(Appendix) Vertebral number of topsmelt, Atherinops affinis, adults and offspring from different latitudes along the North-American Pacific coast

Organisms that are distributed across spatial climate gradients often exhibit adaptive local variations in morphological and physiological traits, but to what extent such gradients shape evolutionary responses is still unclear. Given the strong natural contrast in latitudinal temperature gradients between the North-American Pacific and Atlantic coast, we asked how increases in vertebral number (VN, known as Jordan's Rule) with latitude would differ between Pacific (Atherinops affinis) and Atlantic Silversides (Menidia menidia), two ecologically equivalent and taxonomically similar fishes with similar latitudinal distributions. VN was determined from radiographs of wild-caught adults (genetic + environmental differences) and its genetic basis confirmed by rearing offspring in common garden experiments. Compared to published data on VN variation in M. menidia (a mean increase of 7.0 vertebrae from 32 to 46°N, VN slope = 0.42/lat), the latitudinal VN increase in Pacific Silversides was approximately half as strong (a mean increase of 3.3 vertebrae from 28 to 43°N, VN slope = 0.23/lat). This mimicked the strong Atlantic (1.11°C/lat) versus weak Pacific latitudinal gradient (0.40°C/lat) in median annual sea surface temperature (SST). Importantly, the relationship of VN to SST was not significantly different between the two species (average slope = -0.39 vertebrae/°C), thus suggesting a common thermal dependency of VN in silverside fishes. Our findings provide novel support for the hypothesis that temperature gradients are the ultimate cause of Jordan's Rule, even though its exact adaptive significance remains speculative. A second investigated trait, the mode of sex determination in Atlantic versus Pacific Silversides, revealed patterns that were inconsistent with our expectation: M. menidia displays temperature-dependent sex determination (TSD) at low latitudes, where growing seasons are long or unconstrained, but also a gradual shift to genetic sex determination (GSD) with increasing latitude due to more and more curtailed growing seasons. Sex ratios in A. affinis, on the other hand, were independent of latitude and rearing temperature (indicating GSD), even though growing seasons are thermally unconstrained across most of the geographical distribution of A. affinis. This suggests that additional factors (e.g., longevity) play an important role in shaping the mode of sex determination in silverside fishes.

Path characteristics and colony arrival-departure statistics of king penguins (Aptenodytes patagonicus) on Possession and Kerguelen Island

Orientation based on visual cues can be extremely difficult in crowded bird colonies due to the presence of many individuals. We studied king penguins (Aptenodytes patagonicus) that live in dense colonies and are constantly faced with such problems. Our aims were to describe adult penguin homing paths on land and to test whether visual cues are important for their orientation in the colony. We also tested the hypothesis that older penguins should be better able to cope with limited visual cues due to their greater experience. We collected and examined GPS paths of homing penguins. In addition, we analyzed 8 months of penguin arrivals to and departures from the colony using data from an automatic identification system. We found that birds rearing chicks did not minimize their traveling time on land and did not proceed to their young (located in creches) along straight paths. Moreover, breeding birds' arrivals and departures were affected by the time of day and luminosity levels. Our data suggest that king penguins prefer to move in and out of the colony when visual cues are available. Still, they are capable of navigating even in complete darkness, and this ability seems to develop over the years, with older breeding birds more likely to move through the colony at nighttime luminosity levels. This study is the first step in unveiling the mysteries of king penguin orientation on land.

List of phytoplankton, zooplankton and zoobenthos species abundances

We studied the effects of changed quality of inflow water of aquaculture ponds on three aquatic communities, phytoplankton, zooplankton and zoobenthos, during two seasons of rearing common carp (Cyprinus carpio). The new water source coming from a deep tube well was markedly different in water chemistry from the surface water sources previously used to maintain the investigated fish ponds. Ponds supplied by the tube well water were characterized by lower oxygen and water hardness, and higher total ammonia and conductivity reaching subsaline conditions. Multivariate analysis (co-inertia) revealed that all investigated groups, except Mollusca (zoobenthos), decreased in species richness, abundance and biomass due to changed water chemistry, but differed in the level of susceptibility to stressors. Assemblages of Rotifera and Cladocera were the most affected showing a sharp decline in density and number of species since 29 out of 44 species disappeared from the ponds. The abundance of Copepoda (Cyclopoida) was relatively high although significantly lower in new environmental conditions (P<0.05), with adults being more tolerant to changed inflow water than larvae. Phytoplankton, except Bacillariophyta, had a highest potential to replace previous species with newcomers more adapted to changed inflow water, providing 36 immigrant species while 49 became extinct. Although mainly influenced by fish predation, Chironomidae (zoobenthos) were undoubtedly affected by changed water chemistry, decreasing from 11 to only 3 species. These results suggest that this pattern was a result of the shift from freshwater to subsaline conditions.

Thermal reaction norms of growth in Atlantic and Pacific silverside fishes

How organisms may adapt to rising global temperatures is uncertain, but concepts can emerge from studying adaptive physiological trait variations across existing spatial climate gradients. Many ectotherms, particularly fish, have evolved increasing genetic growth capacities with latitude (i.e. countergradient variation (CnGV) in growth), which are thought to be an adaptation primarily to strong gradients in seasonality. In contrast, evolutionary responses to gradients in mean temperature are often assumed to involve an alternative mode, 'thermal adaptation'. We measured thermal growth reaction norms in Pacific silverside populations (Atherinops affinis) occurring across a weak latitudinal temperature gradient with invariant seasonality along the North American Pacific coast. Instead of thermal adaptation, we found novel evidence for CnGV in growth, suggesting that CnGV is a ubiquitous mode of reaction-norm evolution in ectotherms even in response to weak spatial and, by inference, temporal climate gradients. A novel, large-scale comparison between ecologically equivalent Pacific versus Atlantic silversides (Menidia menidia) revealed how closely growth CnGV patterns reflect their respective climate gradients. While steep growth reaction norms and increasing growth plasticity with latitude in M. menidia mimicked the strong, highly seasonal Atlantic coastal gradient, shallow reaction norms and much smaller, latitude-independent growth plasticity in A. affinis resembled the weak Pacific latitudinal temperature gradient.

Biomarker and radiogenic isotopes from sediment core POS362-2_33

The termination of the African Humid Period in northeastern Africa during the early Holocene was marked by the southward migration of the rain belt and the disappearance of the Green Sahara. This interval of drastic environmental changes was also marked by the initiation of food production by North African huntergatherer populations and thus provides critical information on human-environment relationships. However, existing records of regional climatic and environmental changes exhibit large differences in timing and modes of the wet/dry transition at the end of the African Humid Period. Here we present independent records of changes in river runoff, vegetation and erosion in the Nile River watershed during the Holocene obtained from a unique sedimentary sequence on the Nile River fan using organic and inorganic proxy data. This high-resolution reconstruction allows to examine the phase relationship between the changes of these three parameters and provides a detailed picture of the environmental conditions during the Paleolithic/Neolithic transition. The data show that river runoff decreased gradually during the wet/arid transition at the end of the AHP whereas rapid shifts of vegetation and erosion occurred earlier between 8.7 and about 6 ka BP. These asynchronous changes are compared to other regional records and provide new insights into the threshold responses of the environment to climatic changes. Our record demonstrates that the degradation of the environment in northeastern Africa was more abrupt and occurred earlier than previously thought and may have accelerated the process of domestication in order to secure sustainable food resources for the Neolithic African populations.

Temperature and photoperiod effects on sex determination in Leuresthes tenuis (fish)

In some gonochoristic species, sex is influenced not only by genotype at conception but also by the environment that offspring experience during early ontogeny (termed environmental sex determination or ESD). ESD is thought to be adaptive when seasonal variations in environmental conditions provide a sex-specific fitness advantage. In vertebrates, temperature is the most common determinant of sex, and seasonal variation in temperature serves as a temporal cue of environmental quality such as length of the growing season. Some environments, however, lack strong seasonal temperature fluctuations and other cues, particularly photoperiod, may provide a more reliable indicator of the environment offspring enter. We tested this hypothesis by rearing the offspring of the California grunion (Leuresthes tenuis, Ayres), which experiences low seasonal temperature variation in nature, under common garden conditions at three temperature and two photoperiod treatments. Our experiments revealed that both temperature and photoperiod significantly affected sex ratios in L. tenuis. More females were produced at cooler temperatures and longer day lengths, which is consistent with female biased sex ratios early in the breeding season, and likely adaptive through increased female size and fecundity. To our knowledge, this is the first documented case of photoperiod-dependent sex determination in a gonochoristic vertebrate.

Seawater carbonate chemistry and auditory behavior of Orange clownfish (Amphiprion percula) during experiments, 2011

Ocean acidification is predicted to affect marine ecosystems in many ways, including modification of fish behaviour. Previous studies have identified effects of CO2-enriched conditions on the sensory behaviour of fishes, including the loss of natural responses to odours resulting in ecologically deleterious decisions. Many fishes also rely on hearing for orientation, habitat selection, predator avoidance and communication. We used an auditory choice chamber to study the influence of CO2-enriched conditions on directional responses of juvenile clownfish (Amphiprion percula) to daytime reef noise. Rearing and test conditions were based on Intergovernmental Panel on Climate Change predictions for the twenty-first century: current-day ambient, 600, 700 and 900 µatm pCO2. Juveniles from ambient CO2-conditions significantly avoided the reef noise, as expected, but this behaviour was absent in juveniles from CO2-enriched conditions. This study provides, to our knowledge, the first evidence that ocean acidification affects the auditory response of fishes, with potentially detrimental impacts on early survival.

Effect of ocean acidification on growth and otolith condition of juvenile scup, Stenotomus chrysops

Increasing amounts of atmospheric carbon dioxide (CO2) from human industrial activities are causing changes in global ocean carbonate chemistry, resulting in a reduction in pH, a process termed "ocean acidification." It is important to determine which species are sensitive to elevated levels of CO2 because of potential impacts to ecosystems, marine resources, biodiversity, food webs, populations, and effects on economies. Previous studies with marine fish have documented that exposure to elevated levels of CO2 caused increased growth and larger otoliths in some species. This study was conducted to determine whether the elevated partial pressure of CO2 (pCO2) would have an effect on growth, otolith (ear bone) condition, survival, or the skeleton of juvenile scup, Stenotomus chrysops, a species that supports both important commercial and recreational fisheries. Elevated levels of pCO2 (1200-2600 µatm) had no statistically significant effect on growth, survival, or otolith condition after 8 weeks of rearing. Field data show that in Long Island Sound, where scup spawn, in situ levels of pCO2 are already at levels ranging from 689 to 1828 µatm due to primary productivity, microbial activity, and anthropogenic inputs. These results demonstrate that ocean acidification is not likely to cause adverse effects on the growth and survivability of every species of marine fish. X-ray analysis of the fish revealed a slightly higher incidence of hyperossification in the vertebrae of a few scup from the highest treatments compared to fish from the control treatments. Our results show that juvenile scup are tolerant to increases in seawater pCO2, possibly due to conditions this species encounters in their naturally variable environment and their well-developed pH control mechanisms.

Seawater carbonate chemistry and Amphiprion melanopus activity during experiments, 2011

Two of the major threats to coral reefs are increasing sea surface temperature and ocean acidification, both of which result from rising concentrations of atmospheric carbon dioxide (CO2). Recent evidence suggests that both increased water temperature and elevated levels of dissolved CO2 can change the behaviors of fishes in ways that reduce individual fitness, however the interacting effects of these variables are unknown. We used a fully factorial experiment to test the independent and interactive effects of temperature (3 levels: 28.5, 30, and 31.5 °C) and pCO2 (3 levels: averaging 420, 530, and 960 µatm) on food consumption and activity level of juvenile anemonefish Amphiprion melanopus (Bleeker 1852). Experimental levels were consistent with current-day ocean conditions and predictions for mid-century and late-century based on atmospheric CO2 projections. Sibling fish were reared for 21 days from the end of their larval phase in each of the nine treatments, at which time behavioral observations were conducted. Food consumption and foraging activity decreased at the highest temperature. In isolation, CO2 level did not significantly affect behavior; however, there was an interaction with temperature. While rearing at high temperature (31.5 °C) and control (420 µatm) or moderate (530 µatm) CO2 resulted in a reduction of food consumption and foraging activity, rearing at high temperature and high CO2 (960 µatm) resulted in an elevation in these behaviors. Maintaining food consumption and foraging activity in high temperature and CO2 conditions may reduce energy efficiency if the thermal optimum for food assimilation and growth has been exceeded. Maintaining foraging effort might increase predation vulnerability. These results suggest that changes in foraging behaviors caused by the interactive effects of increased SST and CO2 could have significant effects on the growth and survival of juvenile reef fishes by late century.

Growth, survival, gene expression, microbiota and tryptic activity during feeding of Scophthalmus maximus first feeding larvae with beta-glucan (MacroGard)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

Stickleback F2 generation transgenerational plasticity experiment: egg and body size data

Nongenetic inheritance mechanisms such as transgenerational plasticity (TGP) can buffer populations against rapid environmental change such as ocean warming. Yet, little is known about how long these effects persist and whether they are cumulative over generations. Here, we tested for adaptive TGP in response to simulated ocean warming across parental and grandparental generations of marine sticklebacks. Grandparents were acclimated for two months during reproductive conditioning, whereas parents experienced developmental acclimation, allowing us to compare the fitness consequences of short-term vs. prolonged exposure to elevated temperature across multiple generations. We found that reproductive output of F1 adults was primarily determined by maternal developmental temperature, but carry-over effects from grandparental acclimation environments resulted in cumulative negative effects of elevated temperature on hatching success. In very early stages of growth, F2 offspring reached larger sizes in their respective paternal and grandparental environment down the paternal line, suggesting that other factors than just the paternal genome may be transferred between generations. In later growth stages, maternal and maternal granddam environments strongly influenced offspring body size, but in opposing directions, indicating that the mechanism(s) underlying the transfer of environmental information may have differed between acute and developmental acclimation experienced by the two generations. Taken together, our results suggest that the fitness consequences of parental and grandparental TGP are highly context dependent, but will play an important role in mediating some of the impacts of rapid climate change in this system.

Gasterosteus aculeatus egg traits and fish lengths vs. experiment setup

1) Our study addresses the role of non-genetic and genetic inheritance in shaping the adaptive potential of populations under a warming ocean scenario. We used a combined experimental approach (transgenerational plasticity and quantitative genetics) to partition the relative contribution of maternal vs. paternal (additive genetic) effects to offspring body size (a key component of fitness), and investigated a potential physiological mechanism (mitochondrial respiration capacities) underlying whole organism growth/size responses. 2) In very early stages of growth (up to 30 days), offspring body size of marine sticklebacks benefited from maternal transgenerational plasticity (TGP): offspring of mothers acclimated to17°C were larger when reared at 17°C, and offspring of mothers acclimated to 21°C were larger when reared at 21°C. The benefits of maternal TGP on body size were stronger and persisted longer (up to 60 days) for offspring reared in the warmer (21°C) environment, suggesting that maternal effects will be highly relevant for climate change scenarios in this system. 3) Mitochondrial respiration capacities measured on mature offspring (F1 adults) matched the pattern of TGP for juvenile body size, providing an intuitive mechanistic basis for the maternal acclimation persisting into adulthood. Size differences between temperatures seen at early growth stages remained in the F1 adults, linking offspring body size to maternal inheritance of mitochondria. 4) Lower maternal variance components in the warmer environment were mostly driven by mothers acclimated to ambient (colder) conditions, further supporting our tenet that maternal effects were stronger at elevated temperature. Importantly, all parent-offspring temperature combination groups showed genotype x environment (GxE) interactions, suggesting that reaction norms have the potential to evolve. 5) To summarise, transgenerational plasticity and genotype x environment interactions work in concert to mediate impacts of ocean warming on metabolic capacity and early growth of marine sticklebacks. TGP can buffer short-term detrimental effects of climate warming and may buy time for genetic adaptation to catch up, therefore markedly contributing to the evolutionary potential and persistence of populations under climate change.