previous studies on CWC, and likely increased their net calcification rates when compared to samples grown *in situ*.

Studies testing the resilience of CWC to ocean acidification used arbitrary feeding rates from no artificial feeding (Maier *et al.* 2012) to five times per week with *Artemia nauplii* and/or frozen Cyclops, Mysidacea, minced mussels, fish flakes (Tsounis *et al.* 2010; Orejas *et al.* 2011; Naumann *et al.* 2011; 2013a,b; Carreiro-Silva *et al.* 2014; Movilla *et al.* 2014a). Sometimes this diet was used for years before the experiment was carried out (e.g. Movilla *et al.* 2014a). According to our findings, corals acclimated to such optimal feeding rates might have their metabolism and nutritional habits completely changed, as well as their energy distribution. For instance, in the presence of artificial food in aquaria CWC tend to be permanently expanded, which is not the case in the field. Corals shown to calcify at reduced seawater Ω<sub>ara</sub> (Maier *et al.* 2013a; Movilla *et al.* 2014a; Carreiro-Silva *et al.* 2014) might only have been able to meet the extra energy demands as they were acclimated to abundant food conditions.

To test whether the OA responses of artificially fed corals differed from naturally fed corals we transplanted them to field conditions with normal and elevated pCO₂ where they were seen feeding naturally. In both cases, the corals calcified at rates that were 44% lower than corals kept at similar temperatures and pH in aquaria but artificially fed. Corals that we held in aquaria calcified faster than those in the field, likely due to greater food availability, but they were able to calcify at predicted levels of increased pCO₂ both in the field and in the laboratory. In conclusion, both corals in aquaria and the field, artificially and naturally fed respectively,

showed the same response to acidification: calcification rates were always unaffected by seawater carbonate chemistry.

To test whether the ocean acidification responses of artificially fed corals differed from naturally fed corals we transplanted them to field conditions with normal and elevated pCO₂ where they were seen feeding naturally. In both cases, the corals calcified at rates that were 44% lower than corals kept at similar temperatures and pH in aquaria but artificially fed. Corals that we held in aquaria calcified faster than those in the field, likely due to greater food availability, but they were able to calcify at predicted levels of increased pCO₂ both in the field and in the laboratory. In conclusion, both corals in aquaria and the field, artificially and naturally fed respectively, showed the same response to acidification: calcification rates were always unaffected by seawater carbonate chemistry.

**Fig. 4.6.2.** (a) Net and (b) gross calcification rates of the three Mediterranean cold-water corals held in aquaria during a one-month acclimation period at normal *pCO₂* levels followed by three months at normal and increased *pCO₂* levels. Net calcification was also measured for *Desmophyllum dianthus* transplanted to 350 m depth (Mooring) and at CO₂ seeps. Data are mean ± SD. Replicates per treatment are: (a) Acclimation, n = 14, 14 and 46; Normal and High *pCO₂*, 8, 8, 40 for *Caryophyllia smithii*, *Dendrophyllia cornigera* and *D. dianthus*, respectively; Mooring, 22 and CO₂ seeps 11 *D. dianthus*. (b) Normal and High *pCO₂*, n = 8 each species.

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>
## 4.7. Physiological advantages of dwarfing in surviving extinctions in high CO₂ oceans

LETTERS

PUBLISHED ONLINE: 20 APRIL 2015 | DOI: 10.1038/NCLIMATE2616

nature
climate change

# Physiological advantages of dwarfing in surviving extinctions in high-CO₂ oceans

Vittorio Garilli¹*†, Riccardo Rodolfo-Metalpa²,³*†, Danilo Scuderi⁴, Lorenzo Brusca⁵,
Daniela Parrinello⁶, Samuel P. S. Rastrick⁷, Andy Foggo⁸, Richard J. Twitchett⁹,
Jason M. Hall-Spencer⁸ and Marco Milazzo¹⁰

Among the different model organisms I have used to test for the effect of OA on the ability of organisms to calcify and face acid stress, this study is at the top of the list. Indeed, during my fieldwork at the CO₂ seeps in Vulcano, I observed, buried in the sediment at very low pH water (and in the sediment where the pH is even lower than in the surrounding seawater), shells of two species of tiny gastropods quite dissolved by the undersaturated seawater but still alive. Thanks to the collaboration with a malacologist, Vittorio Garilli, we compared their size to other populations around Sicily and found that their sizes were reduced. Further measures for their calcification at the IAEA lab and their shell structure and periostracum composition at the Centre Scientifique de Monaco, allow us to demonstrate that these species (i) despite the very low pH calcified, although at lower levels; (ii) had a periostracum that protected the inner shell from dissolution (as we previously found for Mytilus galloprovincialis in Ischia (Rodolfo-Metalpa et al. 2011); (iii) adapted to such conditions by a mechanism called Lilliput effect.

Here, we show that two gastropod species adapted to acidified seawater at shallow water CO₂ seeps were smaller than those found in normal pH conditions and had higher mass-specific energy consumption but significantly lower whole animal metabolic energy demand. These physiological changes allowed the animals to maintain calcification and to partially repair shell dissolution. These observations of long-term chronic effects of increased CO₂ levels forewarn of changes we can expect in marine ecosystems as CO₂ emissions continue to rise unchecked and support the hypothesis that ocean acidification contributed to past extinction events. The ability to adapt through dwarfing can confer physiological advantages as the rate of CO₂ emissions continues to increase.

### Introduction to the study

In the immediate aftermath of the mass extinction events, many of the survivors were smaller than before (e.g. brachiopods, gastropods, bivalves and shelled cephalopods); a phenomenon termed the ‘Lilliput effect’. After the most severe Late Permian extinction, gastropod species remained relatively small for millions of years. One hypothesis is that this dwarfing was an adaptation to ocean acidification to mitigate against the increased energy cost of carbonate secretion. To test for this hypothesis, we studied two nassariid gastropods Nassarius corniculus and *Cyclope neritea*, which were abundant on coarse sand and gravel at ca 100 m from the main seeps in Vulcano. These species are widespread in coastal lagoons and salt marshes in the Mediterranean as well as at shallow water hydrothermal seeps (e.g. off Milos and Pantelleria).

We know that populations of *C. neritea* and *N. corniculus* had developed at the CO₂ seeps since their shells had paucispiral protoconches so these snails lack a planktotrophic larval stage.

Seawater off Vulcano has been acidified since the late Pleistocene and a dwarf population of *N. corniculus* has been present for at least 30 years providing an opportunity to study the chronic effects of ocean acidification on gastropods submitted to high CO₂ levels over multiple generations.
## The experiment

Here, we compared gastropods living in naturally acidified shallow-water conditions near CO2 seeps off Vulcano with those at sites with ambient seawater pH to test for their ability to cope with acidification and, potentially, adapt. Shell morphology, dissolution and repair were examined using scanning electron microscopy. Animals collected in September and November 2011 were incubated in aquaria at CO2 levels similar to those measured at reference and CO2 seep sites and their gross calcification rates were measured using $ ^{45}Ca $ at both seawater pH levels. Rates of metabolic oxygen uptake of individuals from CO2 seeps and a reference site were determined by Stop-Flow respirometry at pH 6.5 or pH 8.1 respectively within 24 hours of collection.

## Main results and discussion

<figure><img src="image_23.png" /></figure>

**Fig. 4.7.1.** On the left side. Shells of *Cyclope neritea* (a, c) and *Nassarius corniculus* (b, d). Samples living at CO2 seeps (a, b) showing shell dissolution and apex truncation when compared to shells collected at reference site C1, ambient pH (c, d). Bare scales are two mm (a, b) and five mm (c, d). On the right side. Shell dissolution, apical truncation and repairing calcification process in *Nassarius corniculus* living at CO2 seeps. A. Dissolution first affects shell periostracum (yellow arrows) and then shell mineralised layers (red arrows). B-c. Scanning electronic microscopy images showing shell periostracum (P) fractures (b), followed by shell dissolution (c). d. Juvenile specimen preserving the paucispiral protoconch, and showing dissolution of the sutural area (arrows) as first step toward shell apical truncation. E. Adult specimen from CO2 seeps showing a dramatic effect of dissolution. Although it lacks shell apex, the animal was able to repair its shell closing the internal cavity. F. Shell cross section of a sample from reference site C1 artificially broken to show the normal empty space between the outer part of the shell and the columella.

By comparing the size of individuals from the seeps and the reference sites, we found that they were significantly smaller at CO2 seeps, showing 1.26 & 1.37 mean (log) volume ratios between ambient and acidified conditions. These shell volume shifts, which approximate to biomass shifts, provide a means of directly comparing experimental and fossil data. In his global study of clade-level size change in gastropods Payne (2005) recorded a shell volume shift of 1.45 mean (log) volume between the Late Permian and Early Triassic, which is of the same order as that recorded by our experimental data.
In *N. corniculus*, shell integrity was affected by the corrosive seawater at CO₂ seeps; pockmarks were present on the teleoconch in almost all samples, corresponding to zones where the periostracum was affected by swelling and breaking. Deterioration of the periostracum is the first step before mineralized shell layers undergo dissolution as previously shown in mussels at shallow and deep CO₂ seeps. For *C. neritea* the early shell whorls were also corroded at the CO₂ seeps, with loss of the protoconch and pitting on the first teleoconch whorl.

Gross calcification rates in *N. corniculus* collected at CO₂ seeps (pH~T~ 7.2) were generally lower than samples from the reference site (pH~T~ 8.0) whereas they were consistently greater in *C. neritea* from CO₂ seeps than those living in normal conditions. Nassarius corniculus from high-CO₂ environments consistently maintained significantly lower calcification rates than those at reference sites even when they were removed from elevated CO₂ and cultured at pH~T~ 8.0 showing adaptation to acidified environments rather than plasticity of calcification in response to pH conditions. Our findings add to a growing body of evidence that calcification responses to acidification are both species-specific, which is unsurprising since there can be a great deal of biological control over calcification, and adaptive rather than plastic.

<figure><img src="image_24.png" /><figcaption>**Fig. 4.7.2.** Gross calcification (GC) and metabolic oxygen consumption (MO₂) in *Nassarius corniculus* and *Cyclope neritea* across normal and acidified sites. **A.** GC in September and November at pH measured at collection sites. **b-c.** GC of *N. corniculus* in September (a) and November (b) to measure the response of samples collected within the seeps and at Reference site and incubated at crossed pH treatments. Arrows show the GC change on samples incubated at pH of collection (CO₂ seeps or Reference C1 sites) and the pH of the incubation (pH~T~ 8.0 and 7.2). Oxygen uptake as an index of MO₂ expressed as body mass adjusted MO₂ (**d**) and whole animal MO₂ (**e**).</figcaption></figure>
## 4.8. Papua New Guinea CO₂ seeps, Normanby and Ambitle Islands

In 2011, Fabricius and collaborators published the first of a series of studies showing the effect of ocean acidification on a coral reef in PNG. This study has been a milestone research since the Authors demonstrated that the effect of a shallow water CO₂ seep, lowering the surrounding seawater pH at the level expected for 2100, was deleterious for the reef. There, branching corals were absent and only massive *Porites* dominated the site. In addition, their growth was reduced confirming model predictions that ocean acidification, together with temperature stress, will probably lead to severely reduced diversity, structural complexity and resilience of Indo-Pacific coral reefs within this century. To support these conclusions, the Authors used the Normanby CO₂ seeps and two more seep sites, the latter actually not at the same ecological value as Normanby because very limited in space and quite sulphuric. Overall, this study has accelerated several studies and collaborations in this field and motivated a decade of research to find out how future reefs will be affected by acidification because according to what was found in Normanby, they certainly will be.

In 2016, within the framework of the ANR-funded project CARIOCA ‘Coral Reefs Acclimatization to Ocean Acidification”, I performed 3 expeditions and studied the Normanby seeps. To do that, I used a local diving boat, the Chertan and invited Prof K. Fabricius (AIMS, Australia) for the first mission. In addition, I found in the literature a promising site, in the East Britain province, previously studied from a geochemistry point of view by the team of Prof T. Pichler (University of Bremen, Germany) because the CO₂ vents were hot and rich in arsenic. In parallel to Normanby, I investigated this site 4 times, the first in collaboration with T. Pichler.

<figure><img src="image_25.png" /><figcaption>**Fig. 4.8.1.** Localisation of the two volcanic sites were the CARIOCA project studied the effect of CO₂ seep emissions on coral reefs.</figcaption></figure>
4.8.1. CO₂ seep of Normanby Island (Upa-Upasina reef)

<figure><img src="image_26.png" /><figcaption>**Fig. 4.8.1.2.** Aerial picture of the Upa-Upasina reef. The shadow area is where the 2-3 vents with important gas emissions were found. Sparse seeps were also found in shallow water closer to the beach. The two yellow lines indicate the area where the seawater pH varied according to the dominant current and weather conditions.</figcaption></figure>

This site has been used by our Australian colleagues from AIMS, JCU and several of the most renewed scientists working on the OA topic from different countries. A quick research on Google Scholar using "Normanby CO2 seeps", and "Ocean acidification + CO2 seeps" as keywords for the research has given more than 40 studies, most of them published in very high IF journals, all performed at the Normanby CO2 seep site. This is a non-exhaustive list of contributions, describing the changes in habitat and community composition, to the effect on fish, forams, bacteria, etc from molecular to the behaviour level. However, only some focused on corals and metabolic changes, including photosynthesis and calcification. Among them, is the first study, by Fabricius et al (2011), where the authors described for the first time the site. They found that the low pH, which average was as expected for the end of the century, caused the reduction in coral diversity, recruitment and abundance of structurally complex framework builders, and shifts in competitive interactions between taxa. However, coral cover remained constant between pH 8.1 and ~7.8, because massive *Porites* corals established dominance over structural corals, despite low rates of calcification. Reef development ceased below pH 7.7. This lack of reef complexity and altered settlement substrata was suggested to become a bottleneck for corals coral communities' establishment under ocean acidification (Fabricius et al. 2014; Sunday et al. 2017) directly, due to the difficulty for recruits to set (Fabricius et al. 2017; Noonan et al. 2018), and indirectly by reducing the habitat used by zooplankton, which is part of the diet of corals, fish and other reef species, therefore affecting their ecological success (Smith et al. 2016). At the molecular level, while Kenkel et al. (2018) showed that corals have undergone long-term acclimatization to natural variation in *pCO₂*, with only 61 genes differentially expressed in response to *pCO₂* environment, a recent study by Leiva et al. (2023) demonstrated on the same data coral adaptation at the high CO₂ conditions. The authors demonstrated that adaptation to ocean acidification involved the three main compartments of the coral holobiont. They identified 441 coral host candidate adaptive genes involved in
calcification, response to acidification, and symbiosis; population genetic differentiation in dinoflagellate photosymbionts; and consistent transcriptional microbiome activity despite microbial community shifts. Corals from natural analogues to future ocean conditions harbour beneficial genetic variants with far-reaching rapid adaptation potential. The most complete studies on the physiology of some coral species at the CO₂ seep were performed by Strahl et al (2015; 2015) on two abundant species *Porites* sp. and *Pocillopora damicornis* and two more rare species *Acropora millepora* and *Seriatopora hystrix* at the seep site. They found that the net photosynthesis rates increased considerably in *Porites* spp. and *A. millepora* and slightly in *P. damicornis* at increased pCO₂, but remained unaltered in *S. hystrix*. Rates of light calcification declined in *S. hystrix* at high pCO₂ but were unaffected by pCO₂ in the other three coral taxa. Dark calcification rates remained unchanged in massive Porites and P. damicornis, but were drastically reduced at high pCO₂ in *A. millepora* and *S. hystrix*. However, skeletal densities were similar at both seep and control sites in all coral taxa investigated. Overall the Authors recognised that pCO₂-tolerant corals were characterized by an increased ability to acclimatize to ocean acidification, e.g. by maintaining net calcification. Thus, robust corals, such as *Porites* spp. and pCO₂, are more likely to persist for longer in a future high pCO₂ world than those unable to acclimatize. What the Author did not highlight is that the two less robust species, which are rarer at the seeps, are also the species more sensitive to heat stress and that have been decimated at the site during repeated bleaching events (pers. observation).

<figure><img src="image_27.png" /><figcaption>**Fig. 4.8.1.3.** Reef dominated by massive corals *Porites* spp. in Upa-Upasina reef and sparse CO₂ seeps. Signs of severe colony mortality were evident.</figcaption></figure>
4.8.2. CO₂ seep of Ambitle Island (Tutum bay)

<figure><img src="image_28.png" /><figcaption>**Fig. 4.8.2.1.** Aerial picture of the Tutum Bay reef. The shadow area is where the 3-4 cratera with important gas emissions were found. Sparse seeps were also found everywhere in the bay. The two yellow lines indicate the area where the seawater pH was lower than the pH measured at the adjacent reference reef.</figcaption></figure>

I added this site in the ANR Carioca, originally based only on the site of Normanby, because I read a paper on the journal Marine Chemistry (Pichler et al. 1999) where some amazing pictures showed massive degassing at 8 m depth in a coral reef. Prof Pichler discovered this site likely in 2016 and performed several expeditions and published very important contributions. Prof Pichler and colleagues paid lower attention to the effect of high CO₂ on the reef, likely because it was too early for this topic, but as a geochemist, they fully described the emitted fluids, their origin and how they change in the seawater column (Pichler & Dix 1996). For this site already exists a complete chemical description of the hydrothermal fluids, sediments pore waters including trace elements contents as well as a general census of some key-habitat species, including archaea and bacteria (e.g., Akerman et al. 2011; Karlen et al. 2010; Meyer-Dombard et al. 2012). These studies showed that the concentrations of Si, Mn, Cs, Fe, Zn, and particularly As were much higher in the hydrothermal fluid and that they were slightly elevated in Tutum Bay seawater when compared to “normal” seawater (Pichler et al. 1999a; Price and Pichler 2005; Price et al. 2013b). While metal pollution, however is not specific to coral reefs around CO₂ vents as many reefs worldwide (e.g. Costa Rica, Panama, Red Sea, Thailand, New Caledonia) can have high metal levels (Ali et al. 2011; Biscéré et al. 2017, 2015; Fujita et al. 2014; Guzmán and Jiménez 1992; Moreton et al. 2009; Tanaka et al. 2013; Whitall et al. 2014), and many experimental studies have emphasized that some of them can play key roles in the functioning of corals (Biscéré et al. 2018, 2017; Ferrier-Pagès et al. 2001), Arsenic could become toxic and therefore be a great bias in using this site as natural lab. With this in mind we performed the first expedition in Ambitle in collaboration with Prof T. Pichler with the aim at providing a full description of the CO₂ variation in the Bay, and more importantly the concentration of As on the bottom cora reef level. During this first cruise, and 3 more we reciprocally transplanted (control and high CO₂ sites) three coral species during 7 months, collected cores *Porites*, skeletons of a series of coral species, perfomed on board incubations
and field incubations for the determination of their metabolic rates, transplanted corals near a hot source, re-transplanted fragments of 7 species between sites, did experiments on the nutritional needed of corals exposed to high pCO2, measured diversity, etc etc. Some of these data have been published, other are waiting for collaborators, and other unfortunately will never be published.

Overall, this site is in my opinion the best CO2 seep available today, unfortunately is in a very
remote geographical position. Indeed, without a good research vessel it is impossible to perform
any research there. It is the best because we clearly showed that there is no effect of arsenic and
that other potential harmful elements and gasses emitted by seeps were not bioavailable at the
bottom seabed, and either dispersed or fixed in the surrounding seawater column (Pichler et al.
1999). We measured several time the variability of the seawater carbonate chemistry in the bay
showing that although seawater pH varied, its variability was tiny when compared to the
variability measured at the Normanby site. This clearly affected the ability of corals to keep
elevated calcification pH values and, as a consequence their ecological success (Comeau et al
2021). This site was surprisingly rich and diverse with around one hundred of coral species
(Shlessinger, unpubl data). This findings was in contrast to what studies reported from the most
famous Normanby site and questioned the validity of some previous findings that established
some corals as the winners vs other losers. I mentioned that I performed two transplantations
between the control and the seep sites. Unfortunately, I have been unlucky on that, and for two
reasons. The first transplanting was a real success and all corals survived to the 7 months. It
was planned to measure their calcification using the alkalinity technique, which measures the
amount of carbonate used to calcify during the incubations we performed *in situ* inside semi-
autonomous respirometric chambers; in addition, their transcriptomic, and the change induced
by the new environment had to be measured by my colleagues, partners of the ANR. Neither
the alkalinity method nor the collaboration with my colleagues worked and these two important
pieces of the puzzle remain missing to date. During a second transplantation, I measured the
coral growth using the buoyant weight technique, and I did it on the land, at the local tribu (a
real experience!), using a balance and a battery. Here again, I was unlucky since the corals
transplanted at the control were lost, probably during a previous storm. Surprisingly, corals at
the CO2 seeps survived and grew a lot, but I do not have the data to compare these results to a
reference condition. More data and findings are still to be published from the expeditions
performed at Tutum Bay.
<figure><img src="image_29.png" /><figcaption>**Fig. 4.8.2.2.** Photomosaic showing the transplatented samples of *Porites* at the beginning and after 7 month at CO₂ seeps in Ambitle; the 'cathedral', i.e., the transplantation we did to study the combined effect of warming (note the tube trapping a hot emission) and OA on transplanted corals; some working on land with the special help from local people; the 6 respirometric chambers we used to measure the coral respiration; the second transplant setting and a nice vent eroding a *Porites* colony.</figcaption></figure>
<figure><img src="image_30.png" /></figure>

**Fig. 4.8.2.3.** One of the large CO₂ degassing (vent) and sparse seeps observed in Tutum Bay at ca. 8-10 m depth.

<figure><img src="image_31.png" /></figure>

**Fig. 4.8.2.4.** Some examples of the reef diversity found in Tutum Bay.
## 4.9. pH variability at volcanic CO₂ seeps regulates coral calcifying fluid chemistry

RESEARCH ARTICLE

<figure><img src="image_32.png" /></figure>

WILEY

# pH variability at volcanic CO₂ seeps regulates coral calcifying fluid chemistry

Steeve Comeau¹,² ⚫

Christopher E. Cornwall²,³ ⚫

Tom Shlesinger⁴ ⚫

Mia Hoogenboom⁵

Ralph Mana⁶

Malcolm T. McCulloch²

Riccardo Rodolfo-Metalpa⁷

This study apparently deviates from my field of research on coral physiology since it applies geochemical techniques on the skeletal composition of scleractinian corals living at the CO₂ seeps in PNG to investigate their ability to calcify at low pH conditions. In reality, although I continue to lack this expertise, I have always looked to collaborate with specialists in that exciting field of research. Indeed, I have been co-author of 9 studies analysing lithium isotope compositions (⁷Li/⁶Li), Li/Ca, Li/Mg, ⁸⁸Sr/⁸⁶Sr, B/Ca ratios of shallow-water and deep-sea corals (Rollion-Bard et al. 2009; Trotter et al. 2011; Montagna et al. 2014; Fruchter et al. 2016), carbon and oxygen-isotope analyses on mussels (Hahn et al., 2012), shell structural limpets (Langer et al. 2014, 2018). Geochemical techniques definitely allow investigating calcification processes at a fine scale and the outcome from using such techniques has greatly improved our knowledge on the effect of OA on the ability of corals (and not only to calcify under past and future environmental conditions. For that, I recently supervised the PhD of Clement Tanvet that coupled geochemistry to the physiology of corals from Bouraké. In the Comeau et al paper I have been lucky to collaborate with a panel of great experts of different fields, from which I learned a lot. This study indeed links boron skeletal composition measurements of 7 coral species living in and out of two CO₂ seeps sites in PNG with their distribution and abundance, and the variability of the main environmental parameters. By combining geochemical, ecological, and chemical approaches, our study demonstrates that even under seawater pH lower than that predicted by the end of the century because of climate change, a variety of corals that exert strong control on their calcifying fluid might still be able to calcify, grow, and persist.

### Introduction to the study

To form their skeleton, corals can modify the chemical conditions of the calcifying fluid to facilitate the mineralization process. Similarly, increasing seawater DIC under OA elevates DICf (Comeau et al. 2018). While this increase in DICf could partially alleviate the negative effects of decreasing pHcf (Cornwall et al. 2018; Schoepf et al. 2017) large uncertainties exist in the literature about the magnitude and physiological controls of these effects. In addition, a large range of coral responses to treatments with different levels of pH variability has been reported in laboratory experiments, ranging from no measurable impacts (Camp et al. 2016) to positive offsets against OA (Comeau et al. 2014b). This range of impacts could arise due to species-specific responses to pH variability, but also because of differences among studies in the frequency and magnitude of pH fluctuations used in the experiments. Studies that attempt to shed light on the reasons coral calcification rates varied under OA conditions in experiments are therefore needed. But the reason that pushed toward this research comes from my observation and comparison of two different CO₂ seeps in PNG, the Normanby site, where only a few coral species resist OA conditions, as reported by Fabricius and colleagues, and the Ambitle site, where we observed a rich and diverse reef (Pichler et al. 2019). In Normanby, the deleterious effects of low pH on the physiology, abundance, and diversity of calcareous organisms were reported (Fabricius et al. 2011, 2017) causing the reduction of species diversity
and evenness compared to control sites. The site indeed is dominated by massive *Porites* spp. likely due to its capacity to maintain elevated pHcf under a large range of seawater pH, as demonstrated both *in situ* (Wall et al. 2016; 2019a) and *ex situ* (Comeau et al., 2019). This could be a general mechanism of certain coral species under low pH that favours their presence in naturally acidified sites (Wall et al. 2019a; 2019b). The main question behind this project was: “Were Fabricius and colleagues really in the right when using the Normanby site they demonstrated that OA has a dramatic effect on corals?”. This study does not respond to this question but contributes to clarifying it, and many results remain to be published.

In this study, we aimed firstly to understand whether changes in mean seawater pH and the magnitude of variability in seawater pH affect the control of coral calcifying fluid in the field for multiple species. Secondly, we aimed to explore whether the capacity of species to better regulate calcifying fluid (CF) chemistry is correlated to species relative abundances at sites with different pH and DIC conditions. To that end, we utilised two natural CO₂ seep locations with distinct CO₂ variability, relatively limited in Ambitle, and large in Normanby. Using physiological measurements and field observations of coral species abundances, we tested three complementary hypotheses: 1) corals growing in acidified sites can maintain chemical conditions optimal for calcification in their calcifying fluid (i.e., pHcf homeostasis), 2) the most abundant corals in acidified sites are the ones with the best control on their calcifying fluid, and 3) seawater pH variability will alter both ecological outcomes and corals’ CF chemistry.

## Main results and discussion

*Seawater pH variability.* Ambient mean pH values were 8.01 and 7.96 in the control sites, and 7.64 and 7.51 in the seeps sites in Ambitle and Normanby, respectively. Seawater pH variability was considerably larger at the Normanby seep site where the pH dropped down as low as 6.64 pH units. During the entire time frame of pH logging, corals were exposed to low pH conditions (i.e., pH ranging between 7.6–7.8) for 60% of the time at Ambitle, and for 31% of the time at Normanby. Very low pH values (i.e., pH < 7.6) were less frequent in Ambitle than in Normanby (24% and 43% of the time, respectively). Similarly, high pH values (> 7.8) were less frequent in Ambitle than in Normanby (16% and 26% of the time, respectively). Despite similar mean pH at both CO₂ seeps, corals in Normanby experienced much larger variations in seawater pH.

<figure><img src="image_33.png" /><figcaption>**Fig. 4.9.1.** Seawater pH measured at the Normanby and Ambitle reef over 6 days.</figcaption></figure>
This includes frequent records of pH as low as 6.6, but also values close to ambient conditions (pH 8.0) very frequently. pH in Normanby occasionally varied by as much as one pH unit in less than one hour. This large variability in pH is likely driven by the shallow topography of the Upa-Upasina reef, where the depth varies between ~1–4 m, which makes seawater pH extremely dependent on water mixing caused by local wind conditions (Fabricius et al. 2011). In contrast, Pichler et al. (2019) showed that in Tutum Bay (Ambitle Island) the main seep and other associated sparse seeps change the seawater carbonate chemistry of the whole bay (1-8 m deep). In Tutum Bay, pH variability is mostly driven by tides, with lower pH associated with low tides.

<figure><img src="image_34.png" /><figcaption>**Fig. 4.9.2.** Violin plot showing the *in situ* pH (n > 15,000 for each site) measured using autonomous pH sensors SeaFET at both Ambitle and Normanby seeps and respective control sites during fieldwork in September 2016 and May 2017.</figcaption></figure>

*pH of coral calcifying fluid.* Across the 14 coral species studied in Normanby average pHcf was higher in the control site (8.43 ± 0.02, mean ± SE, n = 42) than in the seeps site (8.39 ± 0.02, mean ± SE, n = 42, t-test, p < 0.05). The highest pHcf was observed in *Galaxea fascicularis* at the control site (8.57 ± 0.01, n = 3), while the lowest was measured in *Favites halicora* at the seeps site (8.27 ± 0.01). pHcf was significantly lower at the seep site compared with the control site in five species (t-test, p < 0.05 for all five), with delta pHcf the lowest in *Acropora samoensis* (-0.15).

In Ambitle, for the 8 species pooled together, pH<sub>cf</sub> was on average significantly higher (t-test, p < 0.05) in the control site (8.46 ± 0.02, n = 24) than in the seeps site (8.35 ± 0.02, n = 24). The highest pHcf was measured in *Acropora tenuis* at the control site (8.54 ± 0.04), while the lowest was found in *Echinopora lamellosa* at the seeps site (8.23 ± 0.03). pH<sub>cf</sub> was higher in the control site compared with the seeps site in all species (t-test, p < 0.05 for all) but one, *Montipora foliosa*, which showed no differences. As a result, delta pH<sub>cf</sub> which is equal to pHcf seeps – pHcf ambient varied between -0.19 in *Echinopora lamellosa* and -0.02 in *Montipora foliosa*. We suggest that the disparity between responses of pHcf in the different locations is most likely due to the larger range and variability in pH at Normanby.
<figure><img src="image_35.png" /></figure>

**Fig. 4.9.3.** Calcifying fluid carbonate chemistry estimates of 8 coral species from the control and seeps sites in Ambitle Island (A, C), and of 14 coral species from the control and seeps sites in Normanby Island (B, D). A, B) pH of the calcifying fluid (pH<sub>cf</sub); C, D) Dissolved inorganic carbon at the site of calcification (DIC<sub>cf</sub>). Blue and red colours indicating the control and seeps data, respectively. Dashed lines represent the pooled pH<sub>cf</sub> and DIC<sub>cf</sub> mean across all species in each site. Asterisks indicating species in which significant differences were found. All data presented as mean ± SE, with n = 3.

*Coral abundance and calcifying fluid chemistry.* In Normanby, most of the studied species were either similarly abundant (absolute abundance) in both sites or more abundant at the control site than at the seeps site. By contrast, this pattern was not observed in Ambitle, where three species were more abundant at the control site than at the seeps site (*G. fascicularis*, *P. damicornis*, and massive *Porites* spp.) but two other species were more abundant at the seeps site than at the control site (*A. nana* and *M. foliosa*). Although being relatively rare in Ambitle, two more species had opposing abundances: *A. tenuis* was more abundant at the seeps site than at the control site (0.22 ± 0.9 and 0.06 ± 0.2 mean abundance per transect, respectively) while *E. lamellose* was more abundant at the control site than at the seeps site (1 ± 1.1 and 0.05 ± 0.2 mean abundance per transect, respectively).
<figure><img src="image_36.png" /><figcaption>**Fig. 4.9.4.** Abundance of the studied species in **A)** Normanby Island; and, **B)** Ambitle Island. The abundance is presented as the number of colonies per belt transect with points indicating individual belt transect data (n = 15 at each site in Normanby, and n = 18 at each site in Ambitle). To aid the visualization of panel B, in Ambitle, three outlying data points were excluded. Two of these points were values > 100 for *G. fascicularis* in the control and the third was a value > 80 for *Porites* spp. in the control. Blue and red colours indicating the control and seeps data, respectively. Center lines of the box plots indicate the medians, boxes indicate the lower and upper quartiles, and whiskers indicate 1.5x interquartile range.</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>**Fig. 4.9.5.** Violin plot of the number of species per transect found in Ambitle and Normanby in May 2017 September 2016, respectively.</figcaption></figure>

Overall, our results support the idea that species-specific coral physiology controls responses to OA *in situ* (as observed with seaweed inorganic carbon use previously; Cornwall et al. 2017) with no or minor relations to coral phylogeny and morphological traits. Moreover, our findings suggest that coral control of carbonate chemistry in the calcifying fluid might influence their ecological success under OA. This manifested in Ambitle, where pH variability is low and where corals with the highest control on their calcifying fluid pH generally had a higher change in relative abundance between the CO₂ seeps and control sites. However, these traits only provide partial information and further research at a more extensive set of sites is now required. In contrast, our study also shows that large pH variability, such as the one found in Normanby, could mask the link between species' physiological traits and ecological success, highlighting the importance of characterizing environmental conditions *in situ* at high temporal resolution.
4.10. The semi-enclosed lagoon of Bouraké

<figure><img src="image_38.png" /><figcaption>**Fig. 4.10.1.** A picture of the semi-enclosed lagoon of Bouraké, surrounded by a rich mangrove forest, taken by M. Nitschke during the landing of the plane at the nearby airport.</figcaption></figure>

In December 2015 David Suggett (University of Technology of Sydney) I worked with in Ischia, asked me if in New Caledonia there was a mangrove area inhabited by corals, which means that the mangrove should not be in a river and estuary area. Thanks to the precious suggestion of the team of specialists in mangrove forests, led at IRD by Dr Cyril Marchand, I visited the Bouraké mangrove and performed the first observations and measurements. At this site, 150 km from Nouméa, lagoon waters flow inside the system with the rising tide, circulate and then exit at the falling tide according to a semi-diurnal cycle. The depth of the system varies from a few centimetres to more than 6 m. The channel, which is more than 80 m wide, penetrates the mangrove and creates large pools over a total area of more than 60,000 m² (without considering the mangrove area). The site is very rich in corals that form true fringing reefs at several points of the system, mostly at the end of the terraces that extend from the mangrove toward the channel. The most dense and compact corals live at the border of these terraces, likely because the terraces are always exposed to the air during the low tide. At each tide, new lagoon water enters through the channel into the vast inner basin of the mangroves. During this journey, the water chemistry changes, due to metabolic reactions in the sediment, coral reefs and mangrove habitats, and it mixes with the already present water which is more acidic, hot and deoxygenated.
<figure><img src="image_39.png" /><figcaption>**Fig. 4.10.2.** Satellite picture of the mangrove area of Bouraké. The blue arrow indicates the direction of the rising tide that brings new seawater from the external lagoon, through the channel and the back reservoir, and finally the mangrove forest. On the contrary, the red arrow indicates the opposite displacement of the hot, acid and deoxygenated seawater from the mangrove toward the external lagoon.</figcaption></figure>

The first measurements of the daily pH fluctuations, during a 24-hour cycle, revealed the value of this unique site for studying the capacity of corals to acclimatize and adapt to extreme conditions. Even at high tide, the water in the system never returns to "normal" values. For example, the maximum pH values are around 7.9 (the normal pH of the ocean is currently 8.05 and is predicted to decrease to 7.7-7.8 in 2100). Only during very high tides does the pH value become 8.0. At falling tide, seawater becomes more acidic and oxygen-depleted as the system begins to drain. The large volumes of water that were inside the mangrove forest enter the system and are then discharged into the lagoon. At low tide, near corals, the pH reaches the extreme value of 7.3 and $O_2$ has a concentration of 2 mg L$^{-1}$ (the normal concentration of $O_2$ at the coast is 4-6 mg L$^{-1}$).
<figure><img src="image_40.png" /><figcaption>**Fig. 4.10.3.** The graph on the top shows the seawater pH~T~, temperature, oxygen changes and tide measured in February 2017 during a 3-day cycle in Bouraké. On the bottom, seawater pH~T~ variations were recorded at the reference (Sts R1 and R2) and Bouraké (Sts B1 and B2) reefs. Data were overlaid at a single tidal phase (12 h). Data are 22, 72, 31, and 72 semidiurnal tidal cycles.</figcaption></figure>

In addition, seawater temperature also is higher than outside the system because it stagnes in the mangrove forest at a very low depth, therefore it is exposed during winter to the atmospheric cold temperature (down to 13°C in the area), and during summer to high temperature due to solar insolation (up to 36°C in the air). This causes the water to be 1°C colder in winter, and 0.5-3°C warmer in summer than outside the system. These changes happen quite suddenly and cause the coral to be exposed to real MHWs all the time with changes in temperature of up to 6°C especially in summer. All these parameters exhibit detectable fluctuations over a 24-hour cycle, which is extremely important in assessing the amount of stress that corals experience over time. This makes this site much more interesting than other natural laboratories in which the duration and intensity of stress (e.g., volcanic vents) are not consistent in time or space.

In a first attempt to describe this site and give the first idea of the ability of corals living there to face the extreme conditions, the team composed of Emma Camp, David Suggett and Matthew
Nitschke worked during two fieldwork periods. From these researches, we published three papers (Camp et al. 2017; 2019 and 2020) that described the seawater environmental conditions; produced a list of coral species, and gave the first analyses of the bacterial community living in symbiosis with the corals. In Camp et al. (2017) we also measured high respiration and increased heterotrophy of corals from Bouraké and interpreted it as an adaptive response of corals to the harsh conditions which was favoured by the autotrophic conditions of seawater (i.e., organic input from the mangrove). However, we did not know at the time of this first fieldwork, in 2016, that seawater temperatures were abnormally high and that corals were under stress, therefore physiological responses partially altered. These precursor papers substantially anticipated further years of research dedicated to reinforcing the environmental measurements with long-term and higher frequency data and diel cycles in the chemical parameters of seawater. All the data collected were analysed in a compelling assessment of this unique natural laboratory (Maggioni et al. 2021). In this study, we also performed a whole dataset of coral, macroalgae and sponge taxonomical list throughout Bouraké, and found 66 species of corals, which was similar to the control reefs but intriguingly more abundant for some species such as *Acropora* (12 species) and *Montipora* (2 species). The site was not dominated by *Porites* as the CO₂ seeps in Normanby. A 3D model of the water circulation completed the study and it was realized using data collected simultaneously by 3 ADCP during 15 days and crossing a cyclon event, although these data have not been yet valorized in a paper. Data showed that the current can be either very strong (up to 1.5 Knt) or almost absent, according to the area considered. The first simulations showed that the water mass moves in and out of the system up to seven times before disappearing. These data are of great value when considering the larval dispersion during spawning, or measuring the DBL of corals at different flow conditions.

Starting from December 2015, I have visited hundreds of times Bouraké, organized fieldwork for whole teams from different institutions and countries, collaborated with tens of people, supervised 3PhD, 1 postdoc, 5 M.Sc, got funds for a series of projects, presented the site for an ambitious ERC Cog project, and finally published a handful of papers from the data obtained from this site. But the most important thing in my eyes is the fact that the site has gained a certain notoriety because it is unique in the world.
<figure><img src="image_41.png" /><figcaption>**Fig. 4.10.3.** Aerial picture of the entrance of the Bouraké semi-enclosed system during low tide.</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>**Fig. 4.10.4.** The reef dominated by *Acropora* spp. in Bouraké with two terracotta tiles positioned on the reef to assess coral recruits, one having CCA and one deprived.</figcaption></figure>
4.11. High coral recruitment despite coralline algal loss under extreme environmental conditions

<figure><img src="image_43.png" /></figure>

High Coral Recruitment Despite
Coralline Algal Loss Under Extreme
Environmental Conditions

Clément Tanvet<fnref n="1" />,²*, Francesca Benzoni<fnref n="3" />, Christophe Peignon<fnref n="1" />, Gérard Thouzeau²
and Riccardo Rodolfo-Metalpa<fnref n="1" />

This study reports the effect of such extreme environmental conditions of the Bouraké semi-
enclosed lagoon on hard coral recruitment and CCA originally settled at a fore reef on artificial
substrates that were transplanted over two years in two fringing reefs and the Bouraké lagoon.
Our data emphasize the negative effects of the extreme conditions in our study sites on the
CCA, which decreased in cover by ca. 80% and lost in the competition with turf algae, which,
in turn, increased up to 162% at the end of the two years. Conversely, hard coral recruitment
remained high at Bouraké throughout the study, three-fold higher than at two sites located
outside Bouraké where environmental conditions were typical for coastal fringing reefs. Our
findings show that while such extreme, climate change conditions have a direct and adverse
effect on CCA abundance, and despite a certain persistence, coral larvae settlement was not
affected. Based on previous findings from Bouraké, and the present observations, both coral
recruits and adults seem to be unaffected despite the extreme environmental conditions. This
study supports previous research illustrating how extreme natural and variable environments
may reveal unexpected and positive insights into the processes underlying coral acclimatization
and adaptation to global change.

**Introduction to the study**

One of the bottlenecks of the reef status is their ability to reproduce, settle and continuously form persistent reefs. Ocean acidification has been predicted to greatly affect this natural and complex mechanism. Scleractinian corals begin their life as pelagic larvae eventually settling to the reef, growing and establishing as adult, long-lasting colonies (Harrison and Wallace 1990). Healthy and functional coral reef communities directly depend on coral recruitment success that, in turn, depends on the abundance of their preferred settling substrate i.e., crustose coralline algae (CCA, family Corallinaceae, Lithophyllaceae, and Porolithaceae) (Heyward and Negri 1999). Indeed, CCA are known as framework binders and act as a preferential settlement substrate for coral larvae (Morse et al. 1988; Heyward and Negri 1999; Harrington et al. 2004; Nelson 2009; Price 2010). Several lab-based experiments addressing the effects of OA on CCA have demonstrated reduced calcification and growth rates (Kuffner et al. 2008; Comeau et al. 2013), increased dissolution (Anthony et al. 2008; Diaz-Pulido et al. 2012), and reduced recruitment (Kuffner et al. 2008; Bradassi et al. 2013). Field studies have shown reduced recruitment (Fabricius et al. 2011, 2015) or impaired capacity to attract coral larval settlement (Doropoulos et al. 2012). However, it has been recently shown that lab-based versus field experiments tend to conclude that OA has a stronger impact on CCA (Page et al. 2022). Indeed, CCA and corals persist at CO₂ seep sites (Kamenos et al. 2016; Enochs et al. 2020; but see also Fabricius et al. 2011 for an opposite finding) and at low pH environments in Palau (Barkley et

<footnote n="1">¹ENTROPIE – UMR 9220 (CNRS, IRD, UR, UNC, IFREMER), Institut de Recherche pour le Développement, Nouméa, New Caledonia, ²LEMAR – UMR 6539 (CNRS, IRD, UBO, IFREMER), Institut Universitaire Européen de la Mer, Plouzané, France,</footnote>

<footnote n="3">³Red Sea Research Center (RSRC), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia</footnote>
al. 2015). For coral recruits, OA has been shown to cause skeletal malformations (Foster et al. 2016), and a decrease in the availability of settlement cues (Albright et al. 2010; Albright and Langdon 2011). Thus, in general, OA could affect coral recruitment by dissolving CCA or changing their microbial biofilm compounds, hence decreasing the availability of a supposedly ideal substrate for coral recruits (Webster et al. 2013) and finally affecting the recruit survival (Doropoulos et al. 2012; Doropoulos and Diaz-Pulido 2013). However, OA is only one of the ongoing threats to coral reefs, and its combination with both ocean warming (OW) and deoxygenation (OD) will likely cause CCA bleaching and mortality (Diaz-Pulido et al. 2012; Cornwall et al. 2019; Hughes et al. 2020), and impairment of coral larvae settlement (Jorissen and Nugues 2021).

Here, we assessed the effects of extreme and fluctuating environmental conditions (i.e., low pH, O₂, and high temperature) on the abundance of CCA, algal turf, and coral recruits. To address this objective, we obtained CCA-covered tiles from a foreereef and moved them to three fringing coastal reef sites with distinct environmental conditions. We investigated the effect of the environmental conditions typical of coastal fringing reefs on the CCA cover twice during the two years experiment and we quantified and identified the coral family recruited on the tiles at the end. Based on the reported effect of OA on CCA cover and coral recruitment rates, we hypothesized that in Bouraké both will be heavily affected by the extreme conditions.

Our findings reinforce the idea that coral recruitment is possible even under extreme conditions and that, particularly for the Bouraké site, corals may have adapted to such variable and extreme environmental conditions.

## Main results and discussion

After 7 months of conditioning at a foreereef, i.e., just before they were moved and randomly shared between the three study stations (T0), the top sides of the tiles were predominantly covered in CCA (median % cover of 69.21%, all tiles pooled), while algal turf was rare (median per cent cover of 11.95%). Two main CCA genera were recognized on the tiles on a simple visual check regarding the conceptacle's size and disposal: *Porolithon* and *Neogoniolithon*. After two months (T2) at the three stations, the CCA median per cent cover was significantly different among stations and significantly decreased at B3 from 69.21% to 41.4%. CCA cover significantly increased at the two stations R2 and M1, respectively 64.4% and 81.3%. In contrast, turf cover was significantly different between stations, reaching the highest values in B3 (20.4%), and only 6.4% and 9.7% at R2 and M1, respectively. After two years of deployment (T24/26), CCA median %cover on the top sides of the tiles was lower than the one at T2 at all stations: this decrease was dramatic at B3 (from 41.4% to 8.4%) and M1 (from 81.3% to 1.5%), but to a much lower extent at R2 (from 64.4% to 49.3%).

CCA cover on the top sides of the tiles remarkably declined at Bouraké after only 2 months of immersion, while remaining stable or slightly higher at the two other reef stations. This suggests that ca. 40% of CCA settled during the tiles pre-deployment, and dissolved during a short-term exposure, therefore further indicating a strong negative effect of the Bouraké natural environmental conditions on CCA. At the same time, turf algae cover was significantly higher at Bouraké than at other reef stations. CCA cover on the top sides of the tiles remarkably declined at Bouraké after only 2 months of immersion, while remaining stable or slightly higher at the two other reef stations.
<figure><img src="image_44.png" /></figure>

**Fig. 4.11.1.** Change in crustose coralline algae (CCA) (A, B, and C) and Turf percent cover (D, E, and F) on top sides of the tiles between the beginning (two months after the time of deployment, T2) and the end (after ca two years of deployment, T24/26) of collector deployment at study stations B3, R2 and M1 (number of tiles n = 15, 20 and 19, respectively). Data are median values ± 25th and 75th percentiles (box), minimum and maximum values (whiskers), and outliers (dots). Stars represent statistical significance.

Coral recruitment significantly differed among stations and sides after two years of tile immersion. It was found to be ca. 3-fold higher at B3 than R2 and M1 with 18.4, 4.6, and 5.5 recruits per tile (all tiles pooled per station), respectively. Regarding where the larvae preferentially settled on the tile, the number of recruits was always higher on the bottom than on the top, and scarce on the lateral sides.

**Fig. 4.11.2.** Number of coral recruits (per tile) found on the different sides of the tiles immersed during ca. two years at Bouraké (B3) and stations R2 and M1. Data are means ± SD (number of tiles n = 15, 20, and 19, for B3, R2, and M1, respectively). Data from the lateral sides were pooled. Stars represent statistical significance.

*Acroporidae* was the dominant family of coral recruits settled in our experiment, with similar relative abundances between stations (65.58%, 73.91%, and 67.33%, at B3, R2, and M1, respectively).

Pocilloporidae was the second most abundant family, with recruits who equally settled at all three stations in terms of relative abundance (14.85%, 17.39%, and 16.83% at B3, R2, and M1, respectively). Only a few *Poritidae* were found at B3 and M1 (17.39%, and 6.52%), and none at M1 which exhibit more unknown genera.

Coral recruitment rates in Bouraké were higher than outside, at both the two fringing reefs. While our findings appear to contradict results from several OA laboratory-based experiments (e.g., Doropoulos et al. 2012), they are in agreement with some field experiments (Kamenos et al. 2016; Enochs et al. 2020) showing in general, a lesser impact of OA (Page et al. 2022).
## 4.12. Corals adapted to extreme and fluctuating seawater pH increase calcification rates and have unique symbiont communities

### RESEARCH ARTICLE

<figure><img src="image_45.png" /></figure>

WILEY

# Corals adapted to extreme and fluctuating seawater pH increase calcification rates and have unique symbiont communities

Clément Tanvet¹,²,³

<figure><img src="image_46.png" /></figure>

Emma F. Camp⁴

<figure><img src="image_47.png" /></figure>

Jill Sutton²

<figure><img src="image_48.png" /></figure>

Fanny Houlbrèque¹,³

<figure><img src="image_49.png" /></figure>

Gérard Thouzeau²

Riccardo Rodolfo-Metalpa¹,³

### Introduction to the study

Although data from our first study (Camp et al. 2017) showed that corals from Bouraké had lower calcification rates, I have transplanted corals several times in Bouraké and found no effect or even increased performance in corals.

<figure><img src="image_50.png" /><figcaption>**Fig. 4.12.1.** Growth rates measured by buoyant weight technique on colonies of the coral *Pocillopora damicornis* reciprocally transplanted between Bouraké and a nearby control reef for 12 months. The arrows indicate the direction of the cross-transplantation (Rodolfo-Metalpa, unpublished data).</figcaption></figure>

For instance, the growth rate of the coral *Pocillopora damicornis* (and two more species) showed that colonies from Bouraké always grew more than control colonies and that these latter increased their growth when moved to Bouraké, which is opposite to what happened to the Bouraké colonies transplanted to the control. These unpublished results suggest that i) although Bouraké has extreme conditions, for some reason its environment favours the growth of this coral species since the control colonies increase their growth rates; ii) the colonies that have lived there all their life have adapted and can perform better even when moved to the control environment.

It seems that chronic environmental changes could make corals more resilient to future changes in pH and/or temperature (Comeau et al. 2022; Enochs et al. 2020; Schoepf et al. 2020). Bouraké and the population of corals living there offer a great opportunity to test several hypotheses from the role of fluctuating environmental parameters to the role of heterotrophic inputs, likely making corals more resistant to face OA, and finally to the host adaptive capacity in the short- and long-term.

In this study, Tanvet et al conducted a 100-day OA experiment in aquaria using three coral species (*Acropora tenuis*, *Montipora digitata* and *Porites* sp.) from the highly variable Bouraké and a less variable adjacent reference reef to assess: i) whether corals adapted to ambient or
fluctuating pH conditions alter their metabolic rates and calcification under different levels of OA (i.e., 7.54, 7.76, 8.11 and 7.56-8.07), and ii) whether Symbiodiniaceae communities are distinct between habitats and treatments at the end of the experiment. In addition, we considered both static pHNBS (7.54 ± 0.08; 7.76 ± 0.07; 8.11 ± 0.05) and variable (ranging from 7.56 ± 0.07 to 8.07 ± 0.07) pHNBS conditions to assess the role that fluctuating pH may play in the success of Bouraké corals, and whether corals adapted to stable pH can acclimate when exposed during 100 days to variable pH. We hypothesized that corals from Bouraké exhibit enhanced physiological traits and a distinct Symbiodiniaceae community, compared with corals from the less variable reef, and that the natural diel fluctuations in seawater pH promote their resilience to OA.

## Main results and discussion

**Growth rate.** In agreement with the consensus on the effect of OA on coral calcification (Gattuso et al. 1999), recently revised by Leung et al. (2022), we found that calcification rates of both *A. tenuis* and *Porites* sp. reference corals were significantly decreased when exposed to future and extreme pH levels. The mean growth rates of the three corals originating from the reference and maintained at control pH, generally decreased when maintained at future, extreme and also variable pH. This decrease was more evident in the two branching corals than in *Porites* sp. In contrast, the mean growth rates of the three corals originating from Bouraké and maintained at future, extreme and variable pH i) were always higher than their counterpart originating from the reference and maintained at the same pH condition; ii) were almost always (with one exception, *A. tenuis* at variable pH) higher than their counterpart originating from the reference and maintained at the control pH condition; iii) were almost always higher or did not change (with one exception, *M. digitata* at variable pH) when maintained at the control pH condition.

These unexpected findings suggest that Bouraké corals have adapted to OA because they have been exposed to extreme conditions throughout their lives, and regain even higher rates of calcification than the same coral counterparts adapted to open-water pH conditions, once at actual open-water pH (i.e., ca. 8.10).

Our data do not consistently suggest that Bouraké corals have acquired a particular mechanism that accounts for better physiological plasticity to cope with low pH conditions, thereby maintaining higher calcification rates. Indeed, we did not find a clear effect of coral origin with a systematic increase in photosynthetic rates, higher contents of Symbiodiniaceae, chlorophylls, and protein in Bouraké corals.

**Effect of variable pH.** One of our hypotheses to explain the success of Bouraké corals was the potential positive effect that diurnal pH fluctuations could have on coral metabolism, as previously found for corals in GBR mangrove lagoons (Camp et al. 2019), St Vincent and the Grenadines CO₂ vents (Enochs et al. 2020), and for others incubated in mesocosms (Brown et al. 2022; Dufault et al. 2012). Our results show that short-term exposure (i.e. acclimation of reference corals during our experiment) to fluctuating pH has a negative effect on coral calcification as all coral species from the reference decreased their growth when incubated at variable pH. In contrast, Bouraké corals, which are acclimatized and/or adapted to local conditions when exposed to variable pH calcified from 7.6% (*A. tenuis*) to 116.2% (*M. digitata*) more than their counterpart from the reference site. Furthermore, Bouraké corals incubated at future and extreme pH maintained these higher growth rates and even increased them when grown under control conditions, confirming their resistance to OA. Although the duration of our experiment was longer than most OA experiments (i.e., 77% lasted 1-11 weeks; Brown et al. 2022; Ziegler et al. 2021), we acknowledge that the duration of variable pH exposure experienced by corals from the reference site during the experiment was too short to compare
with that experienced by corals at Bouraké. Corals exposed to a variable environment throughout their life are physiologically more plastic than corals adapted to stable environments (Kenkel & Matz 2017); such plasticity is probably time-dependent. Future experiments should take into account the length of exposure to variable pH (as well as other environmental parameters) that the corals experienced before the collection.

<figure><img src="image_51.png" /><figcaption>**Fig. 4.12.2.** Reaction norm of the growth rates of corals from Bouraké and Reference site maintained during 100 days at four pH conditions (Control (8.11), Future (7.76), Extreme (7.54), and Variable (7.56-8.07)). The arrow indicates the direction of the change from the origin to the pH condition. Data represented as dots are means of the three replicate tanks ± SE (overlapping SEs are in red) (n = 13-16, depending on species and pH condition).</figcaption></figure>

By assessing the holobiont physiological responses and the Symbiodiniaceae profiles of three coral species from both Bouraké and a reference reef to a large range in pH, we found that corals from Bouraké always exhibited higher growth rates and had a specific and more consistent ITS2 majority sequence than corals from the reference reef both under low and variable pH conditions. Our data suggest that seawater pH level, whether stable or variable, at future or extreme levels, does not affect the Symbiodiniaceae major ITS2 type profiles of corals (at least over 100 days), since at the end of the experiment we found no significant effect among treatments. Microbiome stability (both for symbionts and/or bacteria) was linked to greater physiological resilience to OA (Ge et al. 2021; Grottoli et al. 2018; Quigley et al. 2017, 2019; Ros et al. 2021), suggesting that consistent major ITS2 type profiles for Bouraké corals under pH treatments could facilitate their success; however, further work will be needed to verify this hypothesis.
# 5. FUTURE RESEARCH"Hope for coral reefs. Can acclimatization and adaptation follow the pace of climate change?"

## 5.1. Abstract

Coral reefs shelter one of the largest biodiversity on earth and provide US$36 Billion per year of ecosystem services. According to current experimental evidence, they could disappear or be drastically decimated within the coming decades if CO2 emissions remain unabated. Yet, I recently discovered a remarkable site, which the only natural analogue of future climatic conditions exhibiting the three main parameters that drive climate change in the oceans: lower pH (<7.8) and oxygen (-20 to 30%), and warmer temperatures (+ 0.5 to 3°C). While these conditions are generally recognized as unfavourable to corals, there, a rich and abundant coral reef has developed. This novel natural analogue brings new hope for the future of coral reefs and provides a unique natural laboratory to explore how corals could keep pace with climate change.

HOPE crosses traditional interdisciplinary boundaries between evolutionary biology, physiology, ecology, and genetics to study the mechanisms by which coral reefs may acclimatize or adapt to suboptimal conditions. Ambitious and unprecedented experiments will be carried out in situ where phenotypic responses will be measured for a diverse array of life-history traits in several species. Within-generation, parental effects and transgenerational plasticity responses to suboptimal conditions will be assessed using specifically designed reciprocal transplantations on adult and juvenile corals. Complementary laboratory experiments and analyses will provide solid evidence of the underlying evolutionary mechanisms that corals might use to face climate change. Results will also identify the species that are more likely to resist in the future, which is particularly important for coral reef conservation and assisted evolution projects that aim to enhance the resilience of corals. Overall, HOPE will provide critical and ground-breaking results that can fundamentally change our understanding of how climate change will impact coral reefs in the future.

## 5.2. State-of-the-art and objectives

Atmospheric carbon dioxide (CO₂) has steadily increased over the industrial period [1] leading to ocean warming (OW) and ocean acidification (OA). Current projections of average global warming by the end of the 21st century range from the most optimistic 1.8°C to the most pessimistic 4.0°C temperature increase, in combination with a 0.2 to 0.3 unit drop in ocean pH [2]. In their latest report, the IPCC panel of scientists raise the alarm about the consequences of climate change [3] and, based on current experimental evidence, significant impacts on key marine organisms and ecosystem services are predicted for the coming decades [e.g., 4-5]. Specifically, 70 to 90% of coral reefs (and associated species) are projected to decline with a global warming of 1.5°C, whereas virtually all reef species (>99 %) would be lost with a 2°C warming.

Although the effects of OA are still debated, there is no doubt that it will negatively impact a range of marine organisms [6, 7]. Projected impacts on reef structures and communities include reduced growth, weaker skeletons, increased bioerosion, dissolution of carbonate substrates, as well as other functional traits [8], with consequent biodiversity loss [9]. In contrast, studies also revealed that responses are species-specific and that some taxa may be able to cope with, or even thrive, under projected acidification conditions, at least for some functional traits [e.g., 10-18]. Responses to OW depend on species thermal tolerances and their thermal history. In general, metabolic rates increase with temperature, sometimes with an apparent benefit for some traits such as growth [19]. However, above a thermal tolerance threshold, growth, body size, behavior, reproductive success and a suite of metabolic functions are hindered [20]. For instance, tropical coral reefs are extremely vulnerable to OW and have undergone repeated massive bleaching events during the last decades [5, 21, 22].

One of the limitations of our current knowledge of the impacts of OA and OW on marine organisms is that most studies are based on deterministic models or short-term laboratory experiments of individual organisms [e.g., 4, 23, 24]. These experiments identify the effects of one or a few variables in isolation,
but they are unable to account for the capacity of species to acclimatize in natural environments. Laboratory experiments are also not ecologically realistic since, for instance, they do not consider the effects of species interactions, natural provision of nutritive supplies, and natural fluctuations in the main environmental parameters [25].

But the most significant gap in the current knowledge is that most studies have neglected the role of adaptation and only within-generation responses to global change have been tested during short-term exposures to stress [reviewed in 26]. Exceptions include experiments using short-lived organisms such as coccolithophores, copepods and polychaetes, coralline algae, sea urchins and fish [e.g., 27-34], or breeding experiments involving sea urchin larvae [35, 36]. For example, Pespeni et al. [35] demonstrated the genetic changes and rapid evolutionary capacity of sea urchin larvae collected from coastal areas exposed to upwelling-driven acidification and reared in aquaria under low pH conditions. Unlike previous laboratory experiments [e.g., 37], larval development and morphology showed little response to high pCO₂ levels, probably because they were already able to tolerate these conditions. Transgenerational studies showed that in some mollusks [38-40], sea urchins [41] and coral reef fishes [42-45], the exposure of adults to OA can promote positive carryover effects facilitating the acclimatization and survival of larvae and juveniles under acidified conditions. While adult metabolic performances of the coral *Pocillopora damicornis* were impacted when exposed to a combination of OW and OA, their larvae exhibited metabolic acclimation when subsequently re-exposed [46, 47]. However, as reported for other taxa [reviewed in 48], the duration and timing of parental precondition may induce a transgenerational response in the offspring, resulting in positive but also null or negative adaptive responses [49, 50, 51]. Evidence of natural intra- and trans-generational plasticity (TGP) to increasing temperature was also shown for some corals (e.g., decreasing bleaching effects [52-58]) and reef fish species (e.g., maintaining performance at high temperatures [59, 60]).

Distinguishing whether a species can acclimatize to changes in its environment via phenotypic plasticity or rapid genetic adaptation is challenging since it requires multi-generational experiments, which are difficult to set up, particularly for species with long generation time such as corals. In addition, these approaches have only been possible under artificial laboratory conditions. While TGP has been studied in the laboratory on short-lived organisms, few studies have addressed this important challenge on corals and only for early life stages [46, 47, 51, 61]. No study has tracked performances on later stages of the same offspring, used adult parent colonies that experienced the experimental conditions during their entire life, or performed experiments in the wild. In general, evolutionary aspects of OA and climate change have been largely overlooked. Current projections ignore the capacity of species for phenotypic buffering and their adaptive potential [62-65].

The overall goal of this project is to explore the likelihood and mechanisms by which coral reefs may withstand environmental stressors like climate change. I hypothesize that non-genetic inherited traits can contribute to long term population adaptive responses and help corals to persist under climate change conditions. To reach this goal, I will assess phenotypic responses for a diverse array of life-history traits in selected coral species, together with their genetic diversity and gene expression. Within-generation, parental effects and TGP response (F0-F2) to suboptimal conditions will be assessed during *in situ* long-term reciprocal transplant experiments using a natural analogue of future conditions as the main laboratory. This will allow for realistic and ecologically relevant projections. Combining designed experiments at the natural analogue with laboratory experiments, is the best approach to provide robust evidence of the evolutionary mechanisms coral reefs might use to face climate change. I expect groundbreaking results that can fundamentally change our understanding of how climate change will affect coral reefs and the underlying biological mechanisms involved.

**5.3. Distinguishing acclimatization from adaptation.** Organismal acclimatization (i.e., the phenotypic plasticity organisms express during their entire life to maximize their fitness in response to multiple environmental factors under natural conditions [64]), can be measured during short- and long-term experiments. Plasticity often consists of three main categories: developmental and reversible, both occurring within a single generation (i.e., within-generation plasticity), and transgenerational plasticity (TGP) which is driven by the interaction of parents and a new generation with their environment. While it is widely accepted that phenotypic plasticity can increase the survival of organisms, there is no general agreement on whether plasticity can lead to rapid evolution under specific conditions [66]). Because
acclimatization requires energy, the crucial role of phenotypic plasticity may not be sustained over the long term [65]. Under such circumstances, phenotypic plasticity may be subject to natural selection resulting in evolution through genetic assimilation or phenotypic accommodation [67]. Adaptation (i.e., genetic changes through natural selection of heritable genotypes that express a phenotype enhancing fitness in a new environment [64]) acts over multiple generations at the population level, while phenotypic plasticity acts on individuals within a generation and is generally not heritable [67]. The evolution in the environment through random occurring natural selection is generally considered to be a slow process, requiring hundreds or even thousands of generations. However, recent evidence demonstrated that evolutionary changes in nature are possible over only a few generations [68]. Adaptive transgenerational (heritable) plasticity may buffer a population against such evolutionary change by allowing existing genotypes to maintain their fitness while coping with climate change [69]. In evolutionary ecology, discriminating between acclimatization and adaptation is essential in determining a species capacity for trans-generational response to a rapid increase in anthropogenic disturbances. Reaction norms (i.e., the pattern of phenotypic expression of a single genotype across a range of environments) provide an excellent means of evaluating the phenotypic plasticity [67, 70] of clones and/or genotypically distinct individuals as a function of the environment. Technically, this can be achieved by transplanting multiple individuals from genetically distinct populations both within and between their respective habitats (i.e., 'reciprocal transplant' [71]). Although these approaches have been used for studying adaptation in terrestrial animals and plants [e.g., 71, 72], they have rarely been applied to marine ecosystems [e.g., 73, 74].

**5.4. Objectives, originality and innovative nature of the project.** The originality of my project arises from my recent discovery of an exceptional habitat at Bouraké, in New Caledonia, where environmental conditions (temperature, pH and oxygen) are below the optimum known for corals (i.e., “suboptimal” conditions), and similar to those forecasted for the future (“natural analogue”). While these conditions are generally recognized as unfavorable to corals, a rich and abundant coral reef has developed. With this project I want to understand how corals can thrive in this hostile environment and derive projections of the fate of corals under similar future conditions worldwide.

As a first step, we will characterize the genetic makeup of corals inside and outside Bouraké using RAD sequencing. Neutral markers will be used to describe population genetic patterns, including population connectivity inside and outside Bouraké, while loci under selection will be used to test and describe the genetic adaptation mechanisms. Significant genomic differences among stations with high levels of connectivity would indicate that strong environmental filtering occurs with each generation, and demonstrate sufficient standing genetic variation in natural populations to cope with environmental differences between stations. Low levels of connectivity between genetically different populations would suggest that adaptation takes place slowly, over several generations. The absence of genomic differences between populations would indicate that non-genetic acclimatization is sufficient for corals to inhabit different environments. Then, reciprocal transplant and transgenerational experiments on corals inside and outside Bouraké will make it possible to reveal the physiological and molecular mechanisms that corals use to cope with environmental changes within and between generations.

To test whether the specific traits that allow corals to survive are heritable over several generations, I will conduct transgenerational experiments and perform reciprocal transplantations of F1 recruits born from adult corals (F0) that have spent their whole life in suboptimal conditions in the wild. The experiment was designed to include an ambitious temporal effect and distinguish TGP from parental effects. For that, the duration of exposure of parents and eggs to climate change-like conditions before spawning will vary.

In the short-term, the project will provide robust data about within-generation responses of adult corals, as well as parental and transgenerational carryover effects throughout the larvae and subsequent early life stages. In the long-term, it will identify responses in the later stages of juveniles, in adult colonies of the F1 generation (> 2 to -3 years old), and possibly in an F2 generation. The project represents a significant step forward in addressing global change challenges. It will perform, for the first time, in *situ* experiments on several coral species, to decipher their abilities to acclimatize and adapt to future conditions. This work will help to fundamentally improve our understanding of the fate of coral reefs within an evolutionary context.
HOPE is structured around three primary aims that explore the following questions:

**Aim 1. Assessing genetic diversity and connectivity.** Are corals living in the suboptimal environment of Bouraké genetically selected, and/or genetically connected to the neighboring lagoon fringing reefs?

**Aim 2. Assessing the role of plasticity.** Is plasticity a sustainable response? Is plasticity heritable in the broad sense? Does plasticity have a genomic basis?

**Aim 3. Assessing the contribution of plasticity to adaptation.** Are parental and transgenerational effects used by parent colonies reflected in the traits of the offspring? Do they persist into adulthood, at least for the F1 generation?

**5.5. Natural analogues to study the effects of climate change.** One way of demonstrating how species acclimatize or adapt to future environmental conditions, such as warming, acidification, or deoxygenation, is to provide evidence from species that already live in "suboptimal" conditions. Natural analogues for future conditions are study sites where at least one or more environmental parameters naturally mimic climate change-like conditions over a large area of the ecosystem, therefore providing an opportunity to simultaneously investigate changes in species responses and their capacity to acclimatize and eventually adapt.

Thanks to my scientific background, my network, my research team and our recent findings, I am particularly well-positioned to develop novel *in situ* approaches that can provide ground-breaking discoveries about the effects of climate change on marine ecosystems. During my career, I have pioneered the study of submarine CO₂ vents to understand how species acclimatize and/or adapt to OA, providing the first studies on the effect of OA at the scale of ecosystems (24 publications using CO₂ vents in the Mediterranean Sea [e.g., 13, 75]). During the last three years, my team has investigated coral reef responses to OA using three CO₂ vents in Papua New Guinea (PNG). At these unique sites (see https://youtu.be/xBQjV7ZbHw4 and https://youtu.be/SAa0vsOChn8), continuous emissions of near-pure CO₂ from below the seafloor alter the chemistry of the surrounding seawater, providing an ideal experimental environment to study the ability of species to acclimatize or adapt to future acidification levels [76]. In the framework of two French ANR projects, I carried out seven expeditions and found unusual metabolic and molecular plasticity in coral species (Biscere et al [77], Rippol et al in prep. [78]). Using cross-transplantation of resistant and sensitive species, I found that these corals were able to grow and survive in high pCO₂ conditions (Rodolfo-Metalpa et al in prep. [79]). Interestingly, while previous studies [76] found only a limited number of branching corals at the Normanby CO₂ vent in PNG when compared to massive corals, I found a very rich and diverse reef at another CO₂ vent (Ambitle Island, [80]). At this site, despite the low seawater pH, I did not find any substantial loss of biodiversity or dominance of massive corals as initially expected [Shlessinger et al in prep. 81]. These ground-breaking findings opened new questions about the real capacity of corals to acclimatize to OA and highlight once again the necessity to repeat experiments using more realistic conditions.

In parallel to the PNG project, I discovered a novel natural analogue in New Caledonia [82]. The Bouraké system is a semi-enclosed lagoon with no freshwater input (Fig. 5.5.1. A-B). A channel of more than 80 m wide and 6 m deep penetrates a dense mangrove forest and creates large pools over an estimated area of more than 60,000 m². The first measurements of the diel pH, temperature and oxygen fluctuations (Fig. 5.5.1. C-D) revealed the value of this site for studying the capacity of corals to acclimatize and/or adapt to suboptimal conditions.
<figure><img src="image_52.png" /><figcaption>**Fig. 5.5.1.** Map showing study sites and stations (A); aerial photograph of the semi-enclosed lagoon of Bouraké (B); physicochemical parameters of seawater at control (C) and suboptimal stations (D) in February 2016.</figcaption></figure>

At each high tide, new water from the lagoon enters through the channel into the vast inner basin of the mangrove. During this journey, the water chemistry changes due to metabolic reactions in the sediment, coral reefs and mangrove habitats, and mixes with more acidic, hot and deoxygenated seawater. Even at high tide, the water rarely returns to “normal” with maximum pH<sub>T</sub> (in total scale) values are around 7.9 (currently, the typical average pH<sub>T</sub> of the ocean is 8.05). At low tide, seawater becomes more acidic and oxygen-depleted as the system begins to drain. The large volume of water that was inside the mangrove forest enters the system and is discharged into the lagoon. At low tide, pH values reach a minimum of 7.3 and O<sub>2</sub> a concentration of 2 mg L<sup>-1</sup> (the average concentration of O<sub>2</sub> at the coast is 4 to 6 mg L<sup>-1</sup>) around corals. Both parameters exhibit predictable fluctuations according to the semi-diurnal tidal cycle (1.2 ± 0.3 m), which is extremely important when assessing the amount of stress that corals experience over time. As a result, corals are exposed 45% of the time to a pH<sub>T</sub> between 7.9 and 7.7, and 53% of the time to pH<sub>T</sub> < 7.6 (see [82]).

Sea surface temperatures (SSTs) are also of particular interest. During the first survey in 2016 [82], when SSTs were particularly hot in the Pacific, including in New Caledonia, I found that, inside the Bouraké system, seawater temperatures were 2 to 3°C higher than in the lagoon outside the system (Fig. 5.5.1, D). The seawater temperature was then monitored inside and outside the system from September 2017 to May 2018, including an unusually cold summer. I found that the average (±sd) temperature difference was 0.57 ± 0.58°C with a peak of 1.46°C. This revealed that the coral reefs inside the system are not only exposed to extremely high temperatures during anomalous thermal events, but they also live at temperatures 0.5 to 1°C higher than other fringing reefs outside the system for most of the year. Although these values slightly differ from those expected by the end of the century, they already correspond to those forecasted for the next 20 to 30 years. Besides, corals in the Bouraké system experience significant and chronic oxygen fluctuations towards lower concentrations. As oceans are warming, projections indicate that the upper oceanic layers will become more oxygen-depleted. In combination with coastal eutrophication, deoxygenation has been put forward as the third most important factor potentially affecting marine life in the future, after pH and temperature (the “deadly trio” sensu [83]). We logged these three environmental parameters since February 2016 using
autonomous multiparametric probes deployed at the study stations. Tides and the unique geomorphology of the site appear to be responsible for the suboptimal conditions. Our preliminary studies also indicate that this unique site has not changed for at least the last 80 to 100 years, and most likely longer. This data, therefore, suggests that corals of the Bouraké lagoon have most likely experienced the current suboptimal conditions for several generations.

To this day, and to the best of my knowledge, the discovery of such a study site is unprecedented. The Bouraké system is the only natural analogue with suboptimal values of the three most ecologically relevant parameters, which are driven by climate change in the oceans: (i) a significant drop in seawater pH with extreme and regular variations below 7.8, (ii) temperatures 0.5 to 3° C warmer, and (iii) 20 to 30% less oxygen than usual.

**5.6. Preliminary experiments demonstrating the value of the Bouraké system as a natural analogue.** Since 2017, I have been leading a consortium of scientists to provide preliminary data and prove the exceptional value of this unique site for studying the effects of climate change on coral reef ecosystems. This research has already produced ground-breaking results, while other promising experiments, still in progress, have been instrumental in testing the feasibility of the proposed project.

We first developed a hydrodynamic model (MARS 3D) and forced it with high-frequency data to simulate the effect of the tide on pH, oxygen and temperature (Rodolfo-Metalpa et al in prep. [84]). This model will help to investigate changes in key environmental parameters as well as nutrients and organic compounds across the system, and their effect on coral species distribution and abundance. In May 2017, I then led the first research cruise in collaboration with ten scientists from different disciplines. During two weeks, onboard the research vessel *Alis*, we recorded species distribution of the three main macrobenthos taxa (macroalgae, sponges and corals), as well as precise measurements of seawater parameters at both suboptimal and reference sites (or “inside” and “outside” the system, hereafter). Despite considerable differences in the main environmental parameters, both sites were equally rich and diverse, and the corals diversity and abundance were very similar between sites (52 and 58 species were found inside and outside the system, respectively).

As the first evidence of acclimatization, we measured a change in corals' gross productivity to respiration ratios, which suggests heterotrophic plasticity as a response to local conditions [82]. Interestingly, we found different microbiomes (both eukaryotic and prokaryotic) between the two types of environment (collaboration with C. Voolstra, KAUST; Camp et al. [85]).

We also investigated the reproductive ability of the brooder coral *Pocillopora damicornis* (Dec. 2017-May 2018) using specimens collected both inside and outside the system and maintained in aquaria under controlled pH and temperature conditions (Prasetia et al submitted [86]). We found that preconditioned parent colonies produced offspring that were not particularly high in fitness and metabolic plasticity. Although recruits from the suboptimal site displayed lateral growth and budding rates that were 2 and 1.2 times higher, respectively, than recruits at the reference site, these differences were not statistically significant. However, unlike coral recruits at the reference site, the skeletal microstructure of the recruits at the suboptimal site was not affected by high $pCO_2$ when combined to high temperature. With this experiment, we also obtained an F1 generation of corals fully acclimatized to suboptimal conditions, which we both transplanted at suboptimal and reference stations where they are currently growing, although they suffered from high mortality rates. The experiment was repeated during the latest coral spawning in New Caledonia (17th & 18th of Nov. 2019) using the broadcast spawner *Acropora tenuis*. We collected gametes from stations inside the Bouraké system, and larvae successfully settled on ceramic plugs in tanks, a method I plan to use during the project. Recruits will be transplanted at the study sites in early 2020 and monitored throughout their growth.

Finally, I tested the hypothesis that the resistance of corals living inside the Bouraké system is due to heterotrophic plasticity [82]. I performed reciprocal transplantation between the suboptimal and reference sites using fragments of the coral *Psammocora contigua*. As there are higher heterotrophic inputs in the Bouraké system, which could explain the higher resistance of corals to suboptimal conditions, I expect the hypothesis to be confirmed if the transplanted corals grow better inside the system, regardless of their origin, thus proving their potential for heterotrophic plasticity. In contrast, if
corals from outside the system do not acclimatize (no heterotrophic plasticity, e.g., no change in calcification rates), this would indicate that the phenotype of the corals inside the system is genetically fixed. Preliminary results, eight months after the start of the experiment, show that fragments of *P. contigua* from the suboptimal environment grow better than those from the reference sites, independently of the transplanting site. The fact that calcification rates of coral fragments from the reference did not decrease after transplantation to the suboptimal sites, raises questions. Although these are preliminary results, they indicate that *P. contigua* has calcified more because it is adapted to the suboptimal conditions, rather than due to an increase in its heterotrophic capacity.

**5.7. Scientific and technical programs, project organization.**

**5.7.1. Study sites.** The project will use the unique Bouraké system as a study site where environmental conditions (temperature, pH and oxygen) are “suboptimal” and mimic conditions forecasted for the future and beyond. Seawater carbonate chemistry, oxygen and temperature are spatially uniform throughout the whole system, i.e., there is no substantial difference between the entrance (near the lagoon) and the inner part (near the mangrove forest) during the same tide. Two replicate stations were selected inside the system and two reference stations were selected outside Bouraké (Fig. 1A-B). Due to the complexity of the experimental design, as well as the number of target species and measurements, replicating stations is an high-risk/high-gain objective. Reference stations were carefully selected according to the following criteria: i) be relatively distant from the site of Bouraké but not too far so that working at both study sites on the same day is possible; ii) be as similar as possible to the site of Bouraké in terms of habitat geomorphology, hydrodynamics and main environmental parameters, except for pH, temperature and oxygen, which need to be in the normal/average range; iii) host the same coral species I found in Bouraké. The selected stations are abundant fringing reefs typical of coastal semi-enclosed shallow water bays.

**5.7.2. Environmental parameters.** Comparing species and population responses to contrasting environmental conditions requires knowledge of fine-scale spatial and temporal variations in seawater physicochemical parameters at the study sites. The data collection, carried out by my team over the past two years, will be continued to provide a consistent and longer-term sequence of environmental data. This data will be instrumental during the project, particularly when analyzing physiological measurements. For at least 15 days each month, SeaFET autonomous pH sensors and YSI multi-parameter probes will be deployed to routinely log temperature, salinity, dissolved oxygen, and pH variations. Diel irradiance will be measured on each fieldwork day using Li-Cor quantum sensors. A substantial effort will be devoted to characterizing the chemistry of the water column, which may change with the tide. Repeated diel cycle measurements (two consecutive days each two months) will assess variations in the seawater carbonate chemistry (including DIC<fnref n="1" />, $A_T$), total suspended organic matter (DOC, NP, POC), nutrients, prokaryote and virus diversity, chlorophyll content, metals and other trace elements, seawater $\delta^{13}$C and $\delta^{15}$N signatures. My team is experienced with managing such data and has the required competencies to analyze environmental parameters and descriptors. However, most analyses have to be processed overseas, which can cause significant delays in the project. Being able to process samples locally would allow us to interpret our results quickly and adjust our experimental design accordingly. This critical task requires a significant investment in terms of instruments, consumables and lab assistance (see budget).

**5.7.3. Study species.** Our pilot studies recorded 65 coral species in the Bouraké system, of which about 30 also occur at the two reference stations. For this project, we selected five of these species for their different life-history strategies and traits, and the availability of relevant molecular reference data: *Acropora tenuis*, *Pocillopora damicornis*, *Montipora digitata*, *Platygyra daedalea* and *Galaxea fascicularis*.

<footnote n="1">¹DIC: dissolved inorganic matter; $A_T$: Total alkalinity; DOC: dissolved organic matter; NP: nitrogen particulate; POC: particulate organic carbon.</footnote>
<table><thead><tr><td>Species</td><td>Genome</td><td>Reproduction</td><td>Morphology</td><td>Thermal tolerance</td><td>Symbiodiniacea transmission</td></tr></thead><tbody><tr><td>Acropora tenuis</td><td>published</td><td>hermaphroditic spawner</td><td>corymbose</td><td>medium</td><td>horizontal</td></tr><tr><td>Pocillopora damicornis</td><td>published</td><td>brooder</td><td>corymbose</td><td>low</td><td>vertical</td></tr><tr><td>Montipora digitata</td><td>ready</td><td>hermaphroditic spawner</td><td>digitate</td><td>high</td><td>vertical</td></tr><tr><td>Platygyra daedalea</td><td>ready</td><td>hermaphroditic sp</td><td>massive</td><td>high</td><td>horizontal</td></tr><tr><td>Galaxea fascicularis</td><td>ready</td><td>hermaphroditic spcannic</td><td>phaceloid</td><td>high</td><td>vertical</td></tr></tbody></table>

## 5.8. Experimental designs.

### 5.8.1. Aim 1 – Assessing genetic diversity and connectivity.

Our first goal is to understand whether corals naturally growing at the site of Bouraké are genetically different from those that live at the reference site and whether their populations are connected via gamete or larval dispersal. To this end, during the first field trip, we will tag and sample 20 colonies of each of our study species at each station (20 colonies x 5 species x 4 stations = 400 colonies). Samples will be snap-frozen in liquid nitrogen and preserved in DMSO for RAD sequencing, gene expression studies and microbiome analyses. RADseq data will be used to identify clonemates among our tagged colonies so that downstream analyses can be adjusted accordingly. RADseq data will then be mapped on the reference genomes and neutral markers will be separated from selected markers. Neutral SNP markers will be analyzed in the non-equilibrium population genomic model *Moments* [87] to assess the magnitude and direction of connectivity among populations. The model-free *StairwayPlot* method [88] will then reconstruct historical demographic patterns. Genetic distances will also be calculated based on selected loci to determine whether corals are genetically adapted to their respective environments. In tandem, the microbial community of corals will be described using ITS2 and 16s metabarcoding.

5.8.1.1. *Expected outcome:* The population genomic patterns described in this component will indicate the mechanisms of adaptation to the suboptimal environment of the Bouraké lagoon. If there are significant genomic differences among stations with high levels of connectivity, strong environmental filtering must occur on every new pulse of recruitment. This would indicate that there is high enough standing genetic variation in the natural populations of the reference stations to create genotypes suitable for highly contrasting environments. This result would suggest that coral populations around the world could possess the inherent capacity for instantaneous adaptation, following strong selective sweeps, e.g., the 2015-17 bleaching events. On the other hand, low levels of connectivity among populations that are significantly different in their allele frequencies on selected loci would suggest that adaptation happens slowly, over several generations. In turn, this would indicate that coral populations worldwide will require decades to adapt to climate change. And finally, if we find no significant genomic differences among populations of the suboptimal lagoon habitat and the reference stations, we can assume that other, non-genetic mechanisms dominate the acclimatization of corals (see Aim 2 and 3). Acclimatization via the microbiome will be evaluated using the ITS2 and 16S metabarcoding data. Results of the transcriptome analyses will reveal if gene expression levels are different for putative adaptive loci even in the absence of allelic differences, indicative of epigenetic modifications.

The results of Aim 1 will set the basis for downstream analyses.

5.8.1.2. *Risks mitigation:* Results from this task are relevant for the whole project outcomes since it will enable to select clonemates and genetically different colonies to be used during the project. However, this task has relatively low risks since RAD sequencing is routinely used, and I selected coral species for which the genome is becoming to be well known. In addition, several expressions of interest in participating in this project have been manifested by some of the most experts in the field such as G. Torda (JCU, Australia), F. Benzoni (KAUST, Saudi Arabia) and C. Voolstra (University of Konstanz, Germany) offering, to some extent, to partially cover the cost of the molecular analyses if necessary.
Another difficulty that could arise is the confusion between cryptic species during underwater sampling. This would result in fewer samples per species than expected, at least for *Pocillopora damicornis* which can be confused in the field with *P. acuta*, as both species can be found in sympatry at the site scale in New Caledonia. If this happens, additional sampling will be organized.

### 5.8.2. Aim 2 – Assessing the role of plasticity.

A long-term reciprocal transplantation experiment, involving fragments from a subset of the colonies tagged in Aim 1 was designed to investigate the corals' phenotypic plasticity. This experiment will evaluate if the phenotypic plasticity is genetically determined or not by investigating whether trait differences between individuals from different environments disappear after transplantation. By comparing the phenotypic responses, the diversity of the associated *Symbiodiniacea* and bacteria, and gene expression levels in transplanted fragments, I will be able to assess the role of plasticity in acclimatization and answer the following questions: Is plasticity a long-term response? Is plasticity heritable in the broad sense? Does plasticity have a genomic basis?

To understand the mechanisms of within-generation plasticity, I will measure several physiological traits because not all traits are expected to have the same plasticity levels. Using semi-autonomous Plexiglas chambers *in situ*, I will measure several physiological traits at both the Bouraké and reference stations, and for each of the selected species, including photosynthesis, respiration, calcification, and carbon excretion as well as host trophic status, proteins, lipids, ATP, mitochondrial activity, chlorophyll contents and *Symbiodiniacea* shifts. Changes in traits will be compared using reaction norms, and the adaptive potential of each functional trait will be approximated using a quantitative genetic approach and calculating broad sense heritability (H²), i.e., the proportion of trait phenotypic variation of the traits that has a genetic basis. This approach does not distinguish the contribution of physiological plasticity from epigenetic or genetic components of variation in the stress response. Indeed, because corals are modular organisms, several clonal fragments can be obtained from a single colony, therefore providing an opportunity to study the response of clones with limited biological variations to environmental conditions.

Corals live in close association with unicellular *Symbiodiniacea* species, which have a central role in the success of tropical reef-builders. They also live in association with a range of bacteria, fungi, and viruses, which have been shown to influence coral immunity, nitrogen fixation, nutrient cycling and can affect the overall fitness of the holobiont (i.e., host and microbiome together). Therefore, it is very likely that the response of corals to climate change strongly depends on these associations. Studies have shown that the response of the microbiome to a change in the environment is more pronounced than for the coral host [89-90]. In contrast to the host, both *Symbiodiniacea* and microbes have short generation times which facilitates rapid responses to altered conditions, likely contributing to the acclimatization and adaptive capacity of their coral hosts. It is, therefore, important to assess qualitative and quantitative measurements of both the host and microbiome separately to properly evaluate the adaptive response of the holobiont. Subsamples of the transplanted corals will be analyzed before transplanting colonies (from their station of origin, tagged and collected during Aim 1) and at the end of the experiment (at least one year long) for metagenomics and transcriptomics.

#### 5.8.2.1. Reciprocal transplant experiment
To tell apart phenotypic from adaptive responses using a quantitative genomic approach, I will conduct reciprocal transplant experiments using coral nubbins from (i) the same colonies, i.e., belonging to the same clone, and (ii) colonies of multiple genotypes from both the suboptimal and reference stations. Similar experiments have compared within-genotype and between-genotype variability and enabled the quantification of heritability in the broad sense or the proportion of variation attributable to genetic factors [74, 91-93]. Nubbins will be sampled to assess metabolic and physiological changes in the short (months) and long terms (> one year). These measurements will be repeated at regular time intervals and at different seasons to account for natural environmental fluctuations, with particular attention to the warm season (Dec. to Apr.), which is a long period when higher SST could significantly impact corals. Indeed, I measured up to 33°C inside the Bouraké system in 2016, which was 3°C warmer than at the reference site, where SST was already 2°C above the average temperature for this period of the year in New Caledonia. As previously mentioned, I found that coral bleaching rates inside the Bouraké system were lower than elsewhere, suggesting the
corals' ability to cope with the combination of higher temperature, lower pH and oxygen. Overall, the experiment will: (i) assess the response of potentially adapted corals to suboptimal conditions; (ii) assess the potential mechanisms of resistance that colonies, which live in reference conditions (reference stations), use to cope with a rapid change to suboptimal conditions, as well as the resilience of colonies that live in suboptimal conditions once transferred to reference conditions (i.e., plasticity vs. adaptation); (iii) test if the cost of maintaining such responses is viable in the long-term (>12 months); (iv) assess the heritability of climate change tolerance for a series of functional traits; and (v) characterize the genomic basis of plasticity and adaptation in both coral hosts and microbiome.

**5.8.2.2. Sampling, handling, and experimental design:** The duration of the transplant experiment is 12 months at minima using the five selected species. For each species, at each station (x2) and for each site (x2), 10 genetically distinct and large coral colonies will be selected among those identified in Aim 1. From each colony, 20 nubbins/clones (i.e., clonemates within the same colony) about 5 cm-long will be collected (Fig. 2A). They will be prepared and transplanted at four stations (including two replicates) following a full reciprocal transplant experiment (Fig. 2B).

<figure><img src="image_53.png" /></figure>

**Fig. 5.8.2.2. (A) Sampling design for each of the five coral species and (B) an example of how nubbins/clones from 10 colonies will be reciprocally transplanted between the 4 stations (both at Bouraké and reference sites). Please, note: the design is oversimplified for graphic convenience; only one level of nubbin distribution is depicted for clarity, e.g., on panel B only clones B1.1 are shown.**

Five nubbins from each of the 10 colonies, and for each of the five species will be cross-transplanted at each of the four stations. In total, at each station, I will transplant 1,000 nubbins for a total of 5,000 transplants (see also the Risk mitigation section for a backup experimental design). This experimental design make it possible to apply all treatments to all genotypes, to compare the results within and between each colony, and to assess broad sense H². All fragments will be weighed before and after being individually glued onto tagged plates (see [13, 14]) and attached to non-invasive structures, which will be anchored to a hard substrate.

**5.8.2.3. Physiological traits:** At the beginning of the transplantation, and every two months, photosynthetic efficiency (Fv/Fm) and electron transport rate (ETR) will be measured *in situ* on the transplanted corals using a Diving-Pam. The measurement frequency will be increased during the warm summer period depending on the amount of the hot spots or in case of bleaching.

Metabolic rates will be measured directly *in situ*, to avoid handling stress, during two seasons: summer (February) and winter (August). For each species and station, five genetically distinct nubbins will be incubated using underwater semi-autonomous Plexiglas chambers. Technically, each nubbins will be placed in one of the 10 experimental respirometry chambers (1-2 L) run simultaneously. A reference chamber– with no coral will account for seawater microbial activity. Each chamber will be hermetically sealed and connected to a YSI 6920 multi-parameter probe recording dissolved oxygen (to assess photosynthetic and respiration rates in the light and dark, respectively), temperature and salinity at 1 min intervals. Seawater will be recirculated between the chamber and the probe at a water flow of 2 L min⁻¹ using an adjustable submersible pump (see [94, 95] for details). At the beginning and at the end of each of incubation, usually lasting 1h, seawater samples will be collected in each chamber using 250
mL syringes to measure pH, $A_T$, nutrient depletion and total organic carbon (for carbon excretion: DIC, DOC, POC, Np and bacterial concentration). Metabolic rates will be calculated as the difference between parameters measured at the beginning and at the end of the incubation, for the highest daily irradiance (maximum photosynthetic rate) and in the dark (respiration rate). Photosynthetically active radiation (PAR, 400-700 nm) will be measured using a LI-1500 light sensor logger connected to a spherical quantum sensor. These measurements will help to quantify corals' heterotrophic and autotrophic rates. Carbon and nitrogen isotope ratios ($\delta^{13}C$ and $\delta^{15}N$) will be determined in *Symbiodiniacea*, host tissue, as well as in the different components of the coral food sources (plankton and particulate organic matter in seawater and sediments). The levels of $\delta^{13}C$ and $\delta^{15}N$ will be analyzed in each sample using an automated nitrogen-carbon analyzer (Flash EA 1112) coupled with an isotope ratio mass spectrometer (IRMS, Delta V Advantage with an interface Conflo IV) located at the University of La Rochelle (collaboration with Dr. Anne Lorrain, IRD-LEMAR). Samples will then be gradually collected, carefully transported to the field laboratory located 15 min away from the study area, and gently cleaned. They will be maintained in thermostatically controlled tanks filled with seawater collected at the sampling stations and regularly replaced. The weight of each nubbin will be measured using the buoyant weight technique and their calcification rates calculated. The time necessary to clean and process all samples from one station is estimated to two-three days. Nubbins will then be returned to their station of origin. Metabolic rates will be normalized by skeleton weight. Univariate data will be analyzed in R using G/LM/ER, or GLS, with correction for autocorrelation to deal with the repeated measures. At the end of the experiment, and after all the measurements have been performed, subsamples of each nubbin will be collected, treated and preserved (-20°C; -80°C) for later analysis of their protein, ATP, lipids, and chlorophyll contents, *Symbiodiniacea* densities and diversity, trophic status, and transcriptomic, while the rest of the coral samples will be maintained alive at each station for further long-term assessment and future projects. All coral parameters will be standardized either by skeleton weight, chlorophyll or protein contents.

#### 5.8.2.4. *Symbiodinium and microbial changes: Symbiodiniacea* diversity will be identified using pyrosequencing of the nuclear ribosomal DNA internal transcribed spacer 2 (ITS2), a technique that allows the best sensitive and quantitative method for *Symbiodiniacea* genotyping. Tag-encoded FLX amplicons will be sequenced using Illumina MiSeq pyrosequencing (Integrated Microbiome Resource (CGEB IMR), Halifax, NS B3H 4R2, Canada) producing 2 x 300 tbp-long reads. Bioinformatics processing and sequence analyses will be performed using QIIME and MOTHUR pipelines, as well as the SymPortal [96]. The annotation will be performed using several reference sequences per type that are represented in our reference library and GenBank. Haplotype diversity and intra-genomic variation will be assessed, as well as quantification of relative abundances of symbiont types. Pairwise comparisons of haplotypes (i.e., within type reference sequence variants), parametric pairwise testing of haplotype abundance and haplotype networks will be built using R packages.

The diversity of bacteria will be assessed using the same nucleic acid extract. The V1-V3 hypervariable regions of 16S rRNA genes (specific of prokaryotes) will be PCR amplified using primers 27F-519R [97, 98], which are mostly specific for bacteria. Sequencing and bioinformatics processing will be the same as above. Species assignment will be performed using SILVA v.128 amended with our own coral sequence database for optimal taxonomic identifications. Direct multivariate statistical analysis (such as CCA, RDA) will also be performed to evaluate the link between prokaryotic diversity and the other variables measured in this project.

The transcriptome of each genotype will be developed at the beginning (Aim 1) and the end of the transplant experiment for comparison. If significant changes in gene expression levels are found, the relevant loci will be assessed using target-capture bisulfite sequencing to determine if changes in gene body or promoter methylation underpin the observed transcriptomic changes.

#### 5.8.2.5. Risk mitigation:
The experiment has the ambition of assessing the largest coral transplantation experiment ever performed so far at a natural analogue, measuring a series of functional traits *in situ* for at least five coral species, and coupling physiological with molecular responses to suboptimal conditions. This ambitious project presents limited risks since the methods have already been successfully tested during the CARIOCA project at CO₂ seeps in PNG (2016-2019). Both molecular and physiological techniques are state of the art and, although the large number of samples is very
challenging, my team has enough experience to achieve this work successfully. Some data interpretation, such as the analysis of microbial diversity, will be carried out by a senior and expert post-doc as well as through our established collaborations. For *in situ* incubations, the experimental design will need to be restricted to two stations (B1 and R1), three replicate clones for each of the five genetically distinct colonies. I plan ca. 960 incubations (3 clones x 5 colonies x 4 treatments x 4 stations x 4 species), which is particularly ambitious but feasible over 20 working days when using a set of 10 chambers (plus one reference). In case of technical problems, or due to difficulties for the measurement of maximum photosynthetic rates during days with low insolation, the number of measures will be adapted.

The availability of a fieldwork laboratory close to the study area is a significant asset. Both the Bouraké and reference site are sheltered areas with no risk of difficulties due to waves or extreme weather events, which could cause sample loss and handling issues or injuries. A preliminary experiment, carried out on the coral *Psammocora contigua*, confirmed the feasibility of the project and the protocol used, particularly handling and transplantation, is tolerated by the corals. However, more fragile species may react differently.

I recognize that our sampling plan is open to criticism, particularly due to the number of replicates and species, which can be considered high-risk. However, this will also be the largest coral transplant experiment ever carried out at a natural analogue mimicking future environmental conditions, and as such, it represents a promise of high knowledge gain. With this in mind, I am aware that natural mortality or unscheduled events could decrease the power of our analyses. The primary phase of fieldwork will be critical to decide whether increasing the number of replicates (distinct colonies and clone nubbins) is feasible or not. The appropriate experimental design will be settled during Aim 1. As the selection of coral species is particularly critical for this ambitious experiment, I made sure to choose species that are abundant at both stations and represent as many life-history strategies, genera and morphologies as possible; they are suitable for transplantation, resist handling, and recover well from breakage. The number of clone replicates (n = 20) is realistic if large colonies are targeted to minimize the impact of repeated sampling. In case of substantial mortality, we will repeat the experiment with a different, more robust set of species.

### 5.8.3. Aim 3 – Assessing the contribution of plasticity to adaptation.

As climate change is likely to occur on timescales that span multiple generations of corals, parental effects and/or TGP could be a viable mechanism for corals to adapt to new environmental conditions. Aim 3 is designed to test if the duration and timing of exposure of a coral to climate change-like conditions can influence the performances of the following generation. This component, therefore, intends to establish if plasticity is adaptive and to assess which mechanism is involved between phenotypic acclimatization, epigenetic inheritance, parental effect and TGP. Parental effects have been demonstrated in the offspring of several short-lived marine benthic species when mature adults are exposed to specific environments during their reproductive period, whereas TGP is observed in the offspring when parents develop in a new environment from early life stages [92, 93]. Discriminating between the two mechanisms is difficult, particularly in corals, because the main difference relies on the timing of parental exposition (and therefore of eggs and larvae), which depends on the experimental design, as well as the reproduction mode and life-span of the organisms considered. The duration and timing of parental exposition are, therefore, critical to distinguish TGP from parental effects. Corals inside the Bouraké system have settled and grown in suboptimal conditions during their entire life, and are consequently fully acclimatized. This is why my approach is particularly innovative compared to previous studies: it uses colonies fully acclimatized in a natural analogue system. Previous studies have mainly assessed the effects of OA or OW in isolation, and only four studies have been published that evaluate the effect of high *pCO*₂ in combination with high temperatures on the performances of coral offspring [46, 47, 51, 61]. In these studies, adult colonies of the brooder *P. damicornis* were exposed to experimental conditions for a maximum to two months before larval release, and their performances were measured during a period of 1 to 6 months.

My experiment is designed to distinguish TGP from parental effects by exposing entire colonies from a reference environment to the suboptimal conditions of the natural analogue of Bouraké. I will set up classic reciprocal transplant experiments at two dates: 11 months and 2 days before the following
predicted spawning period (i.e., 3-4 days after the full moon in November-December) (Fig. 3). As a consequence, eggs, sperms and larvae/planulae will be exposed (inside their parental colonies) to suboptimal and/or reference conditions during different periods: never (mother colonies from the reference stations), two days, 11 months and during their entire development (mother colonies from the suboptimal stations). The TGP of one generation can be determined from its developmental plasticity because the duration of the offspring exposition to the environmental conditions of their parents will be different. Larvae will be settled in aquaria and, once the recruits are of sufficient size (at least 4 to 6 months-old) they will be transplanted *in situ* at the Bouraké and reference stations following a full orthogonal experimental design (Fig. 3). My ambition is to follow the performance of these recruits after 6 months, 12 months and beyond, and to assess their functional and fitness-related traits, as well as gene expression change from the larvae to the adult stage. The F1 colonies will be kept until sexual maturity (generally 2 to 3 years), and the experiment repeated to obtain an F2 generation.

**5.8.3.1. Experimental design for larvae and recruits:** This experiment will use four hermaphroditic spawner species (Table 1), genetically identified in Aim 1, by set of two over two years. Each treatment will use six adult colonies (Fig. 3); i.e., six colonies will not be moved (“never”) at both the Bouraké and reference stations, six colonies will be cross-transplanted from the reference to Bouraké in December (11 months before the spawning period), and six colonies will be collected two days before the spawning period from the reference stations. Two days before the expected spawning day, all colonies will be transferred to the *Aquarium des lagons* in Nouméa where the experiment will take place. Each tank will reproduce a similar environmental conditions as measured *in situ* at each stations. In the evening of the spawning day, which usually is three to five days after the full moon of November (i.e., synchronous to the GBR spawning event) each set of colonies (4 treatments x 6 colonies x 2 species) will be maintained in individual large tanks with static seawater.

<figure><img src="image_54.png" /></figure>

**Fig. 5.8.3.1. Experimental design for Aim 3.** From the top: **a)** colonies are selected at both the natural analogue and the reference stations, and **b)** colonies from the reference stations are transplanted (11 months and 2 days before spawning) to the natural analogue. All colonies are collected the day before spawning and maintained in tanks in conditions similar to those experienced *in situ*. After coral spawning, cross-fertilization, and metamorphosis, larvae are allowed to settle and grow during 4 to 6 months. **c)** recruits are then cross-transplanted *in situ*. Once mature, colonies are collected again, and the experiment repeated with the new generation.

Gametes will be cross-fertilized within each treatment and the resulting lineage will be tracked. Larvae will be collected and maintained in eight 20 L conic tanks explicitly designed for larvae maintenance (details in Prasetia et al. subm. [86]). Seawater will be filtered (50 µm), renewed and maintained at the same temperature and pH than previously experienced by parental colonies. For each treatment, larvae
respiration rates will be measured in 8 mL glass vials (n=10 per each set of colonies) equipped with an O₂ sensor spot for optical measurement of dissolved oxygen concentration using a Fibox 4. Oxygen consumption (i.e., dark respiration) will be measured for 30 min in darkness. The larvae size will be measured and subsamples preserved for later assessment of gene expression as well as protein and lipid contents.

Larvae will then be allowed to settle on ceramic and aragonite plugs (Aquasonics, n=40 for each set of colonies), previously deployed *in situ* for at least 7 months to allow for biofilm development. Once the primary polyps are observed, plugs (n=10 per each set of colonies per species) with one or more recruits will be photographed under a graduate stereomicroscope and recruits lateral growth will be monitored for 5 to 8 months. Settlement and survival rates will be recorded during the first two weeks. According to the number of recruits available, some will be sacrificed to compare skeleton integrity using scanning electron microscopy (SEM), and to measure lipid, protein and ATP contents.

At this point, all the plugs with recruits (F1) will be labeled and transplanted to the station where their parent colonies were sampled, following a full-orthogonal experimental design (Fig. 3). Plugs with recruits for each set of colonies/treatment will be equally transplanted between the Bouraké and reference stations. This will be achieved by attaching several plugs from the same treatment to PVC plates protected with plastic nets to avoid fish grazing and recruit disturbance. This stage is critical because, in the wild, young coral recruits suffer high levels of mortality due to several factors such as competition with algae and encrusting invertebrates or smothering by sediments [99]. In a preliminary TGP experiment on the offspring of the coral *P. damicornis* [86], I observed high mortality rates at both the suboptimal and reference stations, likely due to the absence of protective nets and high sedimentation rates during the month following transplantation. Although all care will be taken to avoid excessive loss, any mortality in transplanted corals will be interpreted as natural selection. After six months, photosynthetic efficiency will be measured *in situ* using a non-intrusive Diving-Pam with a 3 mm optical probe. Plugs will then be collected, carefully cleaned, photographed and the lateral growth rate of recruits measured. This will be repeated every six months together with the measurement of a series of functional traits (see Aim 1) and fitness-related traits (e.g., survival, fecundity). Changes in fitness values, the correlation between fitness and the reaction norm slope, adaptive plasticity strategies, and intergenerational inheritance of best traits, as well as transcriptomics, will be compared between generations. If significant changes in gene expression levels are found, the relevant loci will be assessed using target-capture bisulfite sequencing to determine if changes in gene body or promoter methylation underpin the observed transcriptomic differences; and if epigenetic marks were inherited from one generation to the next. Changes in microbiome across generations will be assessed by the microbial profiling of the recruits (see Aim 2). The contribution of physiological and genetic changes will be assessed in a Quantitative Trait Locus analysis framework, and using boosted regression trees.

**5.8.3.2. Risk mitigation:** Aim 3 is very ambitious, and several delicate stages can impact the success of the experiment. However, this section of the project can provide unprecedented results as very limited information is currently available on the transgenerational plasticity of coral species. Our preliminary experiments using the brooder coral *Pocillopora damicornis* and the broadcast spawner *Acropora tenuis* were encouraging, suggesting that our protocol is efficient. Among the limitations, I estimated that six is the minimum number of colonies per treatment to use in such an experimental design, and this is already challenging. In addition, it would be ideal, but logistically impossible, starting with the 4 species at the same time. Because corals are long-lived organisms, it will not be possible to go beyond an F2 generation, at least during the duration of the HOPE project. Reaching an F2 generation before the end of the project will be an incredible opportunity and will, at the same time, greatly facilitate access to new funds to continue the project.

A possible risk is that only a few sexually mature corals may be found at the study stations, as sexual reproduction in corals does not occur every year for all colonies. This can only be anticipated one week before spawning by searching for bundles on coral fragments and selecting colonies accordingly. But, of course, that will not be possible for colonies transplanted 11 months before. To prevent, or at least mitigate, the risk due to the absence of parent colonies, I will transplant more than six colonies per station but will use only six in the final crosses. In the worst scenario, I will use the brooder coral *P. damicornis*, which reproduction period extends to May making it possible a 'rescue' experiment starting from December. I have been working with this easy to reproduce species, and if I have to use it, the
"temporal effect" will be reduced from 11 to 5 months. Our pilot studies indicated that the transportation of colonies between the study stations and the aquarium does not impact the reproductive activity. If transportation was to affect other species, colonies could be kept in large tanks near the study for the duration of the spawning event, or eggs and sperm could be collected directly in situ using underwater mesh tents. I will test this in a pilot study during the spawning in 2020 using the R/V *Alis* (cruise planned 28 Nov. to 08 Dec.).

**The Gantt chart below illustrates the schedule of the project.** Black boxes indicated the whole period of the project devoted to the specific Aim. Grey boxes indicate the provisional period necessary for each task.

<table><thead><tr><th></th><th>202X</th><th>202x+1</th><th>202x+2</th><th>202x+3</th><th>202x+4</th></tr></thead><tbody><tr><td><strong>Aim 1- Coral identification</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sampling & labeling</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Coral colony genotyping</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Aim 2- Within-generation plasticity</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Corals transplantation (RT)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Physiology</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>Symbiodinium</em> and microbiome genotyping</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Gene expression</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Aim 3- Multi-generation plasticity (TGP)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Reproduction and offspring</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Physiology on offspring, juveniles & adult corals</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**5.9. Feasibility and mitigation strategies.** I have been studying natural analogues for more than ten years, and have built strong collaborations with EU research partners, as well as with other research groups studying the effects of climate change on marine ecosystems. Based on my experience, I do not foresee any major obstacles to implement the proposed research and build the dataset. I have an in-depth knowledge of working on the effects of OA at the scale of ecosystems [e.g., 75], particularly on species traits [e.g., 14, 100]. My main interest is calcification response [e.g., 12-14], body size metrics [e.g., 101] and, more recently, I became interested in incorporating a larger selection of traits and using – *omics* approaches (e.g., CARIOCA project). My growing confidence on this topic is demonstrated through the popularity of my studies, which are increasingly cited.

The project is very ambitious in terms of objectives and experimental design, but from my extensive experience of working with natural analogues, I am confident that it is feasible in the framework of this grant, which will provide enough time and funds. For each experiment and aim, I estimated the risks and anticipated alternatives. I also carried out preliminary experiments on the coral *P. contigua* (transplantations), *A. tenuis* and *P. damicornis* type α (reproduction and settlement), which demonstrated the feasibility of our protocols. Technically, our experimental design is innovative and dense, but both molecular and physiological techniques are state of the art and, although there is a large number of samples to process, which is challenging, my team will have enough experience to achieve this work successfully. Besides, I will select the best students and post-doctoral candidates with relevant expertise in genetics, microbiome analysis, and coral reproduction. Given that the project plans a significant amount of laboratory work, I will also appoint a research assistant.

**5.10. Expected results of the project: high-risk/high-gain research.** HOPE represents a significant step forward in addressing the global change challenge, and understanding the evolutionary response of coral reefs. Previous studies have been performed in the laboratory using short-lived species only. Only a few studies have been applied to corals, but their outcomes remained limited due to the constraints of working in a laboratory set-up. Studies at CO₂ vents and other natural analogues have only been able to draw conclusions about the effect of acidification.
My approach will use a novel natural analogue where seawater pH, temperature and oxygen concentrations are suboptimal for corals and mimic conditions forecasted for the future. The validity of this unique site lies in that the extreme environmental conditions at the natural analogue are: i) entirely predictable according to tide; ii) variations are similar from one day to the other; ii) the site geomorphology has not changed during at least the last century, indicating that the environmental conditions experienced by the corals have been similar for a very long time. Although this system is not perfect, like any other of the known natural analogues, it offers unique advantages for exploring the evolutionary mechanisms that corals might develop in a changing world. Using my proposal offers to explore a new model system using an unprecedented strategy. This project has the potential to deliver ground-breaking results that will boost our understanding of how coral reefs respond to the combined effects of several suboptimal environmental parameters, in combination and in nature. My approach is the next logical step forward in this field of science, using existing methodologies combined into ambitious experimental designs. The technical risk is relatively high, particularly due to the high number of species, samples and replicates, but whether we can complete the full experimental design or have to use alternatives, the knowledge gain will be huge. For the first time, *in situ* experiments will be performed on several coral species, and over the long-term, to decipher their ability to acclimatize and adapt to future environmental conditions. I expect ground-breaking results that can fundamentally change our understanding of how climate change will affect coral reefs and the underlying biological mechanisms involved. My project will deliver unprecedented data on the physiological response of coral species to climate change and their ability to “evolve” in this context. By revealing that corals from the natural analogue of Bouraké are already resistant to climate change, and the mechanisms they have used to do that, HOPE will contribute critical knowledge to future projects, particularly those aiming at using assisted evolution to improve coral reef resilience [112]. And finally, after five years, the project will conclude with an unprecedented F2 generation of coral colonies grown in the wild under climate change-like conditions, offering incredible opportunities to leverage a synergy grant.

**5.11. Size and nature of the research team and host institute.**

My project offers an innovative way of tackling fundamental knowledge of the evolutionary trajectories that coral reefs might develop to face climate change. Thanks to its high degree of interdisciplinaryity, the project will explore the physiological responses of different species through the measurements of genes, proteins, and metabolic variability. This comprehensive scientific approach calls for a team able to address various fields of research, techniques and philosophical methods. The team will be composed of a core of high-class and motivated members, including postdocs and PhDs mentored by expert scientists. My vast network of collaborations with internationally renowned experts will be called upon to support and possibly collaborate if necessary.

**Principal investigator:** I have a permanent research scientist position at the French Institute for Research and Development (IRD) in the research group ENTROPIE ("Tropical Marine Ecology of the Pacific and Indian Oceans", http://umr-entropie.ird.nc/index.php/home), which is based in New Caledonia. Since 2015, thanks to several grants, I have established a team (https://rodolfo-metalpalab.com) to study the effects of climate change on coral physiology. I am particularly interested in the responses of a range of reef-forming species to extreme conditions such as experienced at CO2 vents, in mangroves, and along turbid coastal reefs. I believe that natural analogues are the best model to improve our knowledge of this research topic. Although most of my research is in the field, I am investing funds in an aquarium laboratory hosted at the *Aquarium des Lagons* in Nouméa. There, several facilities and gear make it possible to maintain corals at finely controlled water temperatures and pCO2, and run experiments to test the corals' physiological mechanisms of response to environmental stresses. As the group leader, I manage the group's budget, write scientific reports, and organize and develop further collaborations and projects. I am responsible for fieldwork, developing methods and the required technological tools needed, as well as organizing the necessary collaborations within our institute, as well as nationally and internationally when needed. During the project, I will play an active role in preparing and submitting manuscripts for publication in high-profile journals, while training graduate students and postdoctoral researchers to independently write papers and grant proposals. I will ensure that group members are provided with opportunities to learn the skills necessary for the project through scheduled lab meetings, seminars, and courses. All members devoted to fieldwork will be HSE professional divers. Undergraduate students will help with routine measurements of the seawater
carbonate chemistry, protein, chlorophyll, etc. The project will have an extensive media coverage (included in the budget) with the creation of a website, publications on social platforms, and documentaries that will be produced by the IRD Image department. Our communication manager will organize press releases and interviews with national and international newspapers and TV.

**The team:** At IRD in New Caledonia, I perform my research projects in collaboration with one staff scientist, Dr. Fanny Houlbreque, who contributes her extensive experience of coral physiology and biochemistry, and one research assistant, who will be dedicated to molecular analyses at the IRD "Plateforme du vivant". Two students will start their PhD project on the Bouraké system. Dr. Hélène Magalon and Cecile Fauvelot are experts on coral phylogeny and population genetics; they will join the team for the duration of Aim 1. One senior postdoc, with relevant expertise, will be appointed and he/she will work for three years on the host and microbiome molecular analyses and data interpretation. Fanny Houlbreque will be committed to transplantation experiments, assessing species physiological responses, and collecting environmental parameters. She will be assisted by the IRD professional divers, and one PhD for three years. One research assistant, with laboratory skills in biochemical and chemical measurements (DIC, nutrients etc) will be appointed for 4 years. One junior post-doc will be appointed for three years to focus on colony transplantation and transgenerational experiments. All participants, a total of 10 persons, will help during coral spawning events.

**Host institute:** The host is the Institute for Research and Development (IRD: https://en.ird.fr/). The IRD is a French public interdisciplinary organization covering a large panel of scientific fields, from terrestrial to marine ecology as well as human, social and economic sciences. One of its goals is to assess the effects of climate change on ecosystems and populations living in southern countries. The IRD has established representations in Africa, the Mediterranean, Latin America, Asia and the French tropical overseas territories including New Caledonia, which is an EU associated country. This large geographical presence is an asset for scientists to develop transcontinental collaborations, research projects, and fieldwork worldwide. In New Caledonia, the main objective of the IRD team ENTROPIE is to better understand Indo-Pacific marine ecosystems in the context of global change. It has access to several facilities and gear for field research including boats (three diving boats and one 30m R/V), diving services, and cutting-edge laboratories for chemical, molecular, microbiological and physiological measurements.

## 5.12. References

[1] Gattuso JP, Magnan A, Billé R, Cheung WWL, et al. (2015) Contrasting futures for ocean and society from different anthropogenic CO₂ emissions scenarios. Science 349(6243): aac4722-1.

[2] IPCC (2014) Summary for policymakers. In: Field CB et al. (Eds), Climate Change 2014: impacts, adaptation and vulnerability. Contribution of working group ii to the fifth assessment report of the intergovernmental panel on climate change. Cambridge Univ. Press, Cambridge, 32 pp.

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# 6. TRADUCTION EN FRANÇAIS DU PARAGRAPHE 2.3 AU PARAGRAPHE 3.5

## 2.3. Historique de la recherche

Mon sujet principal a toujours été l'effet du changement climatique, avec une attention particulière sur l'acidification des océans (AO), sur une série d'organismes marins calcifiants, en particulier les coraux. Mon approche à cet égard a été classique dans mes premières études, c'est-à-dire en utilisant des expériences en laboratoire, et innovante au cours de la carrière suivante puisque probablement j'ai été le premier à utiliser des laboratoires naturels où des conditions proches à celles prevues à l'avenir, caractérisaient naturellement l'environnement. J'ai publié 71 articles à ce jour : 5 avant mon doctorat et 8 pendant mon doctorat. Au cours de ma carrière post-doctorale, 41 des 58 articles que j'ai publiés étaient effectués dans des laboratoires naturels simulant des conditions futures, tandis que 14 utilisaient une approche en laboratoire.

Quelques étapes préliminaires de mon parcours scientifique peuvent expliquer la dynamique de mes recherches et la continuité du thème principal qui a caractérisé ces 23 dernières années. En 1997, lorsque j'ai commencé mon master au Centre de recherche ENEA S. Teresa, et pendant les quatre années de recherche qui ont suivies, mes superviseurs C.N. Bianchi, A. Peirano et C. Morri m'ont fait découvrir le monde de la recherche scientifique. J'ai réalisé ma première expérience en aquarium, participé à quelques croisières océanographiques, effectué mes premières transplantations de coraux sur le terrain et produit des données pour les cinq premiers articles. Pendant mon doctorat au Centre Scientifique de Monaco sous la supervision de C. Ferrier-Pagès et D. Allemand, j'ai continué sur le sujet que j'étudiais en Italie pendant ma période de recherche pré-doctorale, c'est-à-dire l'effet du réchauffement sur les coraux méditerranéens. Ce sujet a été élargi au thème émergent de l'AO grâce à la collaboration avec J-P Gattuso du laboratoire CNRS LOV à Villefranche-sur-mer. Pendant ma thèse de doctorat, j'ai publié 8 articles.

Au cours de mes deux années de post-doctorat avec le professeur J-H Spencer de l'université de Plymouth, qui ont commencé immédiatement après mon doctorat, j'ai eu la chance de participer à EPOCA et MedSeA, les deux premiers projets de recherche européens sur l'AO. Cela a beaucoup aidé et stimulé ma carrière et ma curiosité scientifique. Avec J-H Spencer, j'ai été le premier à utiliser des resurgences de CO₂ sous-marins pour étudier la réponse des communautés benthiques des zones tempérées à des conditions de faible pH, fournissant ainsi les premières études sur l'effet de l'AO à l'échelle de l'écosystème. Les resurgences naturels de CO₂, où l'émission volcanique de CO₂ modifie la chimie de l'eau de mer environnante, constituent un outil expérimental puissant pour étudier la capacité des espèces à s'acclimater ou à s'adapter aux futurs niveaux d'acidification. Depuis notre premier article en 2008, les sites de CO₂ ont été fréquemment utilisés comme analogues naturels pour étudier les effets de l'AO sur des espèces et/ou des communautés entières. Cette approche a fortement caractérisé mes recherches futures, au cours desquelles j'ai utilisé à plusieurs reprises ces sites spéciaux et en ai découvert d'autres. J'ai décrit pour la première fois les resurgences de CO₂ d'Ischia et de Vulcano (Hall-Spencer, Rodolfo-Metalpa et al. 2008 *Nature* ; Boatta et al. 2013 *Mar Poll Bull*), les resurgences profonds du Golfe de Californie (Prol-Ledesma et al. 2016, *Nature Comm*), et l'île d'Ambitle (Pichler et al. 2019, *Mar Poll Bull*). J'ai travaillé sur les plus célèbres resurgences de CO₂ à Normanby (PNG, Fabricius et al. 2011) et White Island (NZ, Brinkman et Smith, 2014), ainsi qu'à Nikko Bay à Palau (Reimer et al. 2023, *Coral Reefs*). En plus des resurgences de CO₂, j'ai découvert un site unique où les trois principaux paramètres environnementaux du changement climatique, à savoir l'AO, le réchauffement et la désoxygénation, se combinent en
un seul site, la baie semi-fermée de Bouraké en Nouvelle-Calédonie (Camp et al. 2017, *Scientific Reports* ; Maggioni et al. 2021). La plupart de mes projets de recherche de 2017 ont été réalisés à Bouraké, et la plupart de mes projets futurs utiliseront ce formidable laboratoire naturel.

Au cours de ce premier voyage scientifique de 26 ans, j'ai établi des collaborations fortes et amicales avec plusieurs collègues travaillant sur le même sujet. Ces collaborations ont été consolidées au sein du consortium ICONA, "International CO₂ Natural Analogues Network (ICONA)", un projet de collaboration internationale qui vise à utiliser des analogues naturels pour étudier les effets de l'AO sur les écosystèmes des récifs coralliens. Ce projet nous a permis de travailler, en 2023, à Nikko Bay (Palau, Micronésie), probablement la baie semi-fermée la plus emblématique où les coraux se sont adaptés à un réchauffement de 1°C et au niveau d'AO prévu pour l'avenir.

Le principal avantage de l'utilisation de laboratoires naturels pour étudier les effets du changement climatique en cours est que les résultats sont écologiquement pertinents, bien qu'il faille toujours garder à l'esprit certaines limites. En effet, la plupart des conclusions relatives à l'impact de l'AO (ainsi que d'autres facteurs tels que le stress dû au choc thermique, la désoxygénation, l'enrichissement en nutriments, etc.) sur les organismes et les extrapolations qui en découlent au niveau de l'écosystème, proviennent d'expériences de laboratoire à court terme sur des organismes individuels. Ces expériences sont certainement instructives, car elles nous permettent d'identifier l'effet d'une ou de quelques variables, mais isolément, elles sont incapables de rendre compte des implications de la capacité d'acclimatation des espèces. Par conséquent, le temps d'exposition aux futures conditions de changement global et les conditions expérimentales elles-mêmes deviennent une question centrale pour améliorer notre compréhension de la capacité d'acclimatation des organismes marins aux conditions futures. Les expériences en laboratoire ne sont pas non plus réalistes d'un point de vue écologique car, par exemple, elles ne tiennent pas compte des effets des interactions entre les espèces, de l'apport naturel de nutriments et des fluctuations des principaux paramètres environnementaux. En outre, la quasi-totalité des études ont négligé le rôle de l'adaptation puisqu'elles n'ont testé que les réponses au sein d'une même génération au changement global et lors d'une exposition à court terme au stress. Les études utilisant des organismes à courte durée de vie, tels que les coccolithophores, les copépodes et les polychètes, les oursins et les poissons, ou utilisant des expériences de reproduction sur des larves d'oursins, constituent des exceptions.

L'utilisation de laboratoires naturels a certainement contribué à mon recrutement à l'IRD, UMR ENTROPIE en octobre 2013 après une brève période de post-doctorat, et a favorisé l'obtention de deux subventions de l'ANR : CARIOCA - *Acclimatation des récifs coralliens à l'acidification des océans au niveau des suintements de CO₂*, et PNG-Vents - *Utilisation des resurgences volcaniques de CO₂ pour évaluer la capacité des récifs coralliens à s'acclimater et à s'adapter à l'acidification et au réchauffement des océans*. Ces deux projets consécutifs ont permis à mon équipe d'effectuer 7 missions de terrain en Papouasie N.Ile Guinée pour étudier les coraux, les éponges et les poissons vivants dans l'eau de mer acidifiée au sein des resurgences de CO₂. Elles m'ont également permises de collaborer avec plusieurs experts sur ce sujet, formant ainsi un réseau de scientifiques, tous experts de l'effet du changement climatique sur les récifs coralliens et les espèces associées. Les laboratoires naturels ont été pour moi l'outil idéal pour tester des mécanismes spécifiques ou des réponses à l'échelle de l'habitat aux changements environnementaux d'une série d'organismes.

Après mon doctorat en 2007, la plupart de mes recherches se sont concentrées sur les coraux (32 articles sur 58), y compris les espèces tempérées et tropicales d'eaux peu profondes, ainsi que les coraux d'eau froide et les anémones, mais aussi sur d'autres taxons (19 sur 58), y compris
les bryozoaires, les algues corallines et les mollusques, pour lesquels ma contribution a été importante, et les foraminifères, les coccolithophores, les algues, les poissons et les éponges, qui ont été étudiés grâce à des collaborateurs externes possédant des expertises spécifiques. La plupart du temps, j'ai étudié l'effet de l'AO, parfois en combinaison avec le réchauffement, sur les taux métaboliques et la symbiose des espèces récifales calcifiantes (par exemple, la photosynthèse, la respiration, les paramètres des tissus, etc.), mais mon intérêt principal a toujours porté sur leur capacité à calcifier car il s'agit de l'effet le plus évident et le plus uniforme sur la plupart des organismes étudiés dans le contexte de l'AO (Kroeker et al. 2013 ; IPCC 2015). Pour cela, au cours de mes études, la calcification a été étudiée en utilisant des techniques classiques et de pointe telles que la pesée dans l'eau, la coloration à l'alizarine, la transplantation, etc. (39 sur 58), mais aussi en utilisant des traceurs isotopiques et des techniques géochimiques (13 sur 58).

Je me rends compte que la plupart du temps, mes résultats vont à l'encontre du consensus concernant l'effet de l'AO sur le métabolisme en général, c'est-à-dire que j'ai rarement mis en évidence un effet dramatique des faibles niveaux de pH de l'eau de mer sur les taux de calcification corallienne, bien que la plupart des études démontrent une réduction allant jusqu'à 83 % de la capacité de calcification corallienne (Langdon et al. 2005). Pourquoi ? J'ai décidé de développer cette question dans ce manuscrit d'HDR, qui vise à démontrer que j'ai travaillé tout au long de ma carrière autour d'une question fondamentale, et que j'ai essayé de l'éclairer et de la résoudre, ce qui n'est probablement pas le cas ! Ainsi, s'il est une question à laquelle j'ai toujours tenté de répondre, c'est bien "Pourquoi l'AO devrait-elle affecter certains mécanismes métaboliques fondamentaux, tels que la calcification, comme on le prétendait initialement ?" Mes résultats divergents sont-ils simplement le fruit de mon désir de me démarquer du courant dominant ? Ou pire encore, l'effet de l'AO a-t-il été quelque peu exagéré ?

Pour tenter de répondre à la question "Pourquoi les coraux devraient-ils se préoccuper de l'acidification des océans ?", je vais brièvement remplacer dans son contexte l'acidification des océans. Je vais brièvement contextualiser le danger que représente l'acidification des océans pour les coraux, faire une description synthétique des principales études concernant l'effet de l'AO sur la calcification corallienne, revoir les principaux points qui ont permis d'établir le consensus sur l'effet de l'AO sur les récifs coralliens, et dans quelle mesure ce consensus a été modifié par rapport aux premières études réalisées il y a une vingtaine d'années. Je ne rapporterai que les études concernant l'effet de l'AO sur la calcification.

Dans ce manuscrit d'HDR, je n'entrerai pas dans les détails des mécanismes chimiques et physiques complexes qui régissent la calcification des coraux et qui n'ont pas encore été entièrement révélés. En effet, ce n'était pas le but de mes recherches. Je terminerai par résumer dans quelle mesure mes études ont contribué à répondre à la question susmentionnée, puis je décrirai brièvement les articles qui, selon moi, ont fait date dans ma recherche. Ce faisant, je ne décrirai que les études que j'ai publiées au cours des 20 dernières années et qui portaient sur l'effet de l'AO sur la capacité de calcification des espèces calcifiantes des récifs, principalement des coraux. Mon intention n'était pas simplement d'énumérer les publications les uns après les autres, car bien qu'ils soient énumérés dans un ordre chronologique, ils ont en fait été progressifs par rapport à la question scientifique initiale : "Pourquoi la calcification corallienne devrait-elle se préoccuper de l'AO ?" Ces études ont été menées à la fois en utilisant une approche classique en laboratoire, c'est-à-dire en maintenant des organismes dans des aquariums dans des conditions artificielles, et en utilisant des analogues naturels de conditions futures, c'est-à-dire des laboratoires naturels où les conditions environnementales imitent déjà les scénarios futurs. Étant donné que j'ai été à l'avant-garde de cette dernière approche et que la plupart de mes recherches ont été menées dans ces laboratoires naturels, je mettrai particulièrement l'accent sur
ce type d'approche qui a profondément marqué ma carrière. Enfin, je décrirai mes recherches actuelles et mes perspectives d'avenir, y compris le projet que j'ai présenté au dernier appel CoG de l'ERC en 2021, qui reste à mon avis un projet innovant et un défi inestimable à relever dans ma carrière.

## 3. POURQUOI LES CORAUX DOIVENT-ILS SE PRÉOCCUPER DE L'ACIDIFICATION DES OCÉANS : VERS UN REVISION DU CONSENSUS ?

L'acidification des océans est l'une des principales menaces pesant sur les habitats marins, susceptible de provoquer des changements dans la biodiversité et la fonction des écosystèmes au cours de ce siècle. L'acidification des océans pourrait affecter divers paramètres physiologiques à différents stades du cycle de vie des animaux, de la reproduction à la croissance des adultes en passant par les phases larvaires. Les espèces calcifiantes, telles que les coraux scléractinaires qui abritent une grande partie de la biodiversité océanique mondiale, seront apparemment les plus touchées par l'AO puisque leurs taux de calcification et de dissolution semblent liés à la chimie des carbonates de l'eau de mer. Des études montrent une diminution des taux nets de calcification en raison de la baisse du pH et de la concentration en ions carbonate, ainsi qu'une augmentation du taux de dissolution des squelettes carbonatés. Toutefois, le consensus sur la projection d'une réduction de la capacité de calcification des espèces est en contradiction avec plusieurs découvertes passées et récentes montrant que certains calcificateurs ne semblent pas être affectés par l'AO. Ces résultats divergents ont clairement montré que nos connaissances actuelles sur la réponse biologique à l'AO sont extrêmement limitées et que certaines hypothèses que nous avons utilisées jusqu'à présent pourraient être inexactes pour prédire la réponse des espèces à l'AO.

Dans ce rapport, le consensus sur la réponse des taux de calcification corallienne à l'acidification, les premières hypothèses erronées et les idées fausses seront révisées à la lumière des contributions récentes qui imposent un ajustement de notre compréhension actuelle des réponses de la calcification corallienne face à l'AO.

### 3.1. Établir un consensus sur la menace que représente l'acidification des océans

Depuis la révolution industrielle, les niveaux de CO₂ dans l'atmosphère ont augmenté et ont presque doublé depuis. Les projections concernant l'avenir de l'AO indiquent que si les émissions de CO₂ continuent d'augmenter au rythme actuel, le pH de l'océan de surface devrait diminuer d'environ 0,4 unité d'ici la fin du siècle par rapport aux niveaux préindustriels (Jiang et al. 2023). Ce taux d'acidification est sans précédent depuis 65 millions d'années et pose des problèmes importants pour les écosystèmes et les organismes marins.

Environ un tiers des émissions anthropiques de CO₂ a été absorbé par les océans, entraînant le processus d'AO au cours duquel le CO₂ absorbé se transforme en acide carbonique, augmentant les concentrations de H⁺, de bicarbonate (HCO₃⁻) et de dioxyde de carbone dissous (CO₂), tout en diminuant la concentration de carbonate (CO₃²⁻), l'état de saturation du carbonate de calcium (Ω) et le pH de l'eau de mer (Orr et al. 2005). Ω est le rapport entre le produit de la concentration d'ions ([Ca²⁺] x [HCO₃⁻]) et le produit de solubilité (K'sp) pour l'aragonite minérale qui compose le squelette corallien.

Les régions côtières, où les activités et les apports humains sont concentrés, peuvent connaître d'autres problèmes localisés de pH (He & Silliman 2019). Des facteurs tels que le ruissellement des nutriments provenant de l'agriculture, les rejets d'eaux usées et la pollution industrielle peuvent exacerber l'acidification dans ces zones. En outre, les eaux côtières connaissent naturellement une variabilité du pH due à divers facteurs, notamment le cycle des marées, les changements de température, l'apport d'eau douce par les rivières et les remontées d'eau
profonde. Ces fluctuations naturelles peuvent se produire sur des échelles de temps diurnes (quotidiennes) et saisonnières. L'augmentation des apports en nutriments provenant des activités agricoles et des rejets d'eaux usées peut stimuler la croissance des algues et du phytoplancton, entraînant une production excessive de matière organique. La décomposition ultérieure de cette matière organique consomme de l'oxygène et libère du CO2, ce qui exacerbe l'acidification future localisée.

L'idée que l'augmentation de la pCO2 atmosphérique prévue pour le 21ème siècle a un effet négatif sur les récifs coralliens est née à la fin des années 1990, lorsqu'il a été démontré qu'une diminution de l'Ωara, et donc du CO3²⁻, avait un effet négatif sur la calcification corallienne (Smith et Buddemeier 1992). Gattuso et al. (1999) ont réalisé la première étude exhaustive des mécanismes de calcification et de photosynthèse des coraux dans la perspective de la menace relativement nouvelle qui pèse sur la santé des récifs coralliens. Sur la base des estimations du GIEC concernant les augmentations de pCO2 et des quelques études publiées jusqu'en 1999 sur ce sujet, une diminution du taux de calcification de 10 % entre 1880 et 1990 a été mesurée et une nouvelle diminution de 9 à 30 % entre 1990 et 2100 a été prédite. Ces estimations ont été extrapolées à partir de quatre études sur les algues coralliennes et de trois autres sur des mésocosmes de récifs coralliens (références dans Gattuso et al. 1999). Bien que les auteurs soutiennent l'hypothèse selon laquelle la calcification est contrôlée par l'Ωara de l'eau de mer et que les preuves expérimentales suggèrent une chute sévère des taux de calcification à l'avenir, ils admettent que les données expérimentales ne sont pas suffisantes pour fournir une prédiction précise des scénarios futurs. En outre, Kleypas et al. (1999), Gattuso et Buddemeier (2000), et Langdon (2000) ont utilisé le même ensemble de données sur les changements prévus de l'Ωara de l'eau de mer pour prédire une diminution de 17 à 35 % de la calcification des récifs au cours du siècle prochain. Ces auteurs ont conclu que "plusieurs études ont montré que les taux de calcification des coraux et des algues corallines qui construisent les récifs sont réduits par l'augmentation des niveaux de CO2...", et que "la diminution de l'état de saturation pourrait entraîner une réduction des taux de calcification".

Sur la base de ces preuves expérimentales, les scientifiques ont envoyé un message alarmant sur l'avenir des récifs coralliens face à l'AO (Hoegh-Guldberg et al. 2007 ; Erez et al. 2011). Les taux de calcification des coraux scléractinaires diminueront considérablement d'ici la fin de ce siècle en raison de la réduction de la concentration en carbonate de l'eau de mer due à un doublement des niveaux préindustriels de CO2 atmosphérique (Kleypas et al. 2006). En outre, un déclin de 40 à 83 % de la calcification des récifs (Langdon et al. 2005) et un changement global des récifs coralliens d'une accrétion nette à une dissolution nette ont été prédits pour l'année 2065 (Silverman et al. 2009). Par exemple, une étude menée par Jury et Thomas (2001) a examiné les effets d'une réduction du pH sur les taux de calcification de *Porites astreoides*, une espèce corallienne commune des Caraïbes. Les chercheurs ont constaté que la diminution du pH de l'eau de mer entraînait une baisse significative du taux de calcification des coraux. Cohen et ses collègues (2009) ont constaté qu'une baisse du pH réduisait le taux de calcification du corail *Porites astreoides*, qui construit les récifs. De même, une étude d'Albright et al. (2018) a démontré que des niveaux de pH plus faibles réduisaient les taux de calcification et modifiaient la structure du squelette du corail *Porites cylindrica*. Les impacts du pH sur la calcification corallienne sont encore aggravés par d'autres facteurs de stress, tels que l'augmentation de la température de l'eau de mer et la pollution par les nutriments, qui peuvent interagir en synergie avec l'acidification des océans. Cependant, les preuves d'une relation systématique entre Ωara et la calcification corallienne n'ont pas toujours été bien étayées et sont en désaccord avec les conclusions de Lough et Barnes (2000) qui n'ont pas détectés de diminution à long terme (1903-1979) de la calcification dans les carottes de *Porites*, un corail à longue durée de vie, prélevées sur la Grande Barrière de Corail. En outre, Atkinson et al. (1999) ont signalé des taux de croissance élevés à long terme pour 57 espèces de coraux
poussant dans de l'eau de mer relativement riche en nutriments et à très faible pH (environ 7,6), ce qui indique que les coraux peuvent continuer à calcifier à des concentrations de $CO_3^{2-}$ extrêmement faibles.

Après plus de 20 ans d'intenses recherches expérimentales dans le domaine de l'AO, il semble que le consensus sur l'effet de l'AO sur la calcification des récifs coralliens ait persisté jusqu'à très récemment, même si plusieurs études ont mis en évidence les idées fausses et les biais méthodologiques du passé, et qu'il a été démontré que plusieurs espèces résistent aux effets néfastes de l'AO. Par exemple, les coraux d'eau froide se développent bien et sans diminuer leur taux de calcification près de l'horizon de saturation en aragonite (c'est-à-dire $\Omega_{ara} = 1$) (références dans Rodolfo-Metalpa et al. 2015) ; certaines espèces tempérées semblent calcifier à des taux normaux ou même plus élevés dans de l'eau de mer acidifiée (e. Rodolfo-Metalpa et al. 2011 ; Ries et al. 2009) ; plusieurs études ont montré des réponses très variables des espèces tropicales, y compris l'absence d'effet (par exemple Houlbrèque et al. 2012 ; Reynaud et al. 2003).

La calcification corallienne est-elle altérée par l'AO? Les récifs coralliens vont-ils se dissoudre comme on le prétend ? Les connaissances croissantes acquises au cours des dernières années, en particulier sur les mécanismes de calcification, ont démontré que les prédictions communément rapportées sur les effets néfastes de l'AO - réduction de la calcification et augmentation de la dissolution - pourraient devoir être révisées (Roleda et al. 2012).

Ici, je révise le consensus sur l'effet de l'AO sur les taux de calcification et de dissolution des coraux à la lumière de quelques études marquantes (par exemple, Venn et al. 2012, 2011 ; McCulloch et al. 2012a, 2012b ; Comeau et al. 2015a, 2015b, 2014a, 2014b, 2013 ; Jokiel 2011a, 2011b ; Jokiel et al. 2014 ; Murillo et al. 2014 ; etc) qui ont grandement amélioré nos connaissances sur les réponses des récifs coralliens à l'AO, et des métanalyses très récentes (e.g. Leung et al. 2022 ; Connell and Leung 2023). Tout au long de cette étude, l'effet délétère du réchauffement ne sera que partiellement mentionné, mais il est clair que les augmentations de température prévues au cours de ce siècle ont le potentiel d'affecter grandement les récifs coralliens, en combinaison ou non avec l'acidification. Bien que les effets exacts de l'AO sur les principaux mécanismes physiologiques fassent encore l'objet de débats, il ne fait aucun doute que les récifs coralliens sont particulièrement sensibles aux augmentations de température et que le réchauffement sera mortel pour la plupart des espèces (p. ex. Rodolfo-Metalpa et al. 2011).

## 3.2. Effet sur les taux de calcification à l'échelle de l'organisme. La calcification corallienne est-elle altérée par l'AO ?

L'une des principales activités physiologiques affectées pendant une période de stress qui définit l'état de santé d'un corail est sa croissance somatique. La calcification corallienne est un processus vital qui joue un rôle crucial dans la croissance et la survie des récifs coralliens. Il s'agit de la capacité des coraux à construire leur squelette de carbonate de calcium, qui constitue la base structurelle du récif. Le maintien d'un taux élevé de calcification corallienne est crucial pour la croissance et la résilience des récifs coralliens, car il leur confère une intégrité structurelle et une protection contre les perturbations physiques. La calcification est étroitement liée à la photosynthèse (Gattuso et al. 1999) et à l'efficacité de certaines Symbiodiniaceae à transférer des composés photosynthétiques à leurs hôtes (Cooper et al. 2011). Il est essentiel de comprendre le rôle des différentes formes de carbonate ($CO_2$, $HCO_3^-$, $CO_3^{2-}$) sur les mécanismes de calcification et le métabolisme en général pour établir des prévisions éclairées sur les futurs impacts de l'AO sur les récifs coralliens. Une mauvaise compréhension des mécanismes de calcification impliqués a entravé les progrès dans ce débat. Même la question fondamentale de savoir quelle source de carbone inorganique est privilégiée par les coraux pour leur
minéralisation reste partiellement sans réponse (Allemand et al. 2011). Le processus de calcification corallienne implique l'absorption de carbone inorganique dissous (CID) de l'eau environnante, la conversion du CID en ions bicarbonate et la précipitation de cristaux de carbonate de calcium dans les tissus du corail (Cohen et McConnaughey 2003). Cette régulation est essentielle au bon fonctionnement des processus cellulaires impliqués dans la calcification et est fortement liée à la capacité du corail à maintenir une gamme de pH interne. L'évaluation des rôles du CO₂, du HCO₃⁻, et du CO₃²⁻ dans l'apport de carbone inorganique dissous (DIC) pour la calcification des coraux représente un défi important pour l'avancement de ce domaine de recherche, notamment en termes de description des mécanismes de calcification sur lesquels les études ultérieures de l'AO peuvent être basées, et de détermination de la réponse des coraux à l'AO.

À ce jour, plusieurs hypothèses ont été proposées concernant l'origine du CID nécessaire à la calcification, mais elles restent controversées. Il semble que la plupart des études aient pris pour acquis que la formation de CaCO₃ résulterait de la combinaison de Ca²⁺ et de CO₃²⁻ dissous dans l'eau de mer. Étant donné que les taux de calcification et de dissolution des espèces marines sont liés à la chimie des carbonates de l'eau de mer, des relations empiriques entre Ωara ou [CO₃²⁻] et le dépôt de CaCO₃ ont été utilisées pour prédire les futurs taux de calcification et de dissolution en réponse à l'AO (par exemple, Erez et al. 2011 ; Kleypas et al. 2006 ; Kleypas et al. 1999). Étant donné que la concentration de Ca²⁺ est presque conservatrice dans l'eau de mer, Ωara est largement déterminé par [CO₃²⁻] dans l'eau de mer, qui à son tour est l'un des éléments constitutifs du carbonate de calcium (Gattuso et Buddemeier 2000).

Néanmoins, au moins trois réactions principales peuvent expliquer la formation de CaCO₃ :

$$Ca^{2+} + CO_3^{2-} = CaCO_3 \quad (1)$$

$$Ca^{2+} + HCO_3^- = CaCO_3 + H^+ \quad (2)$$

$$Ca^{2+} + CO_2 + H_2O = CaCO_3 + 2H^+ \quad (3)$$

Il est rare que les ions bicarbonate (éq. 2) ou le dioxyde de carbone (éq. 3), qui peuvent se déplacer librement à travers les membranes cellulaires et sont disponibles grâce au processus de photosynthèse, aient été proposés comme substrat pour la calcification dans les études sur l'AO. Un réexamen de la littérature antérieure montre que l'éq. 1 ne contribue pratiquement pas à la formation de CaCO₃ (Allemand et al. 2011) car le rapport [CO₃²⁻]/[HCO₃⁻] à un pH physiologique (entre 7,5 et 9,0 unités de pH) est extrêmement faible (Venn et al. 2009).

Il est connu que les coraux accumulent leurs structures de CaCO₃ dans un milieu rempli de fluide appelé milieu subcalicoblastique (Allemand et al. 2011) sous-jacent à l'épithélium calicoblastique qui est le tissu de calcification. Dans l'espace de calcification de l'organisme, une élévation de plus d'une unité de pH par rapport au pH ambiant rend la calcification possible (Allemand et al. 2011 ; Trotter et al. 2011 ; Venn et al. 2011). Le pH interne est élevé par l'activité de l'antiport Ca²⁺/H⁺ ATPase, qui transfère le Ca²⁺ vers le site de calcification en échange de protons. L'échange de H⁺ entraîne l'équilibre vers la formation de CaCO₃, convertissant HCO₃⁻ en CO₃²⁻ et augmentant ainsi l'état de saturation (Ω) dans le fluide calcifiant (Cohen et McConnaughey 2003). En utilisant différentes approches, Trotter et al. (2011), McCulloch et al. (2012a), et Venn et al. (2011) ont montré que certains coraux tropicaux, tempérés et profonds maintiennent de forts gradients de pH entre l'eau de mer environnante et l'espace corallien interne de calcification pour une gamme de valeurs de pH de l'eau de mer, y compris les valeurs projetées à la fin du siècle. Par conséquent, la précipitation du carbonate dans les structures calcifiées ne provient pas directement du carbonate de l'eau, mais est générée ou modulée par plusieurs réactions à partir de HCO₃⁻ et/ou de CO₂ dans le compartiment alcalin aux taux de calcification. La manière dont les deux autres formes de CID (CO₂ et HCO₃⁻) peuvent être acheminées vers les sites de calcification et utilisées pour la
calcification a été largement discutée (Allemand et al. 2011 ; Jokiel 2011a, 2011b), et leurs effets directs sur les taux de calcification ont été prouvés pour certaines espèces (par exemple Comeau et al. 2013 ; Jury et al. 2010 ; Marubini et al. 2008 ; Herfort et al. 2008 ; Schneider et Erez 2006).

Dans une première tentative de séparer provisoirement la contribution des différents composants du système de carbonate de l'eau de mer, les coraux ont été incubés dans de l'eau de mer où les paramètres de carbonate ont été manipulés en ajoutant de l'acide, des bases ou les deux. Certaines études (par exemple Schneider et Erez 2006) ont montré une augmentation de la croissance des coraux après une augmentation du CID, suggérant que la concentration ambiante en CID de l'eau de mer peut limiter les taux de calcification des coraux hermatypiques. Pour cela, Marubini et al. (2008) ont manipulé séparément la concentration de HCO₃⁻ et le pH pour étudier les mécanismes physiologiques qui permettent la calcification des coraux. Ils ont constaté que *Stylophora pistillata* se développait plus rapidement dans une eau de mer enrichie en bicarbonate, indépendamment des conditions de pH (pH 7,6 à 8,2), ce qui suggère que la calcification est limitée par le carbone. Bien que le CO₃²⁻ soit l'espèce ionique déposée dans le site de calcification, il semble clair que ce dépôt dans le site de calcification dépend du transport de HCO₃⁻ à partir du milieu extérieur. Les coraux peuvent obtenir le carbone nécessaire à la calcification à partir du HCO₃⁻ présent dans l'eau de mer ou du CO₂ produit par leur métabolisme (Furla et al. 2000). L'anhydrase carbonique située dans le cytoplasme du corail convertit le HCO₃⁻, qui constitue la majorité du CID intracellulaire de l'hôte (Venn et al. 2009), en CO₂ et HCO₃⁻, qui sont ensuite utilisés pour la photosynthèse et la calcification, respectivement. En manipulant la concentration en HCO₃⁻ à pH constant (8,2) sur les coraux Acropora sp. et *Porites porites*, Herfort et al. (2008) ont confirmé le rôle du HCO₃⁻ dans la stimulation de la calcification à la fois sur les coraux exposés à la lumière et à l'obscurité, et pas seulement en stimulant la photosynthèse. Cependant, dans cette expérience, HCO₃⁻ et CO₃²⁻ covarent et leur effet ne pouvait pas être séparé. C'est ce qu'ont fait Jury et al. (2009), qui ont constaté que la calcification du corail *Mandracis mirabilis* non seulement n'était pas affectée par l'AO, mais était renforcée par l'ajout de HCO₃⁻. Comeau et al. (2013) ont conçu une expérience de manipulation pour tester les rôles de HCO₃⁻ et CO₃²⁻ sur la calcification du corail *Porites rus* et de l'algue corallienne crustose *Hydrolithon onkodes* à la lumière et à l'obscurité. Les auteurs ont démontré que les deux formes sont impliquées dans la calcification, et de nouveaux modèles conceptuels de la calcification ont été proposés. En mesurant la géochimie du bore du squelette, Allison et al. (2014) ont confirmé que pour les *Porites* spp. massives, HCO₃⁻ contribue principalement au pool CID utilisé pendant la calcification.

Par conséquent, la calcification ne semble pas toujours dépendre de la concentration en ions carbonate de l'eau de mer, comme on le supposait auparavant (voir Erez et al. 2011). Roleda et al. (2012) ont souligné que, bien que la compréhension globale des mécanismes de calcification corallienne ait été difficile au début de l'ère de la recherche sur l'AO, les connaissances étaient suffisantes pour affirmer que la calcification des coraux est biologiquement modulée et ne dépend pas simplement de la combinaison du CO₃²⁻ et du Ca²⁺ dans un milieu d'eau de mer. Il semble que les études pionnières sur l'AO aient négligé les connaissances les plus récentes pour expliquer, de manière plus simpliste, comment la diminution du CO₃²⁻ et du Ω ara a entraîné la diminution observée des taux de calcification des coraux.

Alors pourquoi plusieurs études ont-elles montré qu'à l'échelle des organismes individuels, les taux de calcification corallienne étaient affectés par des diminutions élevées ou même modérées du pH, du CO₃²⁻ ou de l'Ω ara ? La calcification corallienne est-elle altérée par l'AO?

Il est important de noter que les études ont utilisé une gamme de conditions physiques et chimiques qui auraient pu donner des résultats différents, et que la sensibilité des coraux à l'AO est plus spécifique à l'espèce que ce que l'on pensait jusqu'à présent. Pandolfi et al. (2011) et