Advancing responsible genomic analyses of ancient mollusc shells.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111-6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.

Marine latitudinal diversity gradients are generally absent in intertidal ecosystems.

Current latitudinal diversity gradient (LDG) meta-analyses have failed to distinguish one of the most widespread marine habitats, the intertidal zone, as a separate system despite it having unique abiotic challenges and spatially compressed stress gradients that affect the distribution and abundance of resident species. We address this issue by revisiting published literature and datasets on LDGs since 1911 to explore LDG patterns and their strengths in intertidal benthic, subtidal benthic, and pelagic realms and discuss the importance of recognizing intertidal ecosystems as distinct. Rocky shorelines were the most studied intertidal ecosystem encompassing 64.2% of intertidal LDG studies, and 62.9% of studies focused on assemblage composition, while the remaining 37.1% of studies were taxa specific. While our analyses confirmed LDGs in subtidal benthic and pelagic realms, with a decrease in richness toward the poles, we found no consistent intertidal LDGs in any ocean or coastline across hemispheres or biodiversity unit. Analyzing intertidal and subtidal zones as separate systems increased the strength of subtidal benthic LDGs relative to analyses combining these systems. We demonstrate that in intertidal ecosystems across oceans in both hemispheres, a latitudinal decrease in species richness is not readily apparent, which stands in contrast with significant LDG patterns found in the subtidal realm. Intertidal habitat heterogeneity, regional environmental variability and biological interactions can create species-rich hot spots independent of latitude, which may functionally outweigh a typical latitudinal decline in species richness. Although previous work has shown weaker LDGs in benthic than pelagic systems, we demonstrate that this is caused by combining subtidal and intertidal benthic ecosystems into a single benthic category. Thus, we propose that subtidal and intertidal ecosystems cannot be combined into one entity as the physical and biological parameters controlling ecosystem processes are vastly different, even among intertidal ecosystems. Thus, the intertidal zone offers a unique model system in which hypotheses can be further tested to better understand the complex processes underlying LDGs.

Ocean acidification increases susceptibility to sub-zero air temperatures in ecosystem engineers and limits poleward range shifts.

Ongoing climate change has caused rapidly increasing temperatures and an unprecedented decline in seawater pH, known as ocean acidification. Increasing temperatures are redistributing species toward higher and cooler latitudes that are most affected by ocean acidification. While the persistence of intertidal species in cold environments is related to their capacity to resist sub-zero air temperatures, studies have never considered the interacting impacts of ocean acidification and freeze stress on species survival and distribution. Here, a full-factorial experiment was used to study whether ocean acidification increases mortality in subtidal Mytilus trossulus and subtidal M. galloprovincialis, and intertidal M. trossulus following sub-zero air temperature exposure. We examined physiological processes behind variation in freeze tolerance using 1H NMR metabolomics, analyses of fatty acids, and amino acid composition. We show that low pH conditions (pH = 7.5) significantly decrease freeze tolerance in both intertidal and subtidal populations of Mytilus spp. Under current day pH conditions (pH = 7.9), intertidal M. trossulus was more freeze tolerant than subtidal M. trossulus and subtidal M. galloprovincialis. Conversely, under low pH conditions, subtidal M. trossulus was more freeze tolerant than the other mussel categories. Differences in the concentration of various metabolites (cryoprotectants) or in the composition of amino acids and fatty acids could not explain the decrease in survival. These results suggest that ocean acidification can offset the poleward range expansions facilitated by warming and that reduced freeze tolerance could result in a range contraction if temperatures become lethal at the equatorward edge.

Glacial meltwater determines the balance between autotrophic and heterotrophic processes in a Greenland fjord.

Global warming accelerates melting of glaciers and increases the supply of meltwater and associated inorganic particles, nutrients, and organic matter to adjacent coastal seas, but the ecosystem impact is poorly resolved and quantified. When meltwater is delivered by glacial rivers, the potential impact could be a reduction in light and nutrient availability for primary producers while supplying allochthonous carbon for heterotrophic processes, thereby tipping the net community metabolism toward heterotrophy. To test this hypothesis, we determined physical and biogeochemical parameters along a 110-km fjord transect in NE Greenland fjord, impacted by glacial meltwater from the Greenland Ice Sheet. The meltwater is delivered from glacier-fed river outlets in the inner parts of the fjord, creating a gradient in salinity and turbidity. The planktonic primary production was low, 20-45 mg C m-2 d-1, in the more turbid inner half of the fjord, increasing 10-fold to around 350 mg C m-2 d-1 in the shelf waters outside the fjord. Plankton community metabolism was measured at three stations, which displayed a transition from net heterotrophy in the inner fjord to net autotrophy in the coastal shelf waters. Respiration was significantly correlated to turbidity, with a 10-fold increase in the inner turbid part of the fjord. We estimated the changes in meltwater input and sea ice coverage in the area for the last 60 y. The long-term trend and the observed effects demonstrated the importance of freshwater runoff as a key driver of coastal ecosystem change in the Arctic with potential negative consequences for coastal productivity.

Molecular Responses to Thermal and Osmotic Stress in Arctic Intertidal Mussels (Mytilus edulis): The Limits of Resilience.

Increases in Arctic temperatures have accelerated melting of the Greenland icesheet, exposing intertidal organisms, such as the blue mussel Mytilus edulis, to high air temperatures and low salinities in summer. However, the interaction of these combined stressors is poorly described at the transcriptional level. Comparing expression profiles of M. edulis from experimentally warmed (30 °C and 33 °C) animals kept at control (23‱) and low salinities (15‱) revealed a significant lack of enrichment for Gene Ontology terms (GO), indicating that similar processes were active under all conditions. However, there was a progressive increase in the abundance of upregulated genes as each stressor was applied, with synergistic increases at 33 °C and 15‱, suggesting combined stressors push the animal towards their tolerance thresholds. Further analyses comparing the effects of salinity alone (23‱, 15‱ and 5‱) showed high expression of stress and osmoregulatory marker genes at the lowest salinity, implying that the cell is carrying out intracellular osmoregulation to maintain the cytosol as hyperosmotic. Identification of aquaporins and vacuolar-type ATPase transcripts suggested the cell may use fluid-filled cavities to excrete excess intracellular water, as previously identified in embryonic freshwater mussels. These results indicate that M. edulis has considerable resilience to heat stress and highly efficient mechanisms to acclimatise to lowered salinity in a changing world.

Freshening increases the susceptibility to heat stress in intertidal mussels (Mytilus edulis) from the Arctic.

Temperatures in the Arctic are increasing at a faster pace than at lower latitudes resulting in range expansion of boreal species. In Greenland, the warming also drives accelerating melt of the Greenland Ice Sheet resulting in more meltwater entering Greenland fjords in summer. Our aim was to determine if increasing summer temperatures combined with lower salinity can induce the expression of stress-related proteins, for example, heat shock protein, in boreal intertidal mussels in Greenland, and whether low salinity reduces the upper thermal limit at which mortality occurs. We conducted a mortality experiment, using 12 different combinations of salinity and air temperature treatments during a simulated tidal regime, and quantified the change in mRNA levels of five stress-related genes (hsp24, hsp70, hsp90, sod and p38) in surviving mussels to discern the level of sublethal stress. Heat-induced mortality occurred in mussels exposed to an air temperature of 30°C and mortality was higher in treatments with lowered salinity (5 and 15‰), which confirms that low habitat salinity decreases the upper thermal limit of Mytilus edulis. The gene expression analysis supported the mortality results, with the highest gene expression found at combinations of high temperature and low salinity. Combined with seasonal measurements of intertidal temperatures in Greenland, we suggest heat stress occurs in low salinity intertidal area, and that further lowered salinity in coastal water due to increased run-off can make intertidal bivalves more susceptible to summer heat stress. This study thus provides an example of how different impacts of climate warming can work synergistically to stress marine organisms.

Global gradients in intertidal species richness and functional groups.

Whether global latitudinal diversity gradients exist in rocky intertidal α-diversity and across functional groups remains unknown. Using literature data from 433 intertidal sites, we investigated α-diversity patterns across 155° of latitude, and whether local-scale or global-scale structuring processes control α-diversity. We, furthermore, investigated how the relative composition of functional groups changes with latitude. α-Diversity differed among hemispheres with a mid-latitudinal peak in the north, and a non-significant unimodal pattern in the south, but there was no support for a tropical-to-polar decrease in α-diversity. Although global-scale drivers had no discernible effect, the local-scale drivers significantly affected α-diversity, and our results reveal that latitudinal diversity gradients are outweighed by local processes. In contrast to α-diversity patterns, species richness of three functional groups (predators, grazers, and suspension feeders) declined with latitude, coinciding with an inverse gradient in algae. Polar and tropical intertidal data were sparse, and more sampling is required to improve knowledge of marine biodiversity.

Resilience in Greenland intertidal Mytilus: The hidden stress defense.

The Arctic is experiencing particularly rapid rates of warming, consequently invasive boreal species are now able to survive the less extreme Arctic winter temperatures. Whilst persistence of intertidal and terrestrial species in the Arctic is primarily determined by their ability to tolerate the freezing winters, air temperatures in the Arctic summer can reach 36 °C in the intertidal, which is beyond the upper thermal limits of many marine species. This is normally lethal for the conspicuous ecosystem engineer Mytilus edulis. Transcriptomic analyses were undertaken on both in situ collected and experimentally warmed animals to understand whether M. edulis is able to tolerate these very high summer temperatures. Surprisingly there was no significant enrichment for Gene Ontology terms (GO) when comparing the inner and outer fjord intertidal animals with outer fjord subtidal (control) animals, representing animals collected at 27 °C, 19 °C and 3 °C respectively. This lack of differentiation indicated a wide acclimation ability in this species. Conversely, significant enrichment for processes such as signal transduction, cytoskeleton and cellular protein modification was identified in the expression profiles of the 22 °C and 32 °C experimentally heated animals. This difference in gene expression between in situ collected and experimentally warmed animals was almost certainly due to the former being acclimated to a fluctuating, but predictable, temperature regime, which has increased their thermal tolerances. Interestingly, there was no evidence for enrichment of the classical cellular stress response in any of the animals sampled. Identification of a massive expansion of the HSPA12 heat shock protein 70 kDa gene family presented the possibility of these genes acting as intertidal regulators underpinning thermal resilience. This expansion has resulted in a modified cellular stress response, as an evolutionary adaptation to the rigour of the invasive intertidal life style. Thus, M. edulis appear to have considerable capacity to withstand the current rates of Arctic warming, and the very large attendant thermal variation.

Biomineralization plasticity and environmental heterogeneity predict geographical resilience patterns of foundation species to future change.

Although geographical patterns of species' sensitivity to environmental changes are defined by interacting multiple stressors, little is known about compensatory processes shaping regional differences in organismal vulnerability. Here, we examine large-scale spatial variations in biomineralization under heterogeneous environmental gradients of temperature, salinity and food availability across a 30° latitudinal range (3,334 km), to test whether plasticity in calcareous shell production and composition, from juveniles to large adults, mediates geographical patterns of resilience to climate change in critical foundation species, the mussels Mytilus edulis and M. trossulus. We find shell calcification decreased towards high latitude, with mussels producing thinner shells with a higher organic content in polar than temperate regions. Salinity was the best predictor of within-region differences in mussel shell deposition, mineral and organic composition. In polar, subpolar, and Baltic low-salinity environments, mussels produced thin shells with a thicker external organic layer (periostracum), and an increased proportion of calcite (prismatic layer, as opposed to aragonite) and organic matrix, providing potentially higher resistance against dissolution in more corrosive waters. Conversely, in temperate, higher salinity regimes, thicker, more calcified shells with a higher aragonite (nacreous layer) proportion were deposited, which suggests enhanced protection under increased predation pressure. Interacting effects of salinity and food availability on mussel shell composition predict the deposition of a thicker periostracum and organic-enriched prismatic layer under forecasted future environmental conditions, suggesting a capacity for increased protection of high-latitude populations from ocean acidification. These findings support biomineralization plasticity as a potentially advantageous compensatory mechanism conferring Mytilus species a protective capacity for quantitative and qualitative trade-offs in shell deposition as a response to regional alterations of abiotic and biotic conditions in future environments. Our work illustrates that compensatory mechanisms, driving plastic responses to the spatial structure of multiple stressors, can define geographical patterns of unanticipated species resilience to global environmental change.

Acute oil exposure reduces physiological process rates in Arctic phyto- and zooplankton.

Arctic shipping and oil exploration are expected to increase, as sea ice extent is reduced. This enhances the risk for accidental oil spills throughout the Arctic, which emphasises the need to quantify potential consequences to the marine ecosystem and to evaluate risk and choose appropriate remediation methods. This study investigated the sensitivity of Arctic marine plankton to the water accommodated fraction (WAF) of heavy fuel oil. Arctic marine phytoplankton and copepods (Calanus finmarchicus) were exposed to three WAF concentrations corresponding to total hydrocarbon contents of 0.07 mg l-1, 0.28 mg l-1 and 0.55 mg l-1. Additionally, the potential phototoxic effects of exposing the WAF to sunlight, including the UV spectrum, were tested. The study determined sub-lethal effects of WAF exposure on rates of key ecosystem processes: primary production of phytoplankton and grazing (faecal pellet production) of copepods. Both phytoplankton and copepods responded negatively to WAF exposure. Biomass specific primary production was reduced by 6, 52 and 73% and faecal pellet production by 18, 51 and 86% with increasing WAF concentrations compared to controls. The phototoxic effect reduced primary production in the two highest WAF concentration treatments by 71 and 91%, respectively. This experiment contributes to the limited knowledge of acute sub-lethal effects of potential oil spills to the Arctic pelagic food web.

Blue mussel shell shape plasticity and natural environments: a quantitative approach.

Shape variability represents an important direct response of organisms to selective environments. Here, we use a combination of geometric morphometrics and generalised additive mixed models (GAMMs) to identify spatial patterns of natural shell shape variation in the North Atlantic and Arctic blue mussels, Mytilus edulis and M. trossulus, with environmental gradients of temperature, salinity and food availability across 3980 km of coastlines. New statistical methods and multiple study systems at various geographical scales allowed the uncoupling of the developmental and genetic contributions to shell shape and made it possible to identify general relationships between blue mussel shape variation and environment that are independent of age and species influences. We find salinity had the strongest effect on the latitudinal patterns of Mytilus shape, producing shells that were more elongated, narrower and with more parallel dorsoventral margins at lower salinities. Temperature and food supply, however, were the main drivers of mussel shape heterogeneity. Our findings revealed similar shell shape responses in Mytilus to less favourable environmental conditions across the different geographical scales analysed. Our results show how shell shape plasticity represents a powerful indicator to understand the alterations of blue mussel communities in rapidly changing environments.

Climate-change-induced range shifts of three allergenic ragweeds (Ambrosia L.) in Europe and their potential impact on human health.

Invasive allergenic plant species may have severe health-related impacts. In this study we aim to predict the effects of climate change on the distribution of three allergenic ragweed species (Ambrosia spp.) in Europe and discuss the potential associated health impact. We built species distribution models based on presence-only data for three ragweed species, using MAXENT software. Future climatic habitat suitability was modeled under two IPCC climate change scenarios (RCP 6.0 and RCP 8.5). We quantify the extent of the increase in 'high allergy risk' (HAR) areas, i.e., parts of Europe with climatic conditions corresponding to the highest quartile (25%) of present day habitat suitability for each of the three species. We estimate that by year 2100, the distribution range of all three ragweed species increases towards Northern and Eastern Europe under all climate scenarios. HAR areas will expand in Europe by 27-100%, depending on species and climate scenario. Novel HAR areas will occur mostly in Denmark, France, Germany, Russia and the Baltic countries, and overlap with densely populated cities such as Paris and St. Petersburg. We conclude that areas in Europe affected by severe ragweed associated allergy problems are likely to increase substantially by year 2100, affecting millions of people. To avoid this, management strategies must be developed that restrict ragweed dispersal and establishment of new populations. Precautionary efforts should limit the spread of ragweed seeds and reduce existing populations. Only by applying cross-countries management plans can managers mitigate future health risks and economical consequences of a ragweed expansion in Europe.

Genetic diversity and connectivity within Mytilus spp. in the subarctic and Arctic.

Climate changes in the Arctic are predicted to alter distributions of marine species. However, such changes are difficult to quantify because information on present species distribution and the genetic variation within species is lacking or poorly examined. Blue mussels, Mytilus spp., are ecosystem engineers in the coastal zone globally. To improve knowledge of distribution and genetic structure of the Mytilus edulis complex in the Arctic, we analyzed 81 SNPs in 534 Mytilus spp. individuals sampled at 13 sites to provide baseline data for distribution and genetic variation of Mytilus mussels in the European Arctic. Mytilus edulis was the most abundant species found with a clear genetic split between populations in Greenland and the Eastern Atlantic. Surprisingly, analyses revealed the presence of Mytilus trossulus in high Arctic NW Greenland (77°N) and Mytilus galloprovincialis or their hybrids in SW Greenland, Svalbard, and the Pechora Sea. Furthermore, a high degree of hybridization and introgression between species was observed. Our study highlights the importance of distinguishing between congener species, which can display local adaptation and suggests that information on dispersal routes and barriers is essential for accurate predictions of regional susceptibility to range expansions or invasions of boreal species in the Arctic.

Long photoperiods sustain high pH in Arctic kelp forests.

Concern on the impacts of ocean acidification on calcifiers, such as bivalves, sea urchins, and foraminifers, has led to efforts to understand the controls on pH in their habitats, which include kelp forests and seagrass meadows. The metabolism of these habitats can lead to diel fluctuation in pH with increases during the day and declines at night, suggesting no net effect on pH at time scales longer than daily. We examined the capacity of subarctic and Arctic kelps to up-regulate pH in situ and experimentally tested the role of photoperiod in determining the capacity of Arctic macrophytes to up-regulate pH. Field observations at photoperiods of 15 and 24 hours in Greenland combined with experimental manipulations of photoperiod show that photoperiods longer than 21 hours, characteristic of Arctic summers, are conducive to sustained up-regulation of pH by kelp photosynthesis. We report a gradual increase in pH of 0.15 units and a parallel decline in pCO2 of 100 parts per million over a 10-day period in an Arctic kelp forest over midsummer, with ample scope for continued pH increase during the months of continuous daylight. Experimental increase in CO2 concentration further stimulated the capacity of macrophytes to deplete CO2 and increase pH. We conclude that long photoperiods in Arctic summers support sustained up-regulation of pH in kelp forests, with potential benefits for calcifiers, and propose that this mechanism may increase with the projected expansion of Arctic vegetation in response to warming and loss of sea ice.

Does acute lead (Pb) contamination influence membrane fatty acid composition and freeze tolerance in intertidal blue mussels in arctic Greenland?

In their natural habitats, organisms are exposed to multiple stressors. Heavy metal contamination stresses the cell membrane due to increased peroxidation of lipids. Likewise, sub-zero air temperatures potentially reduce membrane functionality in ectothermal animals. We tested if acute lead (Pb) exposure for 7 days would influence survival in intertidal blue mussels (Mytilus edulis) after exposure to realistic sub-zero air temperatures. A full factorial experiment with five tissue Pb concentrations between 0 and 3500 µg Pb/g and six sub-zero temperatures from 0 to -17 °C were used to test the hypothesis that sub-lethal effects of Pb may increase the lethality caused by freezing in blue mussels exposed to temperatures simulating Greenland winter conditions. We found a significant effect of temperature on mortality. However, the short-term exposure to Pb did not result in any effects of Pb, nor did we find interactions between Pb and temperature. We analysed the relative abundance of major phospholipid fatty acids (PLFAs) in the gill tissue, but we found no significant effect of Pb tissue concentration on PLFA composition. Results suggest that Pb accumulation has limited effects on freeze tolerance and does not induce membrane damage in terms of persistent lipid peroxidation.