(Supplement) Regeneration of Pinus sylvestris, disturbance residuals and soil properties 5 years after a mixed-severity wildfire in the inner Italian Alps

We analyzed the abundance of Scots pine regeneration in a 257 ha wildfire in an inner-alpine forest. We sampled regeneration, percent soil cover by classes, physical and chemical properties of topsoils (A horizon, 0-5 cm) under four fire severity levels (unburned, moderate, moderate/high, high severity). 5 plots per severity level, circular (R= 3m). Analysis methods for soil properties as described in the paper.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and ambient magnesium concentration in early life history stages of Paralomis granulosa (Decapoda, Anomura, Lithodidae)

Marine brachyuran and anomuran crustaceans are completely absent from the extremely cold (-1.8 °C) Antarctic continental shelf, but caridean shrimps are abundant. This has at least partly been attributed to low capacities for magnesium excretion in brachyuran and anomuran lithodid crabs ([Mg2+]HL = 20-50 mmol/L) compared to caridean shrimp species ([Mg2+]HL = 5-12 mmol/L). Magnesium has an anaesthetizing effect and reduces cold tolerance and activity of adult brachyuran crabs. We investigated whether the capacity for magnesium regulation is a factor that influences temperature-dependent activity of early ontogenetic stages of the Sub-Antarctic lithodid crab Paralomis granulosa. Ion composition (Na+, Mg2+, Ca2+, Cl-, [SO4]2-) was measured in haemolymph withdrawn from larval stages, the first and second juvenile instars (crabs I and II) and adult males and females. Magnesium excretion improved during ontogeny, but haemolymph sulphate concentration was lowest in the zoeal stages. Neither haemolymph magnesium concentrations nor Ca2+:Mg2+ ratios paralleled activity levels of the life stages. Long-term (3 week) cold exposure of crab I to 1 °C caused a significant rise of haemolymph sulphate concentration and a decrease in magnesium and calcium concentrations compared to control temperature (9 °C). Spontaneous swimming activity of the zoeal stages was determined at 1, 4 and 9 °C in natural sea water (NSW, [Mg2+] = 51 mmol/L) and in sea water enriched with magnesium (NSW + Mg2+, [Mg2+] = 97 mmol/L). It declined significantly with temperature but only insignificantly with increased magnesium concentration. Spontaneous velocities were low, reflecting the demersal life style of the zoeae. Heart rate, scaphognathite beat rate and forced swimming activity (maxilliped beat rate, zoea I) or antennule beat rate (crab I) were investigated in response to acute temperature change (9, 6, 3, 1, -1 °C) in NSW or NSW + Mg2+. High magnesium concentration reduced heart rates in both stages. The temperature-frequency curve of the maxilliped beat (maximum: 9.6 beats/s at 6.6 °C in NSW) of zoea I was depressed and shifted towards warmer temperatures by 2 °C in NSW + Mg2+, but antennule beat rate of crab I was not affected. Magnesium may therefore influence cold tolerance of highly active larvae, but it remains questionable whether the slow-moving lithodid crabs with demersal larvae would benefit from an enhanced magnesium excretion in nature.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and magnesium in adult Paralomis granulosa (Decapoda, Anomura, Lithodidae)

A low capacity for regulation of extracellular Mg2+ has been proposed to exclude reptant marine decapod crustaceans from temperatures below 0°C and thus to exclude them from the high Antarctic. To test this hypothesis and to elaborate the underlying mechanisms in the most cold-tolerant reptant decapod family of the sub-Antarctic, the Lithodidae, thermal tolerance was determined in the crab Paralomis granulosa (Decapoda, Anomura, Lithodidae) using an acute stepwise temperature protocol (-1°, 1°, 4°, 7°, 10°, and 13°C). Arterial and venous oxygen partial pressures (Po2) in hemolymph, heartbeat and ventilation beat frequencies, and hemolymph cation composition were measured at rest and after a forced activity (righting) trial. Scopes for heartbeat and ventilation beat frequencies and intermittent heartbeat and scaphognathite beat rates at rest were evaluated. Hemolymph [Mg2+] was experimentally reduced from 30 mmol/L to a level naturally observed in Antarctic caridean shrimps (12 mmol/L) to investigate whether the animals remain more active and tolerant to cold (-1°, 1°, and 4°C). In natural seawater, righting speed was significantly slower at -1° and 13°C, compared with acclimation temperature (4°C). Arterial and venous hemolymph Po2 increased in response to cooling even though heartbeat and ventilation beat frequencies as well as scopes decreased. At rest, ionic composition of the hemolymph was not affected by temperature. Activity induced a significant increase in hemolymph [K+] at -1° and 1°C. Reduction of hemolymph [Mg2+] did not result in an increase in activity, an increase in heartbeat and ventilation beat frequencies, or a shift in thermal tolerance to lower temperatures. In conclusion, oxygen delivery in this cold-water crustacean was not acutely limiting cold tolerance, and animals may have been constrained more by their functional capacity and motility. In contrast to earlier findings in temperate and subpolar brachyuran crabs, these constraints remained insensitive to changing Mg2+ levels.

Establishment, expansion and regeneration of aspen (Populus tremula) in the Torneträsk area, subarctic Sweden

In subarctic Sweden, recent decadal colonization and expansion of aspen (Populus tremula L.) were recorded. Over the past 100 years, aspen became c. 16 times more abundant, mainly as a result of increased sexual regeneration. Moreover, aspen now reach tree-size (>2 m) at the alpine treeline, an ecotone that has been dominated by mountain birch (Betula pubescens ssp. czerepanovii) for at least the past 4000 years. We found that sexual regeneration in aspen probably occurred seven times or more within the last century. Whereas sexual regeneration occurred during moist years following a year with an exceptionally high June-July temperature, asexual regeneration was favored by warm and dry summers. Disturbance to the birch forest by cyclic moth population outbreaks was critical in aspen establishment in the subalpine area. At the treeline, aspen colonization was less determined by these moth outbreaks, and was mainly restricted by summer temperature. If summer warming persists, aspen spread may continue in subarctic Sweden, particularly at the treeline. However, changing disturbance regimes, future herbivore population dynamics and the responses of aspen's competitors birch and pine to a changing climate may result in different outcomes.

Seawater carbonate chemistry, physiology, morphology of larval sea urchins, Strongylocentrotus purpuratus in a laboratory experiment

Ocean warming and ocean acidification, both consequences of anthropogenic production of CO2, will combine to influence the physiological performance of many species in the marine environment. In this study, we used an integrative approach to forecast the impact of future ocean conditions on larval purple sea urchins (Strongylocentrotus purpuratus) from the northeast Pacific Ocean. In laboratory experiments that simulated ocean warming and ocean acidification, we examined larval development, skeletal growth, metabolism and patterns of gene expression using an orthogonal comparison of two temperature (13°C and 18°C) and pCO2 (400 and 1100 µatm) conditions. Simultaneous exposure to increased temperature and pCO2 significantly reduced larval metabolism and triggered a widespread downregulation of histone encoding genes. pCO2 but not temperature impaired skeletal growth and reduced the expression of a major spicule matrix protein, suggesting that skeletal growth will not be further inhibited by ocean warming. Importantly, shifts in skeletal growth were not associated with developmental delay. Collectively, our results indicate that global change variables will have additive effects that exceed thresholds for optimized physiological performance in this keystone marine species.

Response of the calcifying coccolithophore Emiliania huxleyi to low pH/high pCO2: from physiology to molecular level, 2011

The emergence of ocean acidification as a significant threat to calcifying organisms in marine ecosystems creates a pressing need to understand the physiological and molecular mechanisms by which calcification is affected by environmental parameters. We report here, for the first time, changes in gene expression induced by variations in pH/pCO2 in the widespread and abundant coccolithophore Emiliania huxleyi. Batch cultures were subjected to increased partial pressure of CO2 (pCO2; i.e. decreased pH), and the changes in expression of four functional gene classes directly or indirectly related to calcification were investigated. Increased pCO2 did not affect the calcification rate and only carbonic anhydrase transcripts exhibited a significant down-regulation. Our observation that elevated pCO2 induces only limited changes in the transcription of several transporters of calcium and bicarbonate gives new significant elements to understand cellular mechanisms underlying the early response of E. huxleyi to CO2-driven ocean acidification.

Coralline algal physiology is more adversely affected by elevated temperature than reduced pH

In this study we analyzed the physiological responses of coralline algae to ocean acidification (OA) and global warming, by exposing algal thalli of three species with contrasting photobiology and growth-form to reduced pH and elevated temperature. The analysis aimed to discern between direct and combined effects, while elucidating the role of light and photosynthesis inhibition in this response. We demonstrate the high sensitivity of coralline algae to photodamage under elevated temperature and its severe consequences on thallus photosynthesis and calcification rates. Moderate levels of light-stress, however, were maintained under reduced pH, resulting in no impact on algal photosynthesis, although moderate adverse effects on calcification rates were still observed. Accordingly, our results support the conclusion that global warming is a stronger threat to algal performance than OA, in particular in highly illuminated habitats such as coral reefs. We provide in this study a quantitative physiological model for the estimation of the impact of thermal-stress on coralline carbonate production, useful to foresee the impact of global warming on coralline contribution to reef carbon budgets, reef cementation, coral recruitment and the maintenance of reef biodiversity. This model, however, cannot yet account for the moderate physiological impact of low pH on coralline calcification.

Acid-base physiology response to ocean acidification of two ecologically and economically important holothuroids from contrasting habitats, Holothuria scabra and Holothuria parva

Sea cucumbers are dominant invertebrates in several ecosystems such as coral reefs, seagrass meadows and mangroves. As bioturbators, they have an important ecological role in making available calcium carbonate and nutrients to the rest of the community. However, due to their commercial value, they face overexploitation in the natural environment. On top of that, occurring ocean acidification could impact these organisms, considered sensitive as echinoderms are osmoconformers, high-magnesium calcite producers and have a low metabolism. As a first investigation of the impact of ocean acidification on sea cucumbers, we tested the impact of short-term (6 to 12 days) exposure to ocean acidification (seawater pH 7.7 and 7.4) on two sea cucumbers collected in SW Madagascar, Holothuria scabra, a high commercial value species living in the seagrass meadows, and H. parva, inhabiting the mangroves. The former lives in a habitat with moderate fluctuations of seawater chemistry (driven by day-night differences) while the second lives in a highly variable intertidal environment. In both species, pH of the coelomic fluid was significantly negatively affected by reduced seawater pH, with a pronounced extracellular acidosis in individuals maintained at pH 7.7 and 7.4. This acidosis was due to an increased dissolved inorganic carbon content and pCO2 of the coelomic fluid, indicating a limited diffusion of the CO2 towards the external medium. However, respiration and ammonium excretion rates were not affected. No evidence of accumulation of bicarbonate was observed to buffer the coelomic fluid pH. If this acidosis stays uncompensated for when facing long-term exposure, other processes could be affected in both species, eventually leading to impacts on their ecological role.