Helena Mosbah

Camille Vatier

Franck Boccara

Isabelle Jéru

Marie-Christine Vantyghem

Bruno Donadille

Karim Wahbi

Corinne Vigouroux
email: corinne.vigouroux@inserm.fr

Corinne Pr

Vigouroux

Cardiovascular complications of lipodystrophic syndromes -focus on laminopathies

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction : lipodystrophy and cardiovascular diseases

Lipodystrophic syndromes are genetic or acquired rare diseases characterized by a partial or generalized lack of adipose tissue leading to insulin resistance, hypertriglyceridemia and hepatic steatosis. These heterogeneous diseases are of familial or sporadic occurrence, of congenital or delayed clinical expression during childhood or adulthood, and are associated or not with different multi-organ comorbidities, including cardiovascular manifestations [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF].

Lipodystrophy-associated cardiovascular involvement is expressed as heterogeneous disorders, either heart muscle diseases, with hypertrophic or dilated cardiomyopathies, rhythm and conduction system diseases, and/or coronary artery disease [START_REF] Akinci | Comorbidities and Survival in Patients With Lipodystrophy: An International Chart Review Study[END_REF]. Cardiovascular alterations, which are important causes of morbi-mortality in patients with lipodystrophy, result from several non-mutually exclusive mechanisms. Besides the deleterious role of insulin resistance, diabetes, and dyslipidemia, some studies point to additional pathophysiological factors such as cardiac fatty infiltration, fibrosis, autonomic neuropathy, precocious cellular senescence, and cell-autonomous consequences to each specific pathogenic molecular variant [START_REF] Hegele | Premature atherosclerosis associated with monogenic insulin resistance[END_REF][START_REF] Paauw | Neonatal cardiac hypertrophy: the of hyperinsulinism-a review literature[END_REF][START_REF] Ponte | Association between cardiovascular autonomic neuropathy and left ventricular hypertrophy in young patients with congenital generalized lipodystrophy[END_REF][START_REF] Liu | Lessons from cavin-1 deficiency[END_REF][START_REF] Kwapich | Cardiometabolic assessment of lamin A/C gene mutation carriers: a phenotype-genotype correlation[END_REF].

The example of laminopathies, due to pathogenic variants in LMNA encoding A-type lamins, illustrates well the challenges of multiple cardiovascular phenotypes associated with lipodystrophy and their consequences for clinical practice.

Laminopathies : a broad spectrum of diseases with different cardiovascular phenotypes

Although LMNA is ubiquitously expressed in all differentiated tissues, laminopathies form tissue-specific diseases that may clinically translate into skeletal and/or cardiac muscular dystrophies (Emery-Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy 1B (LGMD1B), congenital muscular dystrophy (L-CMD)), dilated cardiomyopathy with conduction defects (DCM-CD), axonal neuropathies (Charcot-Marie-Tooth disease type 2), lipodystrophies or premature ageing syndromes (mandibuloacral dysplasia, Hutchinson-Gilford progeria syndrome (HGPS), atypical progeroid syndromes, and restrictive dermopathy) [START_REF] Worman | Nuclear lamins and laminopathies[END_REF]. Dunnigan syndrome, also known as familial partial lipodystrophy type 2 (FPLD2), is due to heterozygous LMNA pathogenic variants, with a mutational hot spot located at residue Arg482 in the C-terminal part of the protein. It is the most frequent form of genetically-determined lipodystrophy, characterized by partial lipoatrophy with insulin resistance, hypertriglyceridemia, hepatic steatosis, and frequent ovarian hyperandrogenism [START_REF] Decaudain | New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome[END_REF][START_REF] Gonzaga-Jauregui | Clinical and Molecular Prevalence of Lipodystrophy in an Unascertained Large Clinical Care Cohort[END_REF].

The heart and the vascular wall appear as recurrent targets in most forms of laminopathies. On the one hand, laminopathies of the striated muscle, including EDMD, LGMD1B, L-CMD and DCM-CD, are frequently associated with dilated cardiomyopathy [START_REF] Lu | LMNA cardiomyopathy: cell biology and genetics meet clinical medicine[END_REF]. On the other hand, in LMNA-linked lipodystrophies, frequent and early atherosclerotic cardiovascular events have been reported [START_REF] Hegele | Premature atherosclerosis associated with monogenic insulin resistance[END_REF][START_REF] Bidault | Lipodystrophy-linked LMNA p.R482W mutation induces clinical early atherosclerosis and in vitro endothelial dysfunction[END_REF]. In addition, HGPS and LMNA-associated premature ageing syndromes are associated with lipoatrophy and with very precocious and severe atherosclerosis and valvular calcifications [START_REF] Hennekam | Hutchinson-Gilford progeria syndrome: review of the phenotype[END_REF].

Complexity of cardiovascular involvement in the different types of lipodystrophic laminopathies

In addition to premature atherosclerosis, cardiomyopathic features have been reported in few patients with typical FPLD2 carrying heterozygous substitutions at the codon 482 of the LMNA gene [START_REF] Kwapich | Cardiometabolic assessment of lamin A/C gene mutation carriers: a phenotype-genotype correlation[END_REF]. Furthermore, lipodystrophic complex laminopathy phenotypes, mostly due to non-codon 482-associated LMNA pathogenic variants have been described, associating lipodystrophy and skeletal and/or cardiac dystrophic features [START_REF] Van Der Kooi | Lamin A/C mutations with lipodystrophy, cardiac abnormalities, and muscular dystrophy[END_REF][START_REF] Garg | Multisystem dystrophy syndrome due to novel missense mutations in the amino-terminal head and alpha-helical rod domains of the lamin A/C gene[END_REF][START_REF] Subramanyam | Overlapping syndrome with familial partial lipodystrophy, Dunnigan variety and cardiomyopathy due to amino-terminal heterozygous missense lamin A/C mutations[END_REF][START_REF] Andre | Metabolic and cardiac phenotype characterization in 37 atypical Dunnigan patients with nonfarnesylated mutated prelamin A[END_REF][START_REF] Francisco | Complex phenotype linked to a mutation in exon 11 of the lamin A/C gene: Hypertrophic cardiomyopathy, atrioventricular block, severe dyslipidemia and diabetes[END_REF][START_REF] Araújo-Vilar | A novel phenotypic expression associated with a new mutation in LMNA gene, characterized by partial lipodystrophy, insulin resistance, aortic stenosis and hypertrophic cardiomyopathy[END_REF]. Frequent dilated or to a lower extent hypertrophic cardiomyopathies are observed in such patients [START_REF] Kwapich | Cardiometabolic assessment of lamin A/C gene mutation carriers: a phenotype-genotype correlation[END_REF].

Lipodystrophy can also reveal LMNA-associated atypical premature ageing syndromes [START_REF] Caux | A new clinical condition linked to a novel mutation in lamins A and C with generalized lipoatrophy, insulin-resistant diabetes, disseminated leukomelanodermic papules, liver steatosis, and cardiomyopathy[END_REF][START_REF] Garg | Atypical progeroid syndrome due to heterozygous missense LMNA mutations[END_REF]. In affected patients, the cardiovascular phenotype mainly results from valvulopathy and/or atherosclerotic features. However, cardiomyopathy has also been described, and could be worsened by insulin resistance and/or diabetes [22, and personal results].

In LMNA-linked lipodystrophies, premature and severe atherosclerosis is not systematically associated with major cardiometabolic risk factors [START_REF] Hegele | Premature atherosclerosis associated with monogenic insulin resistance[END_REF][START_REF] Bidault | Lipodystrophy-linked LMNA p.R482W mutation induces clinical early atherosclerosis and in vitro endothelial dysfunction[END_REF][START_REF] Hegele | Laminopathies and atherosclerosis[END_REF]. Recent studies showing mesodermal and endothelial differentiation defects upon expression of the LMNA p.Arg482Trp variant suggest that developmental alterations could contribute to both lipodystrophy and premature atherosclerosis in FPLD2 [START_REF] Briand | The lipodystrophic hotspot lamin A p.R482W mutation deregulates the mesodermal inducer T/Brachyury and early vascular differentiation gene networks[END_REF]. Other pathophysiological mechanisms could include defects in prelamin A post-translational maturation leading to both myocardial inflammation and premature senescence of endothelial and smooth vascular muscle cells [START_REF] Bidault | Lipodystrophy-linked LMNA p.R482W mutation induces clinical early atherosclerosis and in vitro endothelial dysfunction[END_REF][START_REF] Brayson | Prelamin A mediates myocardial inflammation in dilated and HIV-associated cardiomyopathies[END_REF][START_REF] Afonso | LMNA mutations resulting in lipodystrophy and HIV protease inhibitors trigger vascular smooth muscle cell senescence and calcification: Role of ZMPSTE24 downregulation[END_REF] and/or laminopathic alterations in extracellular matrix leading to fibrosis [START_REF] Chatzifrangkeskou | ERK1/2 directly acts on CTGF/CCN2 expression to mediate myocardial fibrosis in cardiomyopathy caused by mutations in the lamin A/C gene[END_REF].

Consequences for clinical practice

In patients diagnosed with a lipodystrophic syndrome, whatever its etiology, cardiovascular investigations, including at least ECG and echocardiography should be systematically performed [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF].

A clear-cut classification of cardiovascular phenotypes among the different tissue-specific laminopathies, involving either heart muscle or the cardiac vasculature, may be challenging. Indeed, in LMNA-linked lipodystrophies, both cardiac muscle and arteries can be affected. Cardiac MRI should also be discussed evaluating cardiac steatosis and fibrosis.

Cardiovascular specific therapies should be discussed with cardiologists trained in the management of LMNA-associated diseases. It should be emphasized that LMNA-associated cardiomyopathy may require the implantation of cardioverter-defibrillator to prevent sudden death [START_REF] Wahbi | Development and Validation of a New Risk Prediction Score for Life-Threatening Ventricular Tachyarrhythmias in Laminopathies[END_REF].

Therapeutic management of lipodystrophy-associated metabolic alterations should follow the current international recommendations, with lifestyle and dietary measures being essential for prevention and treatment of metabolic complications [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF]. Exercise should be strongly encouraged in the absence of cardiovascular contraindications. Dyslipidemia should be managed in accordance with guidelines for the general population. However, even in patients without diabetes, strict lipid targets (LDL-cholesterol 100 mg/dL, non-HDL-cholesterol 130 mg/dL, triglycerides 200 mg/dL) are recommended in the context of lipodystrophyassociated increased cardiovascular risk [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF]. The association of statins and fibrates should be used with caution in the case of associated muscular dystrophy or inflammatory muscular involvement. The cardiovascular effects of metformin, used as a first-line drug therapy in most patients with lipodystrophy-associated diabetes, and of other non-specific antidiabetic drugs, have not been specifically evaluated in patients with lipodystrophy. The orphan drug metreleptin, a leptin analog, has recently obtained an European Marketing Authorization and is available in France for the treatment of metabolic complications associated with leptin deficiency in selected lipodystrophic patients. In addition to its beneficial effects on insulin sensitivity, insulin secretion and hypertriglyceridemia, metreleptin could decrease LDLcholesterol and PCSK9 serum levels, and improve the atherogenic lipid profile in patients with lipodystrophy [START_REF] Levenson | Effect of Leptin Replacement on PCSK9 in ob/ob Mice and Female Lipodystrophic Patients[END_REF][START_REF] Vatier | One-year metreleptin therapy decreases PCSK9 serum levels in diabetic patients with monogenic lipodystrophy syndromes[END_REF][START_REF] Kinzer | Advanced Lipoprotein Analysis Shows Atherogenic Lipid Profile That Improves After Metreleptin in Patients with Lipodystrophy[END_REF].

Conclusion -Cardiovascular evaluation and management : a systematic requirement for patients with lipodystrophic syndromes

The clinical and molecular heterogeneity of lipodystrophic syndromes requires a multidisciplinary expertise for each affected patients. At the cardiovascular level, it translates into several manifestations, including cardiomyopathies, rhythm and conduction disturbances and/or coronary artery disease. It is particularly the case for laminopathies, for which cardiovascular evaluation and management requires a close collaboration between endocrinologists and cardiologists.

Funding and Acknowledgments

The French Reference Center of Rare Diseases of Insulin Secretion and Insulin Sensitivity (PRISIS) is supported by the French Ministry of Solidarity and Health and Assistance Publique-Hôpitaux de Paris, and our research group at Saint-Antoine Research Center (Paris, France) by the Institut National de la Santé et de la Recherche Médicale, Sorbonne Université, and the Fondation pour la Recherche Médicale. We thank Dr Sonja Janmaat for her work as the project manager of the Reference Center PRISIS.

References :

  Therefore, in each patient diagnosed with lipodystrophy due to a LMNA pathogenic variant, in addition to systematic ECG and echocardiography and even in the absence of any specific sign, other cardiovascular investigations should be systematically discussed, based on genotype, cardiovascular family history, age at diagnosis, and other cardiovascular risk factors. These include a 24-hour Holter ECG monitoring to detect rhythm and/or conduction disturbances, and stress test and/or coronary CT angiogram to detect coronary artery disease.


C E Cornwall
email: steeve.comeau@imev-mer.fr

pH variability at volcanic CO2 seeps regulates coral calcifying fluid chemistry running title: Coral calcifying fluid chemistry at CO2 seeps

Keywords: Calcifying fluid, ocean acidification, abundance, coral, Dissolved inorganic carbon, Coral reefs, Papua New Guinea, Calcification

Coral reefs are iconic ecosystems having immense ecological, economic and cultural value, but globally their carbonate-based skeletal construction is threatened by ocean acidification. Identifying coral species that have specialised mechanisms to maintain high rates of calcification in the face of declining seawater pH is of paramount importance to predicting future species composition, and growth of coral reefs. Here, we studied multiple coral species from two distinct volcanic CO2 seeps in Papua New Guinea to assess their capacity to control their calcifying fluid chemistry. Several coral species living under conditions of low mean seawater pH but with either low or high variability in seawater pH were examined and compared with those living under 'normal' (non-seep) ambient seawater pH. We show that when mean seawater pH is low but highly variable, corals have a greater ability to maintain constant pHcf in their calcifying fluid, but this characteristic was not linked with changes in abundance.

Under less variable low seawater pH, corals with limited reductions in pHcf at the seep sites compared to controls tended to be more abundant at the seeps site than at the control site. However, this finding was strongly influenced by a single species (Montipora foliosa), able to maintain complete pHcf homeostasis. Overall, while our findings indicate that there might be an association between ecological success and greater pHcf homeostasis, further research with more species and at more sites with differing seawater pH regimes is required to solidify inferences regarding coral ecological success under future ocean acidification.

Introduction

Ocean acidification (OA) is caused by a shift in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations, and is one of the major threats to the future of coral reefs [START_REF] Hoegh-Guldberg | Coral reef ecosystems under climate change and ocean acidification[END_REF]. Declining seawater pH and altered relative concentrations of the different forms of dissolved inorganic carbon are expected to reduce the capacity of corals, the main reef-building taxon, to precipitate calcium carbonate [START_REF] Kleypas | Coral reefs and ocean acidification[END_REF]. Indeed, laboratory experiments demonstrate that decreases in pH expected by the end of the century will cause an average ~ 15-20% decrease in coral calcification [START_REF] Chan | Sensitivity of coral calcification to ocean acidification: A meta-analysis[END_REF][START_REF] Cornwall | Global declines in coral reef calcium carbonate production under ocean acidification and warming[END_REF]. However, coral responses are highly species-specific, with some species being more resistant to OA, while others are highly sensitive (Comeau, Cornwall, DeCarlo, et al., 2019;Comeau et al., 2014a;[START_REF] Kornder | Thresholds and drivers of coral calcification responses to climate change[END_REF]. Lack of understanding of why certain species are resistant to OA, while others are not, limits reliable projections of how the species composition and ecological functioning of reefs is likely to change in the future.

The physiological mechanisms that control species' capacity to tolerate OA are still unclear. Coral calcification is a key physiological and ecological process that enable corals to form large three-dimensional structures. New coral skeleton made of aragonite is formed via biomineralization of the calcifying fluid (CF) that lies between aboral coral tissues layers and the existing calcium carbonate skeleton [START_REF] Tambutté | Coral biomineralization: From the gene to the environment[END_REF]. To form their skeleton, corals have the ability to modify the chemical conditions of the calcifying fluid to facilitate the mineralization process. In the CF, pH is maintained at values well above that in seawater (pHcf ~ 8.2-8.9, McCulloch et al., 2012;[START_REF] Venn | Effects of light and darkness on pH regulation in three coral species exposed to seawater acidification[END_REF], and the dissolved inorganic carbon (DIC) is increased to values about 1.5-2 times higher than in ambient seawater [START_REF] Sevilgen | Full in vivo characterization of carbonate chemistry at the site of calcification in corals[END_REF]. As a result, the saturation state of calcium carbonate in the CF is elevated to values that thermodynamically favor its precipitation (i.e., Ωcf ~ 12, DeCarlo et al., 2017), which is being catalyzed by a set of organic molecules (e.g., CARPs, [START_REF] Drake | Molecular and geochemical perspectives on the influence of CO2 on calcification in coral cell cultures[END_REF][START_REF] Mass | Cloning and characterization of four novel coral acid-rich proteins that precipitate carbonates in vitro[END_REF]. Decreasing seawater pH under OA generally decreases pHcf [START_REF] Comeau | Decoupling between the response of coral calcifying fluid pH and calcification to ocean acidification[END_REF][START_REF] Holcomb | Coral calcifying fluid pH dictates response to ocean acidification[END_REF][START_REF] Venn | Impact of seawater acidification on pH at the tissue-skeleton interface and calcification in reef corals[END_REF]. Similarly, increasing seawater DIC under OA elevates DICcf [START_REF] Comeau | Similar controls on calcification under ocean acidification across unrelated coral reef taxa[END_REF]. While this increase in DICcf could partially alleviate the negative effects of decreasing pHcf [START_REF] Cornwall | Resistance of corals and coralline algae to ocean acidification: Physiological control of calcification under natural pH variability[END_REF][START_REF] Schoepf | Coral calcification mechanisms facilitate adaptive responses to ocean acidification[END_REF] large uncertainties exist in the magnitude and physiological controls of these effects. Physiological compensating mechanisms under OA, which are being used to maintain optimal conditions within the CF (i.e., pH homeostasis vs DIC upregulation vs calcium upregulation), are species-specific and can be modulated by environmental conditions [START_REF] Comeau | Flow-driven micro-scale pH variability affects the physiology of corals and coralline algae under ocean acidification[END_REF]. In this study we quantify the capacity of different coral species to control their pHcf, and assess whether and how this capacity changes among locations subject to either stable or variable seawater pH.

Both the average pH of seawater, and the magnitude of pH variability, have been suggested to modulate the response of marine organisms to OA on coral reefs [START_REF] Rivest | The role of natural variability in shaping the response of coral reef organisms to climate change[END_REF]. However, 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 [START_REF] Camp | Mangrove and seagrass beds provide different biogeochemical services for corals threatened by climate change[END_REF] to positive offsets against OA [START_REF] Comeau | Fast coral reef calcifiers are more sensitive to ocean acidification in short-term laboratory incubations[END_REF]. 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. To date, only one laboratory study has specifically addressed the effect of a regular diel pH variability on the CF of corals and coralline algae, where mean seawater pH was the main driver of the CF chemistry [START_REF] Cornwall | Resistance of corals and coralline algae to ocean acidification: Physiological control of calcification under natural pH variability[END_REF]. However, resolving these apparently conflicting results requires an understanding of the physiological mechanisms involved in regulating the composition of the CF, and the factors that constrain those mechanisms.

Field observations at naturally acidified sites such as volcanic CO2 seeps, semi-enclosed lagoons, and upwelling regions provide unique opportunities to investigate the effects of ocean acidification and pH variability on time scales that cannot be matched by laboratory experiments (i.e., years to decades). At most of these sites, the pH level, alongside other abiotic parameters, fluctuate around mean pH values similar to those predicted for the global ocean by the end of this century (pH ~ 7.7-7.8, Fabricius et al., 2011;[START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF]. So far, contradictory results describing the effects of OA on benthic marine calcifying taxa have emerged from these naturally acidified sites. For instance, in a semi-enclosed lagoon in New Caledonia with persistent low pH (mean pH ~ 7.6), coral communities are diverse and some species can maintain calcification rates as high as the ones from control sites [START_REF] Camp | Reef-building corals thrive within hot-acidified and deoxygenated waters[END_REF]. In contrast, deleterious effects of decreasing pH on the physiology, abundance, and diversity of calcareous organisms were reported at seep sites in Papua New Guinea [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF][START_REF] Fabricius | Low recruitment due to altered settlement substrata as primary constraint for coral communities under ocean acidification[END_REF]. Specifically, at the CO2 seeps in Normanby, naturally acidified reef areas have reduced species diversity and evenness compared to control sites, and were reported to be mostly dominated by resistant species such as massive Porites [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF]. This dominance of Porites spp. could be due to its capacity to maintain elevated pHcf under a large range of seawater pH, as demonstrated both in situ [START_REF] Wall | Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions[END_REF]Wall et al., 2019a) and ex situ (Comeau, Cornwall, DeCarlo, et al., 2019). Furthermore, the ability to elevate DICcf under low pH could be an additional mechanism that favours the presence of certain coral species in naturally acidified sites (Wall et al., 2019a;Wall et al., 2019b).

Both mechanisms lead to constant Ωcf in corals from low pH conditions. Some locations with naturally-occurring low pH conditions host abundant and diverse hard-coral assemblages, such as in Palau [START_REF] Barkley | Mechanisms and thresholds for pH tolerance in Palau corals[END_REF][START_REF] Golbuu | Long-term isolation and local adaptation in Palau's Nikko Bay help corals thrive in acidic waters[END_REF][START_REF] Shamberger | Low and variable ecosystem calcification in a coral reef lagoon under natural acidification[END_REF], Papua New Guinea [START_REF] Pichler | Suitability of the shallow water hydrothermal system at Ambitle Island (Papua New Guinea) to study the effect of high pCO2 on coral reefs[END_REF], West Australia [START_REF] Dandan | Resilience of coral calcification to extreme temperature variations in the Kimberley region, northwest Australia[END_REF][START_REF] Schoepf | Limits to the thermal tolerance of corals adapted to a highly fluctuating, naturally extreme temperature environment[END_REF], New Caledonia [START_REF] Camp | Reef-building corals thrive within hot-acidified and deoxygenated waters[END_REF], and the Virgin Islands [START_REF] Yates | Diverse coral communities in mangrove habitats suggest a novel refuge from climate change[END_REF]. At one of the CO2 seep sites in Papua New Guinea (Upa-Upasina, Normanby), more than 100 coral species were observed near the seep during our expeditions (Hoogenboom M. and Rodolfo-Metalpa R., Pers. Obs.) coexisting with the dominant mound-shaped massive Porites colonies. This high species richness under OA conditions was confirmed also at the CO2 seeps of Ambitle Island, where around 100 species were found in a large, acidified bay in addition to massive Porites spp. (Shlesinger T. and Rodolfo-Metalpa R., Pers. Obs.).

Previous studies have shown that corals cannot acclimatise within one year to low pH conditions, and that the ability to resist changes in pHcf under low seawater pH (i.e., pH homeostasis) is a species-specific inherent trait (Comeau, Cornwall, DeCarlo, et al., 2019). Therefore, environments with regular high pH could promote species that calcify at enhanced rates during periods of elevated pH. Accordingly, we hypothesize that corals living in consistently low pH their entire lifetime will show stronger control over their calcifying fluid chemistry than corals living under variable or different pH regimes.

While laboratory research has enabled us to understand more of the physiological mechanisms responsible for resistance to low pH, they are unable to provide information regarding how or if this translates to ecological success under OA. Here, we aimed firstly to understand whether changes in mean seawater pH and in the magnitude of variability in seawater pH affects 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 CF chemistry is correlated to species relative abundances at sites with different pH and DIC conditions. To that end, we utilised two natural CO2 seeps locations with distinct pH characteristics. Using physiological measurements and field observations of coral species abundances, we tested three complementary hypotheses: 1) corals growing in acidified sites have the capacity to 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.

Materials and Methods

Study sites, surveys, and sample collections

Two reefs surrounding CO2 seeps and adjacent control reefs (i.e., sites with ambient conditions) in Papua New Guinea were repeatedly visited between September 2016 and October 2019: Upa-Upasina Reef (Normanby Island, Milne Bay Province) and Tutum Bay (Ambitle Island, New Ireland Province) (Fig. 1).

2019).

The seawater carbonate chemistry of the seep areas, and of the adjacent control reefs, at both locations was characterized continuously during each of the seven 10-day trips performed between 2016 and 2019 (i.e., 4 trips to Ambitle and 3 trips to Normanby). Most of the seawater carbonate chemistry data from Normanby has been already reported [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF][START_REF] Fabricius | Ecological effects of ocean acidification and habitat complexity on reef-associated macroinvertebrate communities[END_REF], and several subsequent studies (e.g., [START_REF] Fabricius | Low recruitment due to altered settlement substrata as primary constraint for coral communities under ocean acidification[END_REF]. Most of these studies were conducted on the same reef areas as visited during this study, and where hundreds of discrete water samplings verified that the median pH was close to 7.8 pHT units as projected for 2100. However, only a limited number of studies to date have logged pH at high frequency to characterise pH variability at the Normanby study site [START_REF] Fabricius | Ecological effects of ocean acidification and habitat complexity on reef-associated macroinvertebrate communities[END_REF][START_REF] Smith | Pontellid copepods, Labidocera spp., affected by ocean acidification: A field study at natural CO2 seeps[END_REF][START_REF] Uthicke | Echinometra sea urchins acclimatized to elevated pCO2 at volcanic vents outperform those under present-day pCO2 conditions[END_REF]. The seeps site in Ambitle Island was only recently studied and an exhaustive dataset of seawater physical and chemical conditions have been published [START_REF] Biscéré | High pCO2 promotes coral primary production[END_REF][START_REF] Pichler | Suitability of the shallow water hydrothermal system at Ambitle Island (Papua New Guinea) to study the effect of high pCO2 on coral reefs[END_REF].

During our seven field works at the Normanby and Ambitle seeps, we measured the extent to which pH fluctuated at the study sites because of its potential importance in affecting the coral ability to control their calcifying fluid chemistry. At both Normanby and Ambitle seeps, and at the reference (control) sites at each location, we used three pH loggers (SeaFET V2, Sea-Bird Scientific, Bellevue, WA 98005, USA) recording every 10 minutes (i.e., Tutum Bay in Ambitle; Fig. 1 in [START_REF] Pichler | Suitability of the shallow water hydrothermal system at Ambitle Island (Papua New Guinea) to study the effect of high pCO2 on coral reefs[END_REF]; see all Supplementary Data, ttps://ars.elscdn.com/content/image/1-s2.0-S0025326X1830780X-mmc1.pdf). At each seeps site, we collected continuous pH data for a duration of 10 days at each of 4 fixed stations located in the area where the coral samples were collected, and an additional 20 24-h measurements all around the seeps area. Seawater samples were filtered through 0.45μm Whatman filters using a Nalgene vacuum system and stored at 4 °C in the dark for further testing at the Institut de Recherche pour le Développement (IRD) in New Caledonia. Total alkalinity (AT) was determined using an auto titrator (TIM865 Titralab, Radiometer). Three replicate 20 mL sub-samples were analysed at 25 °C using an open cell potentiometric method. Total alkalinity was calculated from the Gran function applied to pH from 4.2 to 3.0, as mEq L -1 from the slope of the pH vs HCl curve. Results were corrected against AT standards provided by A.G. Dickson (batch #155). Parameters of the carbonate system were calculated from pHT (median, 5 th and 95 th percentile); median AT, temperature and salinity (34) using the R package seacarb. A full description of the carbonate chemistry at the two locations and sites is presented in the Table S1.

During the expedition of 2016, we collected fragments of 14 and 8 coral species at Normanby Island and Ambitle Island, respectively, from both the seeps and control sites (CITES collection permits n. 016132 and 017027). The 14 species sampled in Normanby Island were: Acropora cytherea, Acropora millepora, Acropora samoensis, Acropora tenuis, Favites halicora, Favites pentagona, Galaxea fascicularis, Merulina ampliata, Pachyseris speciosa, Pocillopora verrucosa, Porites rus, Seriatopora caliendrum, Tubastraea sp., and Turbinaria reniformis. The 8 species sampled in Ambitle Island were: Acropora nana, Acropora tenuis, Echinopora lamellosa, Galaxea fascicularis, Montipora foliosa, Pocillopora damicornis, Porites lutea, Psammocora contigua. For each species, one branch tip or a piece of skeleton (3-7 cm long) was collected from three spatially separated colonies (5-20 m distant) using a bone cutter or a hammer and chisel for branching or massive and foliose species, respectively. Coral tissues were removed using high pressure water and they were dried in air over 48 h before being carefully preserved in individual bags.

To quantify the abundance of the focal species mentioned above, field surveys were performed in May 2017 in Normanby Island and in June 2018 in Ambitle Island.

We used 10 x 1 m belt transects haphazardly positioned at a depth range of ~2-10 m at both seeps and control sites in each location. Every colony identified as one of the studied species (except for massive or encrusting Porites that were grouped as Porites spp.) was counted along 15 belt transects at each site in Normanby Island, and along 18 belt transects at each site in Ambitle Island, and the species abundances are presented as the number of colonies per transect.

Analyses of calcifying fluid pH and DIC

Calcifying fluid pH (pHcf) and dissolved inorganic carbon (DICcf) were estimated respectively using the δ 11 B proxy method [START_REF] Trotter | Quantifying the pH 'vital effect' in the temperate zooxanthellate coral Cladocora caespitosa: Validation of the boron seawater pH proxy[END_REF] and the δ 11 B and B/Ca method [START_REF] Holcomb | Factors affecting B/Ca ratios in synthetic aragonite[END_REF]M. T. McCulloch et al., 2017). Fragments of coral colonies were collected from 3 to 4 colonies per species, and the tissues were removed by water pressure before being sun dried. The skeletons were then shipped to the University of Western Australia, where they were cleaned with mQ water, bleached for 24 hours in 6.25 % NaClO to remove any tissue left at the surface of the samples, and dried in a drying oven for 48 h at 50 °C.

For branching corals (e.g., Acropora, Seriatopora, Pocillopora), geochemical analyses were performed on a ~ 1 cm long piece of skeleton from the tip of each of three branches per colony. The three tips were then crushed together to smooth potential differences in calcifying fluid chemistry within colonies and were considered as one sample (for a total of three samples per species and condition). For massive corals (e.g., Favites, Galaxea), a fragment of 0.5 cm 3 located ~ 0.5 cm below the surface was selected using a dental drill to avoid the area of the skeleton where tissues were present (first mm of the skeleton). For the foliose corals (e.g., Echinopora, Pachyseris, Turbinaria), a section of the skeleton close to the growing edge of laminae of the colonies was sampled using cutting pliers. The selected portions of skeletons were bleached for an additional 24 hours in 6.25 % NaClO and then rinsed three times with mQ water to remove any residual traces of organic matter and bleach. Bleached samples were dried for 48h at 50°C. Skeleton samples from all colonies were then crushed in a mortar with a pestle to powder prior to analysis.

In the clean laboratory of the Advanced Geochemical Facility for Indian Ocean Research (AGFIOR, University of Western Australia), 10 mg of powdered skeleton from each coral sample was prepared for dissolution and dilution to 10-ppm Ca solutions. The 10 mg samples were dissolved in 0.51 N HNO3, and the boron was quantitatively separated on ion exchange columns. δ 11 B was measured on a multicollector inductively coupled plasma mass spectrometer (NU II). Measurements of the international carbonate standard JCP-1 yielded a mean value of 24.43 ± 0.08 ‰ (mean ± SE, n = 10), which was similar to the 24.33 ± 0.11 ‰ (SE) reported previously. pHcf was estimated from δ 11 B using the calculations described by [START_REF] Trotter | Quantifying the pH 'vital effect' in the temperate zooxanthellate coral Cladocora caespitosa: Validation of the boron seawater pH proxy[END_REF], as:

pH !" = pK # -log ) (% !! # "# & % !! # $%&' ) )* ()*+),) % !! # $%&' &% !! # "# +,---.* ()*+),) &,/0 * (1),
where pKB is the dissociation constant dependent on temperature and salinity as measured at the site of coral collection, δ 11 Bsw = 39.61 [START_REF] Foster | Boron and magnesium isotopic composition of seawater[END_REF], and α(B3-B4)

is the boron isotopic fractionation factor for the pH dependent equilibrium of the borate (B(OH)4 -) relative to the boric acid (B(OH)3) species in the calcifying fluid, with a value of 1.0272 [START_REF] Klochko | Experimental measurement of boron isotope fractionation in seawater[END_REF].

B/Ca ratios were determined on the same aliquot of the solution used for δ 11 B.

Both B/Ca and δ 11 B were utilized to determine [CO3 2-] and then [DIC] at the site of calcification [DIC]cf following [START_REF] Mcculloch | Coral calcification in a changing World and the interactive dynamics of pH and DIC upregulation[END_REF]. Estimates of carbonate ion concentrations in the calcifying fluid were calculated using the following equation:

[CO 1 2& ] !" = K 3 [B(OH) 4 & ] !" 2 B Ca 4 5 5657 * 4 (2),
where KD = KD.0 exp(-kKD[H + ]T) with KD.0= 2.97 ± 0.17×10 -3 ( ± 95% CI) and kKD = 0.0202 ± 0.042. The concentration of DICcf was then calculated from estimates of pHcf and [CO3 2-]cf.

T-tests were used to assess differences in the estimates of pHcf and DICcf between the seeps and the control samples at both locations, with locations analysed separately. Ttests were used separately for each species because we were interested in whether there is a CO2 effect for each species rather than an effect between species. All of the analyses and visualizations were done in R v4.0.2. All data are presented as mean ± SE. The data that support the findings of this study are archived in the Pangaea database (https://doi.pangaea.de/10.1594/PANGAEA.939651).

Relationship between coral abundance and carbonate chemistry

We used two complementary approaches to determine if the most abundant coral species at the seeps sites were the species with the best control on their calcifying fluid. First, we investigated the relationships between coral abundance at the seeps sites and corals pHcf or DICcf. Second, to consider the differences in species abundance between the seeps and control sites, we calculated the proportional change in their abundance at the seeps relative to the control at each location. We then examined the relationship between the differences in the corals' abundance among sites and their calcifying fluid carbonate chemistry using a linear regression.

Differences for each species in mean abundance between the sites were calculated as the relative change in mean abundance as follows:

Relative change = (mean abundance in the seeps -mean abundance in the control) / mean abundance in the control Principal Component Analyses were used to evaluate how relative change in mean abundance, vent pHcf and DICcf, delta pHcf (pHcf vent -pHcf control) and delta DICcf (pHcf vent -pHcf control) were correlated.

Results

Seawater pH variability

All seawater carbonate chemistry and seawater pH variability measured at fixed stations are in Supplementary Table S1. 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 (Fig. 2). 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). 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 seeps site compared with the control site in five species (Fig. 3A; t-test, p < 0.05 for all five), with delta pHcf the lowest in Acropora samoensis (-0.15, Fig. 3B). There was no difference in pHcf between sites for eight species, while pHcf was higher at the seeps site in Seriatopora caliendrum (Fig. 3A; t-test, p < 0.001), with a positive delta pHcf of 0.07 (Fig. 3B). Species pHcf Species pHcf

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Vents Control A c
• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Vents Control

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carbon at the site of calcification (DICcf); and D) Difference in mean DICcf. Blue and red colours indicating the control and seeps data, respectively. Dashed lines in (A) and (C) represent the pooled pHcf and DICcf 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.

In Ambitle, for the 8 species pooled together, pHcf 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). pHcf 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 (Fig. 4A). As a result, delta pHcf which is equal to pHcf seeps -pHcf ambient varied between -0.19 in Echinopora lamellosa and -0.02 in Montipora foliosa (Fig. 4B).

Dissolved inorganic carbon in coral calcifying fluid

In Normanby, for the 14 species pooled together, mean DICcf was 3833 ± 105 μmol kg -1 and 4022 ± 173 μmol kg -1 at the control and seeps site (n = 42 for both), respectively, with no statistical differences (t-test, p = 0.268). DICcf was more elevated at the seeps site compared with the control site in four species (A. samoensis, F. pentagona, Tubastraea sp., and F. halicora; t-test, p < 0.05 for all), while it was more elevated in the control site for S. caliendrum (t-test, p < 0.05). There was no difference in DICcf between sites in the nine other species (Fig. 3C). There was a large range of species-specific delta DICcf = DICcf seeps -DICcf ambient with the maximal increase in delta DICcf at the seeps found in Favites pentagona (1200 μmol kg -1 , Fig. 3D). In contrast, delta DICcf 233 μmol kg -1 at the seeps site in Seriatopora caliendrum (Fig. 3D), which was also one of the only species with higher pHcf at the seeps.

Across the 8 coral species studied in Ambitle, mean DICcf was 3957 ± 144 μmol kg -1 and 4112 ± 143 μmol kg -1 at the control and seeps site (n = 24 for both), respectively, with no statistical differences (t-test, p = 0.206). DICcf was more elevated at the seeps site compared with the control sites in five species (E. lamellosa, G. fascicularis, A. tenuis, Psammocora sp., and P. damicornis; t-test, p < 0.05 for all), while it was lower at the seeps sites for only massive Porites spp. (t-test, p < 0.05) despite lower pHcf also found at the seeps. There was no difference between sites in DICcf for Acropora nana and Montipora foliosa (Fig. 4C). The delta DICcf ranged from -506 μmol kg -1 in massive Porites spp. to 751 μmol kg -1 in Echinopora lamellosa (Fig. 4D).

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 (Fig. 5A). By contrast, this pattern was not observed in Ambitle (Fig 5B ), 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. lamellosa 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). There was no relationship between delta pHcf or delta DICcf and the abundance of corals at the seeps sites of both Normanby and Ambitle (Fig. S1) (Table S2).

Similarly, there was also no significant relationship between delta pHcf and the relative change in species abundance between the seeps site and the control site in Normanby (Fig. 6A). By contrast, a relationship between delta pHcf and the relative change in abundance was found in Ambitle (Fig. 6B; linear regression, p-value of the slope = 0.05, p-value of the intercept = 0.02, R 2 = 0.55), although it was appears to be largely driven by one species, M. Foliosa. The delta DICcf had no significant relationship with the relative change in abundance at both Normanby and Ambitle (Fig. S2). These results were confirmed by the PCA, which demonstrated no association between proportional abundance and the calcifying chemistry parameters in Normanby (Fig. S3A). In contrast, the PCA for Ambitle showed a clear negative association between delta pHcf at the seep site and relative change in abundance there (Fig. S3B).

Discussion

We observed major site-specific differences in the responses of corals' calcifying fluid chemistry. At the consistently low pH seeps site (i.e., at Ambitle Island) most coral species had lower pHcf at the CO2 seeps compared to the control site. This mirrors observations made on laboratory grown corals under well-controlled and consistently low pH [START_REF] Mcculloch | Coral resilience to ocean acidification and global warming through pH up-regulation[END_REF]. Conversely, this pattern was less clear at the inconsistently low pH seeps site (i.e., at Normanby Island) when compared to the control site. Additionally, at Ambitle, coral species with limited reductions in pHcf at the seep sites compared to controls (i.e. greater pH homeostasis) also tended to be more abundant at the seeps site than at the control site. This association was influenced heavily by the species with complete pHcf homeostasis, which also increased in abundance more than any other species between the control and seep site at Ambitle. However, three out of four of the species that increased in abundance also had the highest pH homeostasis. Three of the four species with the least pHcf homeostasis also had the greatest declines in abundance between the control and seeps. Such trends were not observed at Normanby. 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. Nonetheless, we cannot rule out other factors such as differences in light, temperature, and flow that can affect the calcifying fluid chemistry [START_REF] Comeau | Flow-driven micro-scale pH variability affects the physiology of corals and coralline algae under ocean acidification[END_REF]. As correlation does not imply causation, further assessments at additional sites with consistently low pH are warranted to assess the logical association between coral pHcf homeostasis and directionality of abundance change under consistently low pH that our study suggests.

Controlling pH in the calcifying fluid well above external seawater pH is one of the key mechanisms that corals have developed to favour the precipitation of calcium carbonate. pH homeostasis, where pH in the calcifying fluid remains constant independent of changes in external seawater pH, has therefore been suggested as a mechanism for corals to cope with ocean acidification [START_REF] Georgiou | pH homeostasis during coral calcification in a free ocean CO2 enrichment (FOCE) experiment, Heron Island reef flat, Great Barrier Reef[END_REF][START_REF] Mcculloch | Coral calcification in a changing World and the interactive dynamics of pH and DIC upregulation[END_REF]. To date, pH homeostasis in corals has been demonstrated only in a handful of species: On fragments of Porites cylindrica during an in situ experiment [START_REF] Georgiou | pH homeostasis during coral calcification in a free ocean CO2 enrichment (FOCE) experiment, Heron Island reef flat, Great Barrier Reef[END_REF], on massive Porites during a flume experiment (Comeau, Cornwall, DeCarlo, et al., 2019), and on massive Porites, Porites astreoides, and Balanophyllia europaea at naturally low pH sites [START_REF] Wall | Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions[END_REF](Wall et al., , 2019a(Wall et al., , 2019b)). Using a large range of taxa and morphologies, our study confirms the difficulties associated with determining the resistance to ocean acidification based on coral phylogeny and/or morphology (Comeau et al., 2014a;[START_REF] Okazaki | Species-specific responses to climate change and community composition determine future calcification rates of Florida Keys reefs[END_REF].

While it has been suggested that certain coral genera, such as Porites, are particularly resistant to OA, we found that some species of other coral genera that are largely regarded as highly susceptible to OA [START_REF] Kavousi | Colony-specific calcification and mortality under ocean acidification in the branching coral Montipora digitata[END_REF][START_REF] Kornder | Thresholds and drivers of coral calcification responses to climate change[END_REF], such as Acropora and Montipora, can also exert strong control over their calcifying fluid chemistry. However, a large range of species-specific responses within a genus was found. For example, Acropora millepora was one of the species with the best apparent control over its pHcf at Normanby while Acropora samoensis was the species exhibiting the largest decline in pHcf at this location. Similarly, coral morphology was not correlated with pH homeostasis in the calcifying fluid. Although massive species have been suggested to be more resistant to OA, we found that many of the species that are able to maintain homeostasis were branching (e.g., Acropora cytherea, Acropora millepora, Pocillopora verrucosa) and foliose (e.g., Merulina ampliata, Montipora foliosa, Pachyseris speciosa, Turbinaria reniformis) while some of the species with massive growth morphologies had poor control over their calcifying fluid chemistry (e.g., Favites pentagona, Galaxea fascicularis). However, these trends might be confounded by the differences in seawater pH variability at the two locations.

The trends in pH control may only be physiologically meaningful for sites where seawater pH was consistently low. Apparent pH homeostasis was much more common in Normanby, where eight out of 14 taxa experienced pHcf homeostasis (and one coral had higher pHcf at the seeps than in the control site). In Ambitle, homeostasis was found only for Montipora foliosa, while pHcf was lower at the CO2 seep site in the other seven species investigated. Montipora foliosa was also the species with the highest proportional increase in abundance between the control and the seeps site. This suggests that the capacity of this species to control its pHcf could represent an ecological advantage. However, it is important to note that this species was relatively rare in Ambitle with a total of three colonies recorded in the surveys at the control site and 29 colonies at the seeps site. Therefore, even small increases in abundance equate to large relative increases. Additionally, three of the four species with the least control over their pHcf also had the greatest declines in relative abundance. Together, these results explain the clear association between pHcf homeostasis and relative change in abundance found with the PCA. They also show that rather than the absolute value of pHcf at the vents, it is a greater control over pHcf that could give some ecological advantage in a future high CO2 ocean. However, further assessment at additional sites where seawater pH is low and not highly variable is now needed to confirm these results.

In contrast, a relationship between pHcf control and proportional change in abundance was not found in Normanby. While different species were studied at both sites, this difference between locations was unlikely to be solely because of taxonomic composition as the two species studied at both locations (Acropora tenuis and Galaxea fascicularis) also exhibited lower pHcf in Ambitle's seeps site compared to [START_REF] Comeau | Flow-driven micro-scale pH variability affects the physiology of corals and coralline algae under ocean acidification[END_REF][START_REF] Guo | Seawater temperature and buffering capacity modulate coral calcifying pH[END_REF].

However, they did not impact the present results, as all environmental parameters other than seawater pH were similar between control and seeps sites (Table S1).

Therefore, the different responses observed in Normanby and Ambitle can likely be attributed to differing seawater pH (and DIC) variability.

Greater pH variability could elicit at least three distinct responses. 1) Periods of elevated seawater pH could allow calcification to occur unabated as pHcf is elevated during these time periods, but pHcf is then decreased when seawater pH is reduced. Since calcification rates are higher when pHcf is higher, a greater proportion of boron would be incorporated during the periods of time when pHcf is elevated, which would be reflected by elevated pHcf at CO2 seeps, as in our data. This might further explain the absence of a relationship between relative change in abundance of species and their ability to maintain constant pHcf at the CO2 seeps in Normanby,

where the reef might be dominated by species that are able to rapidly calcify in brief periods when seawater pH is high. This hypothesis is also supported by the two cooccurring species at Ambitle/Normanby possessing lower pHcf at Ambitle, indicative of calcification occurring more rapidly in higher seawater pH. (Cornwall et al. 2013). The only study on the effect of pH variability on coral CF chemistry has shown that pHcf of the coral Goniopora sp. was driven by the mean seawater pH and was not affected by diurnal variability in seawater pH [START_REF] Cornwall | Resistance of corals and coralline algae to ocean acidification: Physiological control of calcification under natural pH variability[END_REF]. However, this supports a null hypothesis 4) that responses at Normanby are equivalent to those that will occur under future ocean acidification. While this contradicts what is found here, it is important to note that this study was restricted to only one species exposed to a regular diurnal variability in pH (low pH at night and high pH during the day), which could elicit much different responses than those occurring at our sites. Collectively, the effects of differences in pH variability on calcification physiology is also highly species-specific [START_REF] Rivest | The role of natural variability in shaping the response of coral reef organisms to climate change[END_REF], and thus extremely high pH, low pH and differences in pH variability itself could have altered both the pHcf and the ecological outcomes at Normanby (and even Ambitle to a lesser extent) in ways that are difficult to predict from our available data.

DICcf is another important parameter of the calcifying physiology of corals [START_REF] Mcculloch | Coral calcification in a changing World and the interactive dynamics of pH and DIC upregulation[END_REF][START_REF] Ross | Mechanisms and seasonal drivers of calcification in the temperate coral Turbinaria reniformis at its latitudinal limits[END_REF]Wall et al., 2019) and is generally inversely correlated with pHcf, whereby DICcf is elevated when pHcf decreases. This trend also persists when corals are grown under low pH in the laboratory (Cornwall et al., 2018;[START_REF] Schoepf | Coral calcification mechanisms facilitate adaptive responses to ocean acidification[END_REF][START_REF] Sevilgen | Full in vivo characterization of carbonate chemistry at the site of calcification in corals[END_REF] and occurs on seasonal cycles in situ [START_REF] Mcculloch | Coral calcification in a changing World and the interactive dynamics of pH and DIC upregulation[END_REF][START_REF] Ross | Active modulation of the calcifying fluid carbonate chemistry (δ 11 B, B/Ca) and seasonally invariant coral calcification at sub-tropical limits[END_REF]. Here, the average DICcf across all coral species in each site was similar between the control and seeps sites at both locations, and only 4 species out of 14 in Normanby and 5 out 8 in Ambitle had higher DICcf at the seeps compared to the control. As for pHcf, the lack of differences in DICcf at the seeps compared to the control at Normanby in most corals could have resulted from the high variability in seawater DIC, because seawater DIC and pH are highly correlated in most conditions, i.e. where total alkalinity is similar. The present results also showed that the relative change in abundance was not associated with either DICcf or delta DICcf. This is not surprising, as previous studies have shown that while increasing DICcf can help to partially mitigate the negative effects of ocean acidification on corals calcification, seawater pH and its impact on pHcf is the main driver of the calcifying fluid chemistry [START_REF] Comeau | Similar controls on calcification under ocean acidification across unrelated coral reef taxa[END_REF]). Nevertheless, there was a significant relationship between pHcf and DICcf at both locations when all the samples were assessed (Fig. S4). Elevation of DICcf by some species has been invoked as one potential mechanism whereby Ωcf could be increased under OA, thereby reducing the negative effects of OA on calcification [START_REF] Cornwall | Resistance of corals and coralline algae to ocean acidification: Physiological control of calcification under natural pH variability[END_REF][START_REF] Schoepf | Coral calcification mechanisms facilitate adaptive responses to ocean acidification[END_REF]Wall et al., 2019). However, we did not find a significant relationship between DICcf elevation and coral abundance or relative change in abundance at either locations. This result therefore suggests that while the control of DICcf could help corals to sustain calcification in low pH under specific circumstances, this mechanism likely plays a minor role compared to the control of pHcf.

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; [START_REF] Cornwall | Inorganic carbon physiology underpins macroalgal responses to elevated CO2[END_REF] 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. 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.

Figure 1 .

 1 Figure 1. Maps showing the sampling and surveyed sites. Corals were collected from Ambitle Island and Normanby Island at both control sites and seeps sites in Tutum

Figure 2 .

 2 Figure 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.

Figure 3 .

 3 Figure 3. Calcifying fluid carbonate chemistry estimates of 14 coral species from the control and seeps sites in Normanby Island. A) pH of the calcifying fluid (pHcf); B) Difference in mean pHcf between control and seeps sites; C) Dissolved inorganic

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Figure 4 .

 4 Figure 4. Calcifying fluid carbonate chemistry estimates of 8 coral species from the control and seeps sites in Ambitle Island. A) pH of the calcifying fluid (pHcf); B) Difference in mean pHcf between control and seeps sites; C) Dissolved inorganic carbon at the site of calcification (DICcf); and, D) Difference in mean DICcf. Blue and red colours indicating the control and seeps data, respectively. Dashed lines in (A) and (C) represent the pooled pHcf and DICcf 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.

  s iv e P o ri te s s p p . M a s s iv e P o ri te s s p p . M a s s iv e P o ri te s s p p .

Figure 5 .

 5 Figure 5. 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, 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.

Figure 6 .

 6 Figure 6. Relationship between the difference in mean pHcf between the control and seeps sites (i.e., Delta pHcf) and the mean relative change in coral abundance between the control and the seeps sites. Positive values indicate an increase in relative abundance between the seeps and the control. There was no relationship in Normanby (A), while there was a linear relationship in Ambitle (B) (p-value of the slope = 0.05, p-value of the intercept = 0.02, R 2 = 0.56). Species names are indicated as initials on the figure.

Figure S2 .

 S2 Figure S2. Relationship between the difference in DICcf between the control and seeps sites (i.e., Delta DICcf) and the relative change in coral abundance between the control and the seeps sites. There was no relationship in Normanby (A) and Ambitle (B).

Figure S3 .

 S3 Figure S3. Principal Component Analyses showing the correlation between change in relative mean abundance (Prop_ab), vent pHcf and DICcf, delta pHcf (pHcf vent -pHcf control; delta pH on the figure) and delta DICcf (pHcf vent -pHcf control; delta DIC on the figure) were correlated.

  Normanby's seep site. Furthermore, these results are based on a large range of coral genera and morphologies representative of the local diversity. Thus, variability in seawater pH and time spent by corals in different pH levels can likely explain the discrepancies between locations. Despite similar mean pH at both CO2 seeps, corals in

	Normanby experienced much larger variations in seawater pH. 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 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. Apparent pHcf homeostasis
	in Normanby in most coral species studied could therefore result from the high
	variability in pH at this location. Moreover, it is likely that this apparent pH
	homeostasis in Normanby might represent calcification occurring predominantly
	during these high pH events associated with high tide. Other environmental
	parameters such as temperature, light, and flow are known to affect the composition
	of the calcifying fluid chemistry

  However, this cannot be tested with the present data, as boron isotopes only provide indication on the mean pHcf during the precipitation of calcium carbonate over several weeks and other methods, such as dyes and microelectrodes, cannot be used in situ presently. 2) Periods of extremely low pH could cause physiological stress and even dissolution of resident organisms, especially for any individuals where skeletal CaCO3 material becomes exposed. For example, the low pH values found in Upa-Upasina reef (Normanby Island) could also explain the deleterious effects of seawater pH on coral skeletal characteristics reported at this site(Prada et al. preprint). In this scenario, dissolution-resistant species would be favoured at Normanby. 3) Greater pH variability itself could cause deleterious effects, such as reduction of calcification as observed in the coralline alga Arthrocardia corymbosa

913 914 Table S1 .

 914S1 Seawater carbonate chemistry in Ambitle and Normanby at the control and 915 the seeps sites. Medians, 5 th and 95 th percentile (between parentheses) of the measured 916 pHT, total alkalinity (AT) and temperature (T) are shown. pCO2, CT, and Ωarag were 917 calculated using the median values of AT and T and the median, 5 th and 95 th percentile

	918
	919	of pHT.
	920
	Site		pHT	AT	T	pCO2	CT	Ωarag
				(µmol kg	-1 )	(µatm)	(µmol kg	-1 )
	Ambitle	8.02
	Control	(7.94 -8.06)

Table S2 .

 S2 Summary of the mean changes in delta pHcf (pHcf seeps -pHcf ambient), delta DICcf (DICcf seeps -DICcf ambient) and relative change in abundance at the two locations.

	Location	Species	Delta pHcf	Delta DICcf Change in
					relative
					abundance
	Normanby	Acropora samoensis	-0.15	581	-0.6
		Favites pentagona	-0.14	1296	-0.71
		Tubastrea sp.	-0.13	306	NA
		Favites halicora	-0.10	726	-0.56
		Galaxea fascicularis	-0.07	90	-0.04
		Pocillopora verrucosa -0.06	150	NA
		Acropora cytherea	-0.05	-83	-1
		Merulina ampliata	-0.03	106	-0.75
		Turbinaria reniformis	-0.03	-154	-0.91
		Porites rus	-0.02	170	0.66
		Acropora tenuis	-0.01	-50	-0.52
		Pachyseris speciosa	0.01	-208	-0.86
		Seriatopora	0.07	-233	-0.70
		caliendrum
		Acropora millepora	0.07	-50	-0.46
	Ambitle	Echinopora lamellosa	-0.19	751	-0.94
		Galaxea fascicularis	-0.17	301	-0.64
		Massive Porites sp.	-0.13	-506	-0.45
		Acropora tenuis	-0.12	290	3
		Acropora nana	-0.10	28	6.85
		Psammocora contigua	-0.09	178	NA
		Pocillopora damicornis -0.09	333	-0.23
		Montipora foliosa	-0.02	-151	8.67

Acknowledgements

This study was partially funded by the French National Research Agency (ANR; project CARIOCA grant agreement no. ANR15CE02-0006-01, 2015), by the French grant scheme Fonds Pacifique (project AMBITLE grant agreement no. 1598, 2016), by the Australian ARC Centre of Excellence in Coral Reef Studies (CE140100020) and by the Flotte Océanographique Française for use of the research vessel Alis. We are grateful to the population of Tutum Bay in Ambitle and Upa-Upasina in Normanby for allowing us to conduct the study on their reefs, and to the National Research Institute, the Milne Bay Provincial Research Committee, the New Ireland Provincial Administration, and the Conservation and Environment Protection

Similarly, there was no relationship with DICcf in Normanby (C) and Ambitle (C).