Compensatory shell thickening in corrosive environments varies between related rocky-shore and estuarine gastropods.

Few studies have considered the capabilities of gastropods living in minerally-deficient acidified coastal waters to compensate for outer shell corrosion or compromised growing edge shell production. We compared inner shell thickening between pristine shells (control) and corroded shells (experiment) of two related intertidal neritid gastropod species from reduced salinity and acidified environments. We predicted that the rocky-shore, Nerita chamaeleon, which has greater access to shell building biomineralization substrates, should better control shell thickness than the estuarine, Neripteron violaceum. Accordingly, N. chameleon was found to compensate perfectly for variation in the thickness of the outer calcitic blocky layer (BL). Optimal shell thickness (OST) was maintained by selective reabsorption of the aperture ridge of the distal shell (aragonitic crossed-lamellar layer, CL) and by increased internal deposition of proximal (older) shell (aragonitic protocrossed lamellar, PCL). Despite greater exposure to acidification and hyposalinity, N. violaceum showed no significant compensatory shell thickening. These findings reveal that shell thickening capability may vary greatly among intertidal gastropods and that this may be constrained by environmental biomineralization substrate availability. Such environmentally-related responses carry implications for predicted future reductions in coastal water pH and salinity.

Quantification, characterization and risk assessment of microplastics from five major estuaries along the northern Bay of Bengal coast.

Microplastics (MPs) as hazardous contaminants has drawn the rapid attention of the general public due to their omnipresence and adverse impacts on ecosystems and human health. Despite this, understanding of MPs contamination levels in the estuarine ecosystems along the Bay of Bengal coast remains very limited. This research focused on the presence, spatial distribution, morpho-chemical characteristics and ecological implications of MPs in water and sediment from five key estuaries (Meghna, Karnaphuli, Matamuhuri, Bakkhali, and Naf rivers) within the Bengal delta. Out of the five estuaries, the Meghna exhibited the least amount of MPs in both surface water (150.00 ± 65.62 items/m3) and sediment (30.56 ± 9.34 items/kg). In contrast, the highest occurrence of MPs was recorded in Karnaphuli river water (350.00 ± 69.22 items/m3) and Matamuhuri river sediment (118.33 ± 26.81 items/kg). ANOVA indicated a statistically significant distinction (p < 0.01) among the examined estuaries. Most identified MPs were fibers and < 0.5 mm in size in both water and sediment samples. Transparent MPs were dominant in both water (42.28%) and sediment (45.22%). Besides violet, red, blue, pink and green colored MPs were also observed. Various polymer types, including PE, PP, PET, PS, Nylon, EVA, and ABS, were detected, with PE being the dominant one. Based on the polymer risk index (PHI), the estuaries were classified as hazard level V, signifying a severe level of MP contamination. However, the potential ecological hazardous index (PHI), potential ecological risk index (RI), and pollution load index (PLI) indicated moderate pollution levels. This study offers initial insights into the pollution caused by MPs in major estuaries of Bengal delta, which policymakers can utilize to implement suitable management strategies.

Organismal Responses to Coastal Acidification Informed by Interrelating Erosion, Roundness and Growth of Gastropod Shells.

urrent understanding of how calcifying organisms respond to externally forced oceanic and coastal acidification (OCA) is largely based on short-term, controlled laboratory or mesocosm experiments. Studies on organismal responses to acidification (reduced carbonate saturation and pH) in the wild, where animals simultaneously interact with a range of biotic and abiotic circumstances, are limited in scope and interpretation. The present study aimed to better understand how gastropod shell attributes and their interrelations can inform about responses to coastal acidification. We investigated shell chemical erosion, shell roundness, and growth rate of Planaxis sulcatus snails, which are locally exposed to acidified and non-acidified rocky intertidal water. We tested a new approach to quantifying shell erosion based on the spiral suture length (EI, erosion index) and found that shell erosion mirrored field acidification conditions. Exposure to acidification caused shells to become rounder (width/length). Field growth rate, determined from apertural margin extension of marked and later recaptured snails, was strongly negatively related to both shell erosion and shell roundness. Since different shell attributes are indicative of different relationships-shell erosion is an extrinsic passive marker of acidification, and shell roundness and growth rate are intrinsic performance responders-analyzing their interrelations can imply causation, enhance predictive power, and bolster interpretation confidence. This study contributes to the methodology and interpretation of findings of trait-based field investigations to understand organismal responses to coastal acidification.

Conservation of thermal physiology in tropical intertidal snails following an evolutionary transition to a cooler ecosystem: climate change implications.

Predictions for animal responses to climate warming usually assume that thermal physiology is adapted to present-day environments, and seldom consider the influence of evolutionary background. Little is known about the conservation of warm-adapted physiology following an evolutionary transition to a cooler environment. We used cardiac thermal performance curves (cTPCs) of six neritid gastropod species to study physiological thermal trait variation associated with a lineage transition from warmer rocky shores to cooler mangroves. We distinguished between functional thermal performance traits, related to energy homeostasis (slope gradient, slope curvature, HRmax, maximum cardiac activity and Topt, the temperature that maximizes cardiac activity) and a trait that limits performance (ULT, the upper lethal temperature). Considering the theory of optimal thermal performance, we predicted that the functional traits should be under greater selective pressure to change directionally and in magnitude than the thermal limit, which is redundant in the cooler environment. We found little variation in all traits across species, habitats and ecosystems, despite a ~20°C reduction in maximum habitat temperature in the mangrove species over 50 million years. While slope gradient was significantly lowered in the mangrove species, the effect difference was negated by greater thermal plasticity in the rocky shore species. ULT showed the least variation and suggested thermal specialization in the warmest habitat studied. The observed muted variation of the functional traits among the species may be explained by their limited role in energy acquisition and rather their association with heat tolerance adaptation, which is redundant in the mangrove species. These findings have implications for the conservation of habitat of intertidal gastropods that transition to cooler environments. Furthermore, they highlight the significance of evolutionary history and physiological conservation when predicting species responses to climate change.

Clay-shielded estuarine gastropods are better protected against environmental acidification than unshielded individuals.

The effects of progressive global acidification on the shells of marine organisms is a topic of much current interest. Most studies on molluscan shell resistance to dissolution consider the carbonate mineral component, with less known about the protective role of the outer organic periostracum. Outer-shell resistance would seem especially important to gastropods living in carbonate-undersaturated and calcium-deficient estuarine waters that threaten shell dissolution and constrain CaCO3 production. We tested this prediction using gastropods from an acidified estuarine population (Neripteron violaceum) that form a clay shield outside the periostracum. Specifically, we aimed to show that the carbonate shell component lacks integrity, that the formation of the clay shield is directed by the organism, and that the clay shield functions to protect against shell dissolution. We found no evidence for any specific carbonate dissolution resistance strategy in the thin, predominantly aragonitic shells of these gastropods. Shield formation was directed by an ornamented periostracum which strongly bonded illite elements (e.g., Fe, Al and S), that become available through suspension in the water column. In unshielded individuals, CaCO3 erosion was initiated randomly across the shell (not age-related) and progressed rapidly when the periostracum was breached. A light reflectance technique showed qualitatively that shield consolidation is negatively-related to shell erosion. These findings support a conceptual framework for gastropod outer-shell responses to acidification that considers both environmental and evolutionary constraints on shell construction. We describe a novel strategy for shell protection against dissolution, highlighting the diversity of mechanisms available to gastropods facing extreme coastal acidification.

Microplastic accumulation in oysters along a Bornean coastline (Brunei, South China Sea): Insights into local sources and sinks.

The number of studies on microplastic accumulation in marine organisms has increased precipitously recently, though information is geographically-skewed and limited in terms of local effects. We characterized microplastic accumulation in oysters (Saccostrea cucullata) along a Bornean coastline, focusing on spatial variation. Comparisons were made between locally-polluted (Brunei Estuarine System, BES) and relatively pristine, open-shore (South China Sea, SCS) coastlines. Sixteen coloured microplastic types were characterized into three shapes (fragments, fibres, pellets). Fragments (74.9%), especially smaller polypropylene black fragments predominated in the samples (<50 µm, 31.7%). Site-specific levels of microplastic accumulation varied from 0.43 to 7.20 particles/g oyster tissue. BES and SCS sites differed qualitatively, indicating limited interaction. In the BES, accumulation was greatest near the predicted source (Bandar) and declined strongly seawards, implying current flow, environmental sequestration (local sinks) and seawater dilution effects. Such local-scale variation in microplastic loading in estuaries cautions against extrapolating from limited sampling.

Tracking coastal acidification from erosion of gastropod shells: spatial sensitivity and organism size effect.

The rapidly changing marine environmental chemistry associated with growing industrialisation, urban population expansion, and the unabated rise in atmospheric CO2 necessitates monitoring. Traditional approaches using metres, dataloggers, and buoys to monitor marine acidification have limited application in coastal oceans and intertidal zones subjected to direct wave action. The present study trialled a system to biomonitor coastal acidification (carbonate ion and pH) based on the dissolution of living gastropod shells. We extended on an approach that ranked shell erosion (SER) in Nerita chamaeleon (Nc) in environments where such erosion was found to correlate with exposure to acidified water. We assessed the spatial scale at which the Nc-SER marker could detect change in acidification along rocky shores, and whether snail body size affected this marker. We found that proportional and unique Nc-SERs not only varied between acidified and non-acidified reference shores at a coarse spatial scale (10 km), but also in predictable ways at fine scales (metres), vertically and horizontally within a shore. Differences between acidified and reference shores in the relationship for snail size and Nc-SER were accentuated by less weathered shells at reference localities, highlighting the value of including small, juvenile snails in monitoring protocols. Gastropod shells are shown to be useful for assessing point sources of acidification and the spatial area of affected coastal zones. This cost-effective and easy-to-use approach (potentially even by citizen-scientists) offers an early warning system of acidification of rocky shore ecosystems, where the deployment of instruments is precluded.

Physiological determinants of biogeography: The importance of metabolic depression to heat tolerance.

A quantitative understanding of physiological thermal responses is vital for forecasting species distributional shifts in response to climate change. Many studies have focused on metabolic rate as a global metric for analyzing the sublethal effects of changing environments on physiology. Thermal performance curves (TPCs) have been suggested as a viable analytical framework, but standard TPCs may not fully capture physiological responses, due in part to failure to consider the process of metabolic depression. We derived a model based on the nonlinear regression of biological temperature-dependent rate processes and built a heart rate data set for 26 species of intertidal molluscs distributed from 33°S to ~40°N. We then calculated physiological thermal performance limits with continuous heating using T 1 / 2 H , the temperature at which heart rate is decreased to 50% of the maximal rate, as a more realistic measure of upper thermal limits. Results indicate that heat-induced metabolic depression of cardiac performance is a common adaptive response that allows tolerance of harsh environments. Furthermore, our model accounted for the high inter-individual variability in the shape of cardiac TPCs. We then used these TPCs to calculate physiological thermal safety margins (pTSM), the difference between the maximal operative temperature (95th percentile of field temperatures) and T 1 / 2 H of each individual. Using pTSMs, we developed a physiological species distribution model (pSDM) to forecast future geographic distributions. pSDM results indicate that climate-induced species range shifts are potentially less severe than predicted by a simple correlative SDM. Species with metabolic depression below the optimum temperature will be more thermal resistant at their warm trailing edges. High intraspecific variability further suggests that models based on species-level vulnerability to environmental change may be problematic. This multi-scale, mechanistic understanding that incorporates metabolic depression and inter-individual variability in thermal response enables better predictions about the relationship between thermal stress and species distributions.

Metabolic Regulation, Oxygen Limitation and Heat Tolerance in a Subtidal Marine Gastropod Reveal the Complexity of Predicting Climate Change Vulnerability.

Predictions for climate vulnerability of ectotherms have focused on performance-enhancing physiology, even though an organism's energetic state can also be balanced by lowering resting maintenance costs. Adaptive metabolic depression (hypometabolism) enables animals to endure food scarcity, and physically extreme and variable environmental conditions. Hypometabolism is common in terrestrial and intertidal marine gastropod species, though this physiology and tolerance of environmental change are poorly understood in subtidal benthic gastropods. We investigated oxygen limitation tolerance, hypometabolism and thermal performance in the subtidal, tropical snail Turritella bacillum. Survival, cardiac activity and oxygen debt repayment were determined when oxygen uptake was limited by gill function impairment (air exposure) or exposure to hypoxic seawater. Thermal performance and tolerance were assessed from survival and cardiac performance when heated. The ability of snails to regulate metabolism during oxygen limitation was demonstrated by their tolerance of air exposure (>36 h) and hypoxia (>16 h), rhythmicity and reversibility of bradycardia, and inconsistent anaerobic compensation. Under acute heating, mean heart rate was temperature-insensitive in water and temperature-dependent in air. Converging or peaking of individual heart rates during heating suggest maximization of thermal performance at 38-39°C, whereas survival and heartbeat flatlining suggest an upper thermal limit exceeding 42°C. Snails survived 16 h in seawater at 38°C. Their metabolic regulation complies with the oxygen-limiting, sediment-burrowing lifestyle of the species. Although a tropical organism, the species' thermal tolerance so far exceeds present habitat temperatures as to question its susceptibility to centennial climate warming. Our findings reveal the importance of knowing the metabolic regulatory capabilities and conserved physiological attributes of species used in climate vulnerability tests. Studies of ectotherm climate vulnerability that identify generalized trends based on physiologically similar animals may be misleading by missing information on physiological diversity.

Assessment of tacrolimus and creatinine concentration collected using Mitra microsampling devices.

BACKGROUND: Current practice requires regular venous blood samples for monitoring of tacrolimus concentrations post renal transplant requiring regular hospital visits. Mitra devices use volumetric absorptive microsampling technology and absorb a fixed amount of blood (10 µL) from a capillary blood sample. They are a viable volumetric alternative to dried blood spots and are able to be posted to the laboratory for analysis. OBJECTIVE: The aim was to develop and validate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for tacrolimus and creatinine analysis using Mitra devices. The usefulness of this approach was assessed in renal transplant patients routinely monitored for tacrolimus and creatinine. METHOD: Routine tacrolimus samples were used to assess the utility and reliability of Mitra sampling. Shared sample preparation for both tacrolimus and creatinine was carried out in a 96-deep well plate; mass spectrometric analysis was then undertaken for tacrolimus followed by re-injection for creatinine analysis. RESULTS: Comparison of 131 Mitra samples with a routine LC-MS/MS assay for tacrolimus showed a minimal bias -5.6% (95% CI -8.5 to -2.7%). Comparison of 135 serum and Mitra samples for creatinine using a fully validated LC-MS/MS assay showed a bias -6.5% (95% CI -8.5 to -4.5%). DISCUSSION: We have developed assays for tacrolimus and creatinine on fingerprick blood using the Mitra device and believe this approach provides a viable alternative to repeated venepuncture for therapeutic drug monitoring. This method could open up the opportunity for patients to perform tacrolimus and kidney function monitoring at home.

Differences in heat tolerance plasticity between supratidal and intertidal snails indicate complex responses to microhabitat temperature variation.

Tropical intertidal gastropods that experience extreme and highly variable daily temperatures have evolved significant and complex heat tolerance plasticity, comprising components that respond to different timescales of temperature variation. An earlier study showed different plasticity attributes in snails from differently-heated coastlines, suggesting lifelong irreversible responses that matched habitat thermal regimes. To determine whether heat tolerance plasticity varied at a finer, within-shore spatial scale, we compared the responses of supratidal (predominantly shade-dwelling) and intertidal (frequently solar-exposed) populations of the tropical thermophilic gastropod, Echinolittorina malaccana. Snails modified lethal temperature (LT50) under warm or cool laboratory acclimation, with the overall variation in LT50 being greater in the supratidal (56.0-58.0 °C) than in the intertidal population (57.1-58.1 °C). Similar maximum LT50s expressed by the populations after warm acclimation suggest a capacity limitation under these temperature conditons. The different minimum LT50s after cool acclimation corresponded with microhabitat temperature and field acclimatization of the snails. Different responses to the same laboratory acclimation treatment imply long-term (and possibly lifelong) thermal acclimatization, which could benefit sedentary organisms that are randomly recruited as larvae from a common thermally-stable aquatic environment to thermally-unpredictable intertidal microhabitats. These findings provide another example of thermal tolerance plasticity operating at microhabitat scales, suggesting the importance of considering microhabitat thermal responses when assessing broad-scale environmental change.

Quantification of testosterone, androstenedione and 17-hydroxyprogesterone in whole blood collected using Mitra microsampling devices.

BACKGROUND: Measurement of testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17OHP) usually requires a venous serum sample which may have implications for sample stability or collection. OBJECTIVE: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for samples collected using Mitra devices. Analytical validation was completed, and sample comparisons were undertaken to assess Mitra versus venous samples. METHOD: Sample was combined with deionized water and internal standard. After mixing, MTBE was added for extraction. The supernatant was transferred to a deep-well plate and dried prior to re-constitution. A HSS T3 column and Waters TQS Micro was used, the detected quantifier transitions were T m/z 289.2 > 96.95, A4 287.2 > 96.95 and 17OHP 331.25 > 96.95. RESULTS: Mean recovery was 102% for T, 98% for A4 and 97% for 17OHP. Lower limit of quantification was 1 nmol/L for T/A4 and 4 nmol/L for 17OHP. T was linear up to 41.6 nmol/L, A4 41.9 nmol/L and 17OHP 72.6 nmol/L. Ion suppression was <10% for all analytes. A4 and 17OHP showed minimal bias for Mitra samples collected from finger prick blood. The bias for T differed between capillary and venous blood, indicating differences in constituency. DISCUSSION: A simple, fast and reproducible LC-MS/MS assay has been developed for measurement of blood collected using Mitra devices for T, A4 and 17OHP. Further comparisons with serum and capillary blood collected onto Mitra devices serum may pave the way for future use in a clinical setting.

Biomonitoring acidification using marine gastropods.

Ocean acidification is mainly being monitored using data loggers which currently offer limited coverage of marine ecosystems. Here, we trial the use of gastropod shells to monitor acidification on rocky shores. Animals living in areas with highly variable pH (8.6-5.9) were compared with those from sites with more stable pH (8.6-7.9). Differences in site pH were reflected in size, shape and erosion patterns in Nerita chamaeleon and Planaxis sulcatus. Shells from acidified sites were shorter, more globular and more eroded, with both of these species proving to be good biomonitors. After an assessment of baseline weathering, shell erosion can be used to indicate the level of exposure of organisms to corrosive water, providing a tool for biomonitoring acidification in heterogeneous intertidal systems. A shell erosion ranking system was found to clearly discriminate between acidified and reference sites. Being spatially-extensive, this approach can identify coastal areas of greater or lesser acidification. Cost-effective and simple shell erosion ranking is amenable to citizen science projects and could serve as an early-warning-signal for natural or anthropogenic acidification of coastal waters.

Substantial heat tolerance acclimation capacity in tropical thermophilic snails, but to what benefit?

The theory for thermal acclimation of ectotherms suggests that (1) heat tolerance is traded off for thermal acclimation in thermophilic species and that (2) plasticity is constrained in tropically distributed ectotherms, which commonly experience relatively thermally stable environments. We observed substantial heat tolerance plasticity in a test of this theory using tropical, thermophilic marine intertidal snails that inhabit extremely hot and highly variable thermal environments. The implication of these results is that plasticity selection is largely driven by habitat temperature conditions irrespective of basal heat tolerance or latitude. However, heat tolerance of field-fresh snails was comparable with that of laboratory warm-acclimated snails, suggesting that snails in the field may often be unable to improve heat hardening with further environmental warming. These findings suggest that field referencing is crucial to using laboratory-measured acclimation capacity when inferring climate warming vulnerability in ectotherms, and overall they question how well current thermal biology theory predicts the outcomes of global change in intertidal environments.

Genetic rescue in a plant polyploid complex: Case study on the importance of genetic and trait data for conservation management.

Knowledge of the biology of rare plant species is indispensable to aid their survival and to inform efficient conservation actions, but in many cases relevant data are lacking. In addition, while studies of conservation genetics have provided a wealth of information on the considerations arising from inbreeding, mate limitation, or local adaptation, the impact of intraspecific polyploidy remains understudied. In this study, we examined the breeding system of the rare Australian daisy Rutidosis lanata (Asteraceae) and screened ten of its populations for their ploidy level to develop recommendations for management actions, in particular, with regard to seed sourcing and genetic rescue. We found R. lanata to represent a polyploid complex, with tetraploid, pentaploid and hexaploid individuals coexisting in the same species. Crossing experiments confirmed R. lanata to be self-incompatible. Mate availability varied from c. 49% to c. 76% across populations. Most populations showed mate availability of c. 50%-70%, suggesting that mate limitation resulting from a lack of local genetic diversity may cause or at least contribute to reduced seed set. Crossing between populations resulted in significantly higher reproductive success for all populations except one, suggesting the possibility of genetic rescue through population mixing. However, the crossing experiments also showed that pentaploids suffer from a severely reduced paternal reproductive fitness. Any additional hybrids between tetraploids and pentaploids, as would be created by mixing populations with different genome copy numbers during conservation work, would consequently exacerbate mate limitation and thus reduce population viability. We conclude that seed set and thus population viability can be maximized by mixing populations with the same number of genome copies, but that populations with different numbers should be kept spatially separated. The case of Rutidosis lanata provides an example and a potential template for examining the conservation genetics of other species that may constitute polyploid complexes.

A combined liquid chromatography tandem mass spectrometry assay for the quantification of urinary oxalate and citrate in patients with nephrolithiasis.

Background Analysis of citrate and oxalate in a 24-h urine sample is important in the screening and monitoring of patients with nephrolithiasis. To streamline the analytical process, it was decided to combine oxalate and citrate and analyse them simultaneously in the same assay. Objective A highly sensitive and specific assay for analysis of urine citrate and oxalate was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a simple weak anion exchange solid phase extraction (WAX SPE) clean-up procedure. Method Premixed calibrator/acidified urine (50 µL) was combined with mixed internal standard (13C2 oxalate/citrate-d4) and 5% v/v formic acid in water and passed through a Waters WAX SPE plate. After clean-up steps, the plate was eluted with 5% NH3 in methanol, the eluent was dried down and re-constituted with 100 µL distilled water. Separation was then performed on an HSS T3 2.1 × 50 mm column (Waters, Manchester, UK), flow rate of 0.5 mL/min using a gradient of aqueous and organic mobile phases. We detected multiple reaction monitoring transitions m/z citrate 191.1>110.9, citrate IS 195.1>112.9, oxalate 88.9>60.85, oxalate IS 90.9>61.9 using a Waters TQD in electrospray-negative mode. Results Oxalate and 13C2 oxalate were eluted at 0.29 min; citrate and citrate-d4 were eluted at 0.52 min. Mean recovery was 100% for oxalate and 103% for citrate; lower limit of quantification of oxalate was 60 µmol/L and 50 µmol/L for citrate. Oxalate was linear up to 1388 µmol/L; citrate was linear up to 4762.5 µmol/L. Oxalate was found to be affected by ion suppression (matrix effect: -23 to +65%) but was compensated for by the internal standard used in all cases. The coefficient of variation of the assay in urine for oxalate was <7% for oxalate and 5% for citrate. Discussion We have developed a rapid assay for LC-MS/MS measurement of urinary oxalate and citrate in a routine clinical laboratory. It is simple, reproducible and easy to perform.

Non-reversible and Reversible Heat Tolerance Plasticity in Tropical Intertidal Animals: Responding to Habitat Temperature Heterogeneity.

The theory for thermal plasticity of tropical ectotherms has centered on terrestrial and open-water marine animals which experience reduced variation in diurnal and seasonal temperatures, conditions constraining plasticity selection. Tropical marine intertidal animals, however, experience complex habitat thermal heterogeneity, circumstances encouraging thermal plasticity selection. Using the tropical rocky-intertidal gastropod, Echinolittorina malaccana, we investigated heat tolerance plasticity in terms of laboratory acclimation and natural acclimatization of populations from thermally-dissimilar nearby shorelines. Laboratory treatments yielded similar capacities of snails from either population to acclimate their lethal thermal limit (LT50 variation was ∼2°C). However, the populations differed in the temperature range over which acclimatory adjustments could be made; LT50 plasticity occurred over a higher temperature range in the warm-shore snails compared to the cool-shore snails, giving an overall acclimation capacity for the populations combined of 2.9°C. In addition to confirming significant heat tolerance plasticity in tropical intertidal animals, these findings reveal two plasticity forms, reversible (laboratory acclimation) and non-reversible (population or shoreline specific) plasticity. The plasticity forms should account for different spatiotemporal scales of the environmental temperature variation; reversible plasticity for daily and tidal variations in microhabitat temperature and non-reversible plasticity for lifelong, shoreline temperature conditions. Non-reversible heat tolerance plasticity, likely established after larvae settle on the shore, should be energetically beneficial in preventing heat shock protein overexpression, but also should facilitate widespread colonization of coasts that support thermally-diverse shorelines. This first demonstration of different plasticity forms in benthic intertidal animals supports the hypothesis that habitat heterogeneity (irrespective of latitude) drives thermal plasticity selection. It further suggests that studies not making reference to different spatial scales of thermal heterogeneity, nor seeking how these may drive different thermal plasticity forms, risk misinterpreting ectothermic responses to environmental warming.

Decoupling of behavioural and physiological thermal performance curves in ectothermic animals: a critical adaptive trait.

Thermal performance curves (TPCs) represent an increasingly popular tool in ecology for anticipating species responses to climate change. TPC theory has been developed using species that experience similar temperatures during activity and at rest and consequently exhibit thermal ranges for activity that closely coincide with their physiological thermal tolerances. Many species, however, experience other stressors, such as desiccation, that limit active behaviour at temperatures below the maximum values experienced. As a result, activity is constrained to a narrow thermal window that is a subset of the range of temperatures that can be tolerated physiologically. This results in a decoupling of behavioural and physiological TPCs that does not conform to the present paradigm. To test the generality of TPC theory, we measured thermal responses for behaviour (crawling speed) and physiological tolerance (heart rate) for six rocky shore gastropods spanning a thermal/desiccation stress gradient. We hypothesized a positive relationship between shore level and the degree of decoupling of behavioural and physiological TPCs. This prediction was confirmed, and was explained by the extension of the physiological TPC beyond the range of the behavioural TPC. Decoupling of behavioural and physiological TPCs is central to predicting accurately the fitness dynamics of ectothermic species subject to multiple stressors. We believe that this decoupling should be explicitly considered as an adaptive trait defining an organism's thermal niche.

Empirical evidence of fixed and homeostatic patterns of polyploid advantage in a keystone grass exposed to drought and heat stress.

A long-standing hypothesis in evolutionary biology is that polyploid plants have a fitness advantage over diploids in climatically variable or extreme habitats. Here we provide the first empirical evidence that polyploid advantage in these environments is caused by two distinct processes: homeostatic maintenance of reproductive output under elevated abiotic stress, and fixed differences in seed development. In an outdoor climate manipulation experiment using coastal to inland Australian populations of the perennial grass Themeda triandra Forssk., we found that total output of viable seed in drought- and heat-stressed tetraploid plants was over four times higher than in diploids, despite being equal under more favourable growing conditions. Tetraploids also consistently produced heavier seeds with longer hygroscopic awns, traits which increase propagule fitness in extreme environments. These differences add to fitness benefits associated with broader-scale local adaptation of inland T. triandra populations to drought stress. Our study provides evidence that nucleotypic effects of genome size and increased reproductive flexibility can jointly underlie polyploid advantage in plants in stressful environments, and argue that ploidy can be an important criterion for selecting plant populations for use in genetic rescue, restoration and revegetation projects, including in habitats affected by climate change.