature. Survival rate was significantly higher in 1/3 SW than in FW and higher in SW than in FW at 23 °C and 28 °C. At the latter part of the experiment, there was no survival in FW and at 28 °C. Survival was higher in lower temperatures, but larval development did not occur in FW. Specific gravity of newly hatched larvae was 1.036 at 28 °C and 1.034 at 23 °C. When exposed to a light source on one side of an aquarium, larval distribution was not affected. Our results indicated larval S. japonicus are more adapted to brackish water and seawater than freshwater, while the adults and eggs are more adapted to freshwater and brackish water than seawater. This is consistent with their amphidromous life history with growth and spawning occurring in freshwater and the larval stage utilizing marine habitats.     

Effects of salinity on the life history and fitness of Daphnia magna: variability within and between populations

The life history traits of Daphnia magna were studied in laboratory experiments under freshwater and brackish (5 salinity) conditions. The variability of responses within and between populations was examined by comparing 11 clones from a brackish lake and 10 clones from a freshwater pond. Experimental clones were hatched from ephippia collected from the sediment and thus represent random samples of the clone banks of each population.Most clones with a high salinity tolerance were from the population of the brackish habitat, but some were also found in the freshwater population. Thus, freshwater populations appear to have the potential to invade brackish habitats. A proportion of clones from the brackish population had very low fitness (measured as e^r) under freshwater conditions. This unexpected result means that freshwater adaptation can be lost by the freshwater cladoceran Daphnia magna. The effects of unfavourable conditions on growth and reproduction varied among clones and were not correlated. This clonal variation in growth and reproduction indicates that the environmental sensitivities of these traits are independent. The pattern of fitness reaction norms showed no trade-off between fitness under brackish and under freshwater conditions for either population. Thus, euryhaline generalists should be favoured in habitats with salinity fluctuations between freshwater and brackish conditions.     

Trade‐offs in osmoregulation and parallel shifts in molecular function follow ecological transitions to freshwater in the Alewife

Adaptation to freshwater may be expected to reduce performance in seawater because these environments represent opposing selective regimes. We tested for such a trade‐off in populations of the Alewife (Alosa pseudoharengus). Alewives are ancestrally anadromous, and multiple populations have been independently restricted to freshwater (landlocked). We conducted salinity challenge experiments, whereby juvenile Alewives from one anadromous and multiple landlocked populations were exposed to freshwater and seawater on acute and acclimation timescales. In response to acute salinity challenge trials, independently derived landlocked populations varied in the degree to which seawater tolerance has been lost. In laboratory‐acclimation experiments, landlocked Alewives exhibited improved freshwater tolerance, which was correlated with reductions in seawater tolerance and hypo‐osmotic balance, suggesting that trade‐offs in osmoregulation may be associated with local adaptation to freshwater. We detected differentiation between life‐history forms in the expression of an ion‐uptake gene (NHE3), and in gill Na⁺/K⁺‐ATPase activity. Trade‐offs in osmoregulation, therefore, may be mediated by differentiation in ion‐uptake and salt‐secreting pathways.     

Testing for beneficial reversal of dominance during salinity shifts in the invasive copepod Eurytemora affinis,and implications for the maintenance of genetic variation

Maintenance of genetic variation at loci under selection has profound implications for adaptation under environmental change. In temporally and spatially varying habitats, non‐neutral polymorphism could be maintained by heterozygote advantage across environments (marginal overdominance), which could be greatly increased by beneficial reversal of dominance across conditions. We tested for reversal of dominance and marginal overdominance in salinity tolerance in the saltwater‐to‐freshwater invading copepod Eurytemora affinis. We compared survival of F1 offspring generated by crossing saline and freshwater inbred lines (between‐salinity F1 crosses) relative to within‐salinity F1 crosses, across three salinities. We found evidence for both beneficial reversal of dominance and marginal overdominance in salinity tolerance. In support of reversal of dominance, survival of between‐salinity F1 crosses was not different from that of freshwater F1 crosses under freshwater conditions and saltwater F1 crosses under saltwater conditions. In support of marginal overdominance, between‐salinity F1 crosses exhibited significantly higher survival across salinities relative to both freshwater and saltwater F1 crosses. Our study provides a rare empirical example of complete beneficial reversal of dominance associated with environmental change. This mechanism might be crucial for maintaining genetic variation in salinity tolerance in E. affinis populations, allowing rapid adaptation to salinity changes during habitat invasions.     

Growth pattern and survival in populations of Poecilia vivipara (Teleostei; Poeciliidae) inhabiting an environmental gradient: a common garden study

We performed a common garden experiment to assess the existence of genetic differences on growth and body size between two populations of Poecilia vivipara inhabiting extremes of an environmental gradient caused by water salinity in lagoons of Northern Rio de Janeiro State, Brazil: the Campelo lagoon (freshwater) and Açu lagoon (brackish/saltwater). The two populations show extreme differences in average phenotypes for body size, shape and life history (freshwater populations with smaller body size, lower fecundity and larger reproductive allotment). Pregnant females were brought to the lab and the offspring from both groups were kept in a common recirculating system with freshwater. Standard length and survival were measured weekly over a period of 200 days and growth models were fitted and selected with information criteria. The offspring originally from the brackish water lagoon presented larger asymptotic length, higher maximum growth rate but lower survival than the offspring originally from the freshwater lagoon. Potential confounding variables such as density differences due to mortality and maternal effects (offspring size) were included as covariates in comparisons of growth rates between groups. The results are consistent with phenotypic differences among populations having some genetic basis, and with the existence of a trade-off between growth and maintenance due to the high growth/low survival observed in the group that changed from salt to freshwater. Comparisons of captive and natural populations suggest that the influence of environmental factors, such as salinity, food availability, fish density and predation should also be considered relevant to explain phenotypic variation in this system.     

Effect of saline water on early success of amphidromous fish

Organisms that migrate between rivers and the sea inevitably pass through estuarine habitats. Despite the potential importance of salinity and temperature fluctuations for metabolic adaptation, little is known about the impact of environmental changes in estuaries on the survival of residents. Ayu (Plecoglossus altivelis) is a migratory fish that inhabits estuarine brackish water in its early life stages. The recent decline in the abundance of populations ascending into rivers is of concern for local biodiversity. The present study aims to elucidate the ecological processes that determine the early success of Ayu larvae under variable environmental conditions. The effects of salinity and water temperature on the endogenous growth of newly hatched larvae from the same brood were examined experimentally based on morphological and metabolic characteristics. High salinity and high water temperature together appeared to require more energy for larval osmoregulation, resulting in the acceleration of yolk depletion and reduced growth of the notochord. Increasing the osmoregulative cost during the yolk-sac stage resulted in the faster induction of a state of starvation. Seawater is considered to have an adverse effect on the survival of newly hatched larvae, as it lowers the efficiency of foraging and predator avoidance due to an energetic tradeoff. More attention should be paid to the significance of estuarine environments involving brackish waters to ensure the early survival of amphidromous fish such as Ayu.     

Metamorphosis of a sesarmid river crab, Armases roberti: stimulation by adult odours versus inhibition by salinity stress

In an experimental laboratory study with the megalopa stage of Armases roberti, a freshwater-inhabiting species of crab from the Caribbean region, we evaluated the combined, potentially antagonistic effects of odours from conspecific adults and of stepwise salinity reductions (simulating upstream migration, reaching within 1 week conditions of 2‰ or freshwater). Neither of these treatments affected the rate of survival, but the duration of development to metamorphosis was significantly (by about 25%) shortened, when odours from conspecific adult crabs were present, regardless of the salinity conditions. Our results indicate that the metamorphosis-stimulating effect of chemical cues from an adult population of A. roberti is far stronger than the potentially retarding effect of increasing hypoosmotic stress. This suggests that the final phase of larval development, including the processes of settlement and metamorphosis, occurs in this species in freshwater habitats, where conspecific populations live.