Diversity and flexibility of algal symbiont community in globally distributed larger benthic foraminifera of the genus Amphistegina.

BACKGROUND: Understanding the specificity and flexibility of the algal symbiosis-host association is fundamental for predicting how species occupy a diverse range of habitats. Here we assessed the algal symbiosis diversity of three species of larger benthic foraminifera from the genus Amphistegina and investigated the role of habitat and species identity in shaping the associated algal community. RESULTS: We used next-generation sequencing to identify the associated algal community, and DNA barcoding to identify the diatom endosymbionts associated with species of A. lobifera, A. lessonii, and A. radiata, collected from shallow habitats (< 15 m) in 16 sites, ranging from the Mediterranean Sea to French Polynesia. Next-generation sequencing results showed the consistent presence of Ochrophyta as the main algal phylum associated with all species and sites analysed. A significant proportion of phylotypes were classified as Chlorophyta and Myzozoa. We uncovered unprecedented diversity of algal phylotypes found in low abundance, especially of the class Bacillariophyta (i.e., diatoms). We found a significant influence of sites rather than host identity in shaping algal communities in all species. DNA barcoding revealed the consistent presence of phylotypes classified within the order Fragilariales as the diatoms associated with A. lobifera and A. lessonii, while A. radiata specimens host predominately diatoms of the order Triceratiales. CONCLUSIONS: We show that local habitat is the main factor influencing the overall composition of the algal symbiont community. However, host identity and the phylogenetic relationship among hosts is relevant in shaping the specific endosymbiont diatom community, suggesting that the relationship between diatom endosymbiont and hosts plays a crucial role in the evolutionary history of the genus Amphistegina. The capacity of Amphistegina species to associate with a diverse array of diatoms, and possibly other algal groups, likely underpins the ecological success of these crucial calcifying organisms across their extensive geographic range.

Integrating morphology and metagenomics to understand taxonomic variability of Amphisorus (Foraminifera, Miliolida) from Western Australia and Indonesia.

Foraminifera are a group of mostly marine protists with high taxonomic diversity. Species identification is often complex, as both morphological and molecular approaches can be challenging due to a lack of unique characters and reference sequences. An integrative approach combining state of the art morphological and molecular tools is therefore promising. In this study, we analysed large benthic Foraminifera of the genus Amphisorus from Western Australia and Indonesia. Based on previous findings on high morphological variability observed in the Soritidae and the discontinuous distribution of Amphisorus along the coast of western Australia, we expected to find multiple morphologically and genetically unique Amphisorus types. In order to gain detailed insights into the diversity of Amphisorus, we applied micro CT scanning and shotgun metagenomic sequencing. We identified four distinct morphotypes of Amphisorus, two each in Australia and Indonesia, and showed that each morphotype is a distinct genotype. Furthermore, metagenomics revealed the presence of three dinoflagellate symbiont clades. The most common symbiont was Fugacium Fr5, and we could show that its genotypes were mostly specific to Amphisorus morphotypes. Finally, we assembled the microbial taxa associated with the two Western Australian morphotypes, and analysed their microbial community composition. Even though each Amphisorus morphotype harboured distinct bacterial communities, sampling location had a stronger influence on bacterial community composition, and we infer that the prokaryotic community is primarily shaped by the microhabitat rather than host identity. The integrated approach combining analyses of host morphology and genetics, dinoflagellate symbionts, and associated microbes leads to the conclusion that we identified distinct, yet undescribed taxa of Amphisorus. We argue that the combination of morphological and molecular methods provides unprecedented insights into the diversity of foraminifera, which paves the way for a deeper understanding of their biodiversity, and facilitates future taxonomic and ecological work.

High dispersal capacity and biogeographic breaks shape the genetic diversity of a globally distributed reef-dwelling calcifier.

Understanding the role of dispersal and adaptation in the evolutionary history of marine species is essential for predicting their response to changing conditions. We analyzed patterns of genetic differentiation in the key tropical calcifying species of large benthic foraminifera Amphistegina lobifera to reveal the evolutionary processes responsible for its biogeographic distribution. We collected specimens from 16 sites encompassing the entire range of the species and analyzed hypervariable fragments of the 18S SSU rDNA marker. We identified six hierarchically organized genotypes with mutually exclusive distribution organized along a longitudinal gradient. The distribution is consistent with diversification occurring in the Indo-West Pacific (IWP) followed by dispersal toward the periphery. This pattern can be explained by: (a) high dispersal capacity of the species, (b) habitat heterogeneity driving more recent differentiation in the IWP, and (c) ecological-scale processes such as niche incumbency reinforcing patterns of genotype mutual exclusion. The dispersal potential of this species drives the ongoing range expansion into the Mediterranean Sea, indicating that A. lobifera is able to expand its distribution by tracking increases in temperature. The genetic structure reveals recent diversification and high rate of extinction in the evolutionary history of the clade suggesting a high turnover rate of the diversity at the cryptic level. This diversification dynamic combined with high dispersal potential, allowed the species to maintain a widespread distribution over periods of geological and climatic upheaval. These characteristics are likely to allow the species to modify its geographic range in response to ongoing global warming without requiring genetic differentiation.

Foraminifera as bioindicators of water quality: The FoRAM Index revisited.

Coral reefs worldwide are degrading at alarming rates due to local and global stressors. There are ongoing needs for bioindicator systems that can be used to assess reef health status, the potential for recovery following destructive events such as tropical storms, and for the success of coral transplants. Benthic foraminiferal shells are ubiquitous components of carbonate sediment in reef environments that can be sampled at minimal cost and environmental impact. Here we review the development and application of the FoRAM Index (FI), which provides a bioindicator metric for water quality that supports reef accretion. We outline the strengths and limitations of the FI, and propose how it can be applied more effectively across different geographical regions.

Evolutionary significance of the microbial assemblages of large benthic Foraminifera.

Large benthic Foraminifera (LBF) are major carbonate producers on coral reefs, and are hosts to a diverse symbiotic microbial community. During warm episodes in the geological past, these reef-building organisms expanded their geographical ranges as subtropical and tropical belts moved into higher latitudes. During these range-expansion periods, LBF were the most prolific carbonate producers on reefs, dominating shallow carbonate platforms over reef-building corals. Even though the fossil and modern distributions of groups of species that harbour different types of symbionts are known, the nature, mechanisms, and factors that influence their occurrence remain elusive. Furthermore, the presence of a diverse and persistent bacterial community has only recently gained attention. We examined recent advances in molecular identification of prokaryotic (i.e. bacteria) and eukaryotic (i.e. microalgae) associates, and palaeoecology, and place the partnership with bacteria and algae in the context of climate change. In critically reviewing the available fossil and modern data on symbiosis, we reveal a crucial role of microalgae in the response of LBF to ocean warming, and their capacity to colonise a variety of habitats, across both latitudes and broad depth ranges. Symbiont identity is a key factor enabling LBF to expand their geographic ranges when the sea-surface temperature increases. Our analyses showed that over the past 66 million years (My), diatom-bearing species were dominant in reef environments. The modern record shows that these species display a stable, persistent eukaryotic assemblage across their geographic distribution range, and are less dependent on symbiotic photosynthesis for survival. By contrast, dinoflagellate and chlorophytic species, which show a provincial distribution, tend to have a more flexible eukaryotic community throughout their range. This group is more dependent on their symbionts, and flexibility in their symbiosis is likely to be the driving force behind their evolutionary history, as they form a monophyletic group originating from a rhodophyte-bearing ancestor. The study of bacterial assemblages, while still in its infancy, is a promising field of study. Bacterial communities are likely to be shaped by the local environment, although a core bacterial microbiome is found in species with global distributions. Cryptic speciation is also an important factor that must be taken into consideration. As global warming intensifies, genetic divergence in hosts in addition to the range of flexibility/specificity within host-symbiont associations will be important elements in the continued evolutionary success of LBF species in a wide range of environments. Based on fossil and modern data, we conclude that the microbiome, which includes both algal and bacterial partners, is a key factor influencing the evolution of LBF. As a result, the microbiome assists LBF in colonising a wide range of habitats, and allowed them to become the most important calcifiers on shallow platforms worldwide during periods of ocean warming in the geologic past. Since LBF are crucial ecosystem engineers and prolific carbonate producers, the microbiome is a critical component that will play a central role in the responses of LBF to a changing ocean, and ultimately in shaping the future of coral reefs.

Bleaching-Associated Changes in the Microbiome of Large Benthic Foraminifera of the Great Barrier Reef, Australia.

Ocean warming is known to cause detrimental effects in coral reef fauna that rely on photo-symbiosis for survival. Microbial associations can facilitate the success of species across a range of environmental conditions, and play a role in the capacity of organisms to respond to climate change. In 2016, the Great Barrier Reef experienced its third mass bleaching event, with sea surface temperature rising to 1.3°C above long-term monthly summer averages. Here, I investigate the effects of ocean warming on the chlorophyll a (chl a) content and microbiome of the large benthic Foraminifera Amphistegina radiata. Samples were collected in January and April 2016, before and after the mass bleaching event. In total, 71 specimens were collected from two different depths (6- and 18-m) to investigate depth-dependant responses associated with changes in chl a and microbiome. Pigment analysis showed a significant reduction in chl a between time points in specimens collected at both depths. Reduction in pigmentation was accompanied by changes in the microbiome, and a significant interaction of depth and time was observed. Genus-level bacterial community associated with A. radiata was significantly different across depth and time. However, ocean warming affected populations at both depths to a similar extent, and resulted in change from a Betaproteobacteria-dominated assemblage in January to a more diverse bacterial community by April. Analysis of presence/absence and relative abundance of bacterial taxa revealed significant differences between time points at both depths analyzed. OTUs classified as Firmicutes, which were either absent, or present in very low relative abundances (<0.1%) across all sample groups in January, were identified in abundances as high as ∼20% in specimens collected from 18-m depth in April. Class-level shifts were observed in shallow-dwelling specimens, from high abundances of Betaproteobacteria to a high abundance and diversity of Actinobacteria. These results demonstrate the sensitivity of LBF to the effects of ocean warming, for which depth did not provide protection, and highlights the capacity of LBF to re-assemble bacterial communities after a disturbance. This study provides the first molecular-based demonstration of changes in foraminifera-associated bacterial assemblages during a bleaching event on a natural reef system.

Variation in sensitivity of large benthic Foraminifera to the combined effects of ocean warming and local impacts.

Large benthic foraminifera (LBF) are crucial marine calcifiers in coral reefs, and sensitive to environmental changes. Yet, many species successfully colonise a wide range of habitats including highly fluctuating environments. We tested the combined effects of ocean warming, local impacts and different light levels on populations of the common LBF Amphistegina lobifera collected along a cross-shelf gradient of temperature and nutrients fluctuations. We analysed survivorship, bleaching frequency, chlorophyll a content and fecundity. Elevated temperature and nitrate significantly reduced survivorship and fecundity of A. lobifera across populations studied. This pattern was exacerbated when combined with below optimum light levels. Inshore populations showed a consistent resistance to increased temperature and nitrate levels, but all populations studied were significantly affected by light reduction. These findings demonstrated the capacity of some populations of LBF to acclimate to local conditions; nonetheless improvements in local water quality can ultimately ameliorate effects of climate change in local LBF populations.

Symbiosis and microbiome flexibility in calcifying benthic foraminifera of the Great Barrier Reef.

BACKGROUND: Symbiosis is a phenomenon that allows organisms to colonise a wide range of environments and occupy a variety of ecological niches in marine environments. Large benthic foraminifera (LBF) are crucial marine calcifiers that rely on photo-endosymbionts for growth and calcification, yet the influence of environmental conditions in shaping their interactions with prokaryotic and eukaryotic associates is poorly known. RESULTS: Here, we used next-generation sequencing to identify eukaryotic photosynthesizing and prokaryotic microbes associated with the common LBF Amphistegina lobifera across a physio-chemical gradient on the Great Barrier Reef (GBR). We collected samples from three reef sites located in the inner-, mid- and outer-shelf regions of the northern section of the GBR. Results showed the consistent presence of Bacillaryophyta as the main eukaryotic taxa associated with A. lobifera across all reef sites analysed; however, the abundance and the diversity of prokaryotic organisms varied among reef sites. Inner-shelf specimens showed the highest diversity of prokaryote associates, with a total of 231 genotypes in their core microbiome. A total of 30 taxa were identified in the core microbiome across all reef sites. Within these taxa, Proteobacteria was the most abundant bacteria present. The presence of groups such as Actinobacteria was significantly correlated with inner-shelf populations, whereas the abundance of Bacteroidetes and Firmicutes was associated with A. lobifera collected from mid- and outer-shelf reef sites. CONCLUSIONS: We found that benthic foraminifera form stable and persistent symbiosis with eukaryotic partners, but flexible and site-specific associations with prokaryotic microbes that likely influence the ecological success of these crucial calcifying organisms on the GBR.

Effects of Elevated Temperature on the Shell Density of the Large Benthic Foraminifera Amphistegina lobifera.

This study investigated the effects of elevated temperature on shell density and Mg-ATPase activity of Amphistegina lobifera. This species is abundant in shallow reef habitats, and can be vulnerable to daily physicochemical fluctuations. To assess potential responses and acclimation mechanisms of A. lobifera to changing temperature conditions, we performed a blocked-design experiment exposing specimens collected from different reef sites (inshore and offshore) to three temperature treatments (Control: 24 °C, + 2 °C: 26 °C and + 5 °C: 29 °C) for 30 days. The final size and shell density of inshore reef foraminifera were unaffected by elevated temperature, and the enzyme activity in these individuals showed that they were able to acclimate to new temperature conditions. In contrast, offshore A. lobifera were more sensitive to changes in temperature, and heat stress caused growth impairment and inhibited Mg-ATPase activity. However, newly added chambers were not affected. These results suggested that Mg-ATPase plays an important role in regulating intracellular Mg2+ ions, but has little influence in the onset of calcification in A. lobifera. Moreover, it suggests that even though A. lobifera can regulate intracellular functions, local habitat seems to play a crucial role in determining how foraminifera respond to environmental changes.

Influence of local habitat on the physiological responses of large benthic foraminifera to temperature and nutrient stress.

Large benthic foraminifera (LBF) are important for reef sediment formation, but sensitive to elevated temperature and nutrients. However, it is possible that conspecific foraminifera living in different reef sites present divergent response to environmental shifts. We investigated how populations of Amphistegina lobifera from reef sites located along a temperature and nutrient gradient of the northern Great Barrier Reef respond and acclimate to elevated temperature and nitrate under lab-controlled conditions. Generalized linear mixed models showed that interaction between reef sites and temperature or nitrate conditions had a significant effect on survivorship, bleaching frequency and growth rates of A. lobifera. Further physiological analyses of antioxidant capacity and Ca-ATPase activity showed that populations collected from the inner-shelf sites (highest nutrient levels, largest temperature variation) were consistently able to acclimate to both parameters after 30 days. In contrast, foraminifera collected from the reef sites located in the mid- and outer-shelfs were significantly more sensitive to elevated temperatures and nitrate. Our results highlight the importance of local habitat in shaping the tolerance of LBF to changing environmental conditions; populations that live in stable environments are more sensitive to elevated temperature and nitrate, even within their fundamental tolerance range, than those that experience fluctuating conditions.

Ocean acidification induces biochemical and morphological changes in the calcification process of large benthic foraminifera.

Large benthic foraminifera are significant contributors to sediment formation on coral reefs, yet they are vulnerable to ocean acidification. Here, we assessed the biochemical and morphological impacts of acidification on the calcification of Amphistegina lessonii and Marginopora vertebralis exposed to different pH conditions. We measured growth rates (surface area and buoyant weight) and Ca-ATPase and Mg-ATPase activities and calculated shell density using micro-computer tomography images. In A. lessonii, we detected a significant decrease in buoyant weight, a reduction in the density of inner skeletal chambers, and an increase of Ca-ATPase and Mg-ATPase activities at pH 7.6 when compared with ambient conditions of pH 8.1. By contrast, M. vertebralis showed an inhibition in Mg-ATPase activity under lowered pH, with growth rate and skeletal density remaining constant. While M. vertebralis is considered to be more sensitive than A. lessonii owing to its high-Mg-calcite skeleton, it appears to be less affected by changes in pH, based on the parameters assessed in this study. We suggest difference in biochemical pathways of calcification as the main factor influencing response to changes in pH levels, and that A. lessonii and M. vertebralis have the ability to regulate biochemical functions to cope with short-term increases in acidity.

Influence of copper pre-exposure on biochemical responses of the sea anemone Bunodosoma cangicum to changes in oxygen availability.

The influence of copper on the ability of the intertidal sea anemone Bunodosoma cangicum to cope with reactive oxygen species generation associated with changes in oxygen availability was evaluated. Sea anemones were kept under control condition or pre-exposed (96 h) to dissolved copper (6.1 µg ± 2.7 µg/L) and then subjected to a 6-h period of hypoxia (0.5mg O2/L) followed by a 6-h period of re-oxygenation (7.5mg O2/L). Antioxidant capacity against peroxyl radicals (ACAP), superoxide dismutase (SOD) activity, reduced glutathione (GSH) concentration, lipid peroxidation (LPO) level, and ATP concentration were evaluated. Control sea anemones showed variations in SOD and LPO while copper pre-exposed sea anemones displayed changes in ACAP, GSH, LPO and ATP. However, no clear pattern of change over time was observed. ACAP was lower in copper pre-exposed sea anemones than in the control ones during hypoxia and recovery. SOD activity was increased during hypoxia and reduced shortly after recovery in control sea anemones. GSH concentration was higher in copper pre-exposed sea anemones than in the control ones in all experimental conditions. The LPO level increased shortly after recovery in both groups of sea anemones, being higher in control sea anemones than in copper pre-exposed ones. ATP concentration showed transient changes in copper pre-exposed sea anemones, being lower in these sea anemones than in control ones during recovery. These findings suggest that B. cangicum possess mechanisms to prevent oxidative stress generated by changes in oxygen availability associated with the tidal cycle, which can be disturbed by pre-exposure to copper.