Núria Teixidó

Erik Caroselli
email: erik.caroselli@unibo.it

Samir Alliouane

Chiara Ceccarelli

Steeve Comeau

Jean-Pierre Gattuso

Pietro Fici

Fiorenza Micheli

Alice Mirasole

Stephen G Monismith

Nuria Teixido
email: nuria.teixido@obs-vlfr.fr

Stazione Zoologica

Anton Dohrn

Anton Dohrn Monismith

Nuria Teixidó

Jean- Pierre Gattuso

Marco Munari

Stephen R Palumbi

Elizabeth Sheets

Lidia Urbini

Cinzia De Vittor

Stefano Goffredo
email: s.goffredo@unibo.it

Maria Cristina Gambi

Ocean acidification causes variable trait-shifts in a coral species

Keywords: corals, calcification, ocean acidification, variable environments, natural CO2 vents, acclimatization/adaptation

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of exposure to elevated pCO 2 on trait-shifts observed throughout natural populations of Astroides calycularis, an azooxanthellate scleractinian coral endemic to the Mediterranean. Unexpected shifts in skeletal and growth patterns were found. Colonies shifted to a skeletal phenotype characterized by encrusting morphology, smaller size, reduced coenosarc tissue, fewer polyps, and less porous and denser skeletons at low pH. Interestingly, while individual polyps calcified more and extended faster at low pH, whole colonies found at low pH site calcified and extended their skeleton at the same rate as did those at ambient pH sites. Transcriptomic data revealed strong genetic differentiation among local populations of this warm water species whose distribution range is currently expanding northward. We found excess differentiation in the CO 2 vent population for genes central to calcification, including genes for calcium management (calmodulin, calcium-binding proteins), pH regulation (V-type proton ATPase), and inorganic carbon regulation (carbonic anhydrase). Combined, our results demonstrate how coral populations can persist in high pCO 2 environments, making this system a powerful candidate for investigating acclimatization and local adaptation of organisms to global environmental change.

Abstract

High pCO 2 habitats and their populations provide an unparalleled opportunity to assess how species may survive under future ocean acidification conditions, and help to reveal the traits that confer tolerance. Here we utilize a unique CO 2 vent system to study the effects of exposure to elevated pCO2 on trait-shifts observed throughout natural populations of Astroides calycularis, an azooxanthellate scleractinian coral endemic to the Mediterranean. Unexpected shifts in skeletal and growth patterns were found. Colonies shifted to a skeletal phenotype characterized by encrusting morphology, smaller size, reduced coenosarc tissue, fewer polyps, and less porous and denser skeletons at low pH. Interestingly, while individual polyps calcified more and extended faster at low pH, whole colonies found at low pH site calcified and extended their skeleton at the same rate as did those at ambient pH sites. Transcriptomic data revealed strong genetic differentiation among local populations of this warm water species whose distribution range is currently expanding northward. We found excess differentiation in the CO 2 vent population for genes central to calcification, including genes for calcium management (calmodulin, calciumbinding proteins), pH regulation (V-type proton ATPase), and inorganic carbon regulation (carbonic anhydrase). Combined, our results demonstrate how coral populations can persist in high pCO 2 environments, making this system a powerful candidate for investigating acclimatization and Understanding the effects of environmental variability and extremes on natural populations and ecosystems is a key priority as global environmental change intensifies [START_REF] Bennett | Integrating within-species variation in thermal physiology into climate change ecology[END_REF][START_REF] Bozinovic | Physiological correlates of geographic range in animals[END_REF]. High local variability in physical and chemical ocean properties can create extreme climatic environments, where marine species persist under suboptimal environmental conditions such as highly variable temperatures, marginal habitats at latitudinal extremes, and acidification at CO 2 vent sites [START_REF] Camp | The future of coral reefs subject to rapid climate change: lessons from natural extreme environments[END_REF][START_REF] Kapsenberg | Ocean acidification refugia in variable environments[END_REF][START_REF] Kroeker | Ecological change in dynamic environments: Accounting for temporal environmental variability in studies of ocean change biology[END_REF]. Populations living in these unique settings experience high environmental variability and can have broad physiological tolerance to environmental stressors that would prevent survival of conspecifics living in less variable micro-environments [START_REF] Bozinovic | Physiological correlates of geographic range in animals[END_REF][START_REF] Thomas | Mechanisms of thermal tolerance in reef-building corals across a fine-grained environmental mosaic: lessons from Ofu, American Samoa[END_REF]. Two important mechanisms for intraspecific variation in tolerance to environmental variability and extremes are adjusting life traits through phenotypic plasticity and local adaptation, and these processes may interact synergistically [START_REF] Hoffmann | Climate change and evolutionary adaptation[END_REF][START_REF] Savolainen | Ecological genomics of local adaptation[END_REF]. Phenotypic plasticity (also referred to as acclimatization) is the ability of a genotype to produce different morphological and physiological responses when exposed to different environmental conditions within an organism's lifespan, resulting in a phenotypic shift that is plastic and often reversible [START_REF] Savolainen | Ecological genomics of local adaptation[END_REF][START_REF] Thomas | Mechanisms of thermal tolerance in reef-building corals across a fine-grained environmental mosaic: lessons from Ofu, American Samoa[END_REF]. Adaptation is the result of natural selection on beneficial genotypes in a population where these changes are heritable and passed on to the next generation [START_REF] Hoffmann | Climate change and evolutionary adaptation[END_REF][START_REF] Savolainen | Ecological genomics of local adaptation[END_REF]. Natural extreme environments are potential locations for climate-adapted populations where, for example, microhabitats experiencing periodic temperature extremes have shown to generate high-tolerance in some reef-building corals [START_REF] Palumbi | Mechanisms of reef coral resistance to future climate change[END_REF][START_REF] Thomas | Mechanisms of thermal tolerance in reef-building corals across a fine-grained environmental mosaic: lessons from Ofu, American Samoa[END_REF]. However, there is still much to learn about Insights into species' tolerance to environmental change may be gained by analyzing traits that directly influence an organism's performance [START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]. Shifts in the occurrence of these traits under variable environmental conditions can reflect patterns of differential survival and growth strategies; for example, different morphological forms (e.g. massive or encrusting), longevity, size, growth rates, physical defenses and dispersal ability [START_REF] Darling | Evaluating life-history strategies of reef corals from species traits[END_REF][START_REF] Teixidó | Functional biodiversity loss along natural CO 2 gradients[END_REF]. These traits provide relevant information about life strategies that are the result of different evolutionary and ecological processes and influence, both the fitness of individuals and the viability of natural populations [START_REF] Darling | Evaluating life-history strategies of reef corals from species traits[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF][START_REF] Teixidó | Functional biodiversity loss along natural CO 2 gradients[END_REF]. However, we still know comparatively little about trait-shifts within natural populations and the capacity to adapt to long-term novel environmental conditions. Natural volcanic CO 2 vents cause local acidification of seawater and are used as a proxy to study future ocean acidification [START_REF] Enochs | Shift from coral to macroalgae dominance on a volcanically acidified reef[END_REF][START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF][START_REF] Hall-Spencer | Volcanic carbon dioxide vents show ecosystem effects of ocean acidification[END_REF].

Ocean acidification reflects a suite of changes in seawater carbonate chemistry due to the uptake of excess anthropogenic CO 2 by the ocean, resulting in a decline in the surface ocean pH, carbonate ion concentration, and saturation state of calcium carbonate minerals (e.g. aragonite), while increasing the partial pressure of carbon dioxide (pCO 2 ) and bicarbonate ion concentrations [START_REF] Doney | Ocean Acidification: The Other CO 2 Problem[END_REF]. Low pH levels in natural CO 2 vents represent future climatic conditions where, relative to 1870, surface pH is projected to decline by -0.14 to -0. [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF][START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO 2 emissions scenarios[END_REF][START_REF] Goffredo | Biomineralization control related to population density under ocean acidification[END_REF][START_REF] Teixidó | Functional biodiversity loss along natural CO 2 gradients[END_REF]. Although these pH conditions can provide some insight into future acidification scenarios, they are not perfect proxies. One important assumption to consider is that variability of seawater pH increases with decreasing means at CO 2 vent systems.

Although variability in pH/pCO 2 will increase with dissolved inorganic carbon due to the thermodynamics of the carbonate system in the future ocean [START_REF] Takeshita | Including high frequency variability in coastal ocean acidification projections[END_REF], it is not possible to disentangle the effects of changes in the mean versus variability in this system. Thus, the conditions in the pH zones should be considered as pH regimes, with decreases in mean pH coinciding with increases in variability. Nevertheless, these high pCO 2 environments and their populations provide an unparalleled opportunity to assess how species may survive into future pH conditions and to reveal if general traits that confer tolerance can be identified.

Corals are key marine organisms that are particularly vulnerable to the impacts of climate change and ocean acidification [START_REF] Brandl | Coral reef ecosystem functioning: eight core processes and the role of biodiversity[END_REF][START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO 2 emissions scenarios[END_REF]. They create habitats for many species, enhancing biodiversity, playing fundamental ecological roles and sustaining ecosystem processes and services such as fisheries, coastal protection and tourism [START_REF] Brandl | Coral reef ecosystem functioning: eight core processes and the role of biodiversity[END_REF][START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO 2 emissions scenarios[END_REF]. Ocean acidification may pose a major threat to corals because their growth relies on the precipitation of calcium carbonate (calcification), a process that is expected to decrease as seawater acidity increases [START_REF] Chan | Sensitivity of coral calcification to ocean acidification: a meta-analysis[END_REF]. Studies conducted at CO 2 vent ecosystems on native corals have reported an overall decline in species abundances, decreases in calcification and skeletal density with increasing acidification [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF][START_REF] Fantazzini | Gains and losses of coral skeletal porosity changes with ocean acidification acclimation[END_REF][START_REF] Goffredo | Biomineralization control related to population density under ocean acidification[END_REF].

Here we utilize a unique CO2 vent system to investigate the effects of exposure to elevate pCO 2 on trait-shifts on Astroides calycularis, an azooxanthellate scleractinian coral endemic to the

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Mediterranean, that naturally occurs in the acidified environment of a newly discovered CO 2 vent system in Ischia, Italy. This CO 2 vent system locally acidifies the seawater with gas comprising 92-95% CO 2 (no sulphur, and no temperature anomaly). A. calycularis is a long-lived coral (large colonies may have a life span of several decades), considered a warm-water species with a narrow temperature tolerance confined to 14°C during winter [START_REF] Bianchi | Biodiversity issues for the forthcoming tropical Mediterranean Sea[END_REF][START_REF] Zibrowius | The "southern" Astroides calycularis in the Pleistocene of the northern Mediterranean-An indicator of climatic changes (Cnidaria, Scleractinia)[END_REF]. A. calycularis has low dispersal capacities, and therefore restricted gene flow (Casado-Amezúa, [START_REF] Casado-Amezúa | Genetic assessment of population structure and connectivity in the threatened Mediterranean coral Astroides calycularis (Scleractinia, Dendrophylliidae) at different spatial scales[END_REF]. Because A. calycularis is a calcifying and long-lived species with low dispersal capacity, and found throughout the CO 2 vents, it is a great model system for investigating variation in local climate phenotypic plasticity and adaptation. Previous research on the effects of ocean acidification on A. calycularis has shown contrasting results: a reduction of net calcification rates was found when colonies growing in ambient pH conditions were transplanted to a vent system with pH below 7.7 [START_REF] Prada | Ocean warming and acidification synergistically increase coral mortality[END_REF], while no change in calcification under acidification was observed in controlled laboratory conditions [START_REF] Movilla | Annual response of two Mediterranean azooxanthellate temperate corals to low-pH and high-temperature conditions[END_REF]. We compare populations living near the vent to two reference areas outside the influence of CO 2 venting to examine the effects of low pH conditions on A. calycularis traits, to characterize the genetic population structure, and to identify differentiation in genes that are central to calcification. Specifically, we addressed the following questions: i) do populations at the CO 2 vent and reference sites exhibit significant trait variation?, ii) do these nearby populations display genetic differentiation?, and iii) does the CO 2 vent population have highly divergent SNP genotypes from calcium-related loci? To answer these questions, we characterized the physical and chemical parameters of the study sites and combined in situ population demographics, skeletal characteristics, computed tomography and transcriptomic approaches to assess changes in population abundance, skeletal properties, age, and genomics of differentiation of A. calycularis.

MATERIAL AND METHODS

Experimental design and study sites

Here we compare natural populations of the scleractinian coral A. calycularis at a volcanic CO 2 vent and two nearby reference sites with ambient pH and no vent activity along the coast of Ischia, Italy (Figure 1). The CO 2 vent system is located at a 5 m depth inside a semi-submerged cave of volcanic origin named Grotta del Mago (Magician's Cave, 40°42'41.87"N, 13°57'51.06"E, hereafter Vent system) (Figure 1). The cave (total length of 110 m) consists of a main outer chamber (10 m wide x 30 m long), connected to an inner chamber by a long narrow passage [START_REF] Cinelli | Distribution of benthic phyto-and zoocoenoses along a light gradient in a superficial marine cave[END_REF]. Published studies and personal observations indicated an increase in the CO 2 vent activity over the last 50 years in the main chamber, with limited vent activity in the 70's [START_REF] Cinelli | Distribution of benthic phyto-and zoocoenoses along a light gradient in a superficial marine cave[END_REF]) and 2000's [START_REF] Dappiano | New data on occurrence of thermophile Scleractinia (Cnidaria, Anthozoa) in the Phlaegrean Island (Ischia, Procida, Vivara, Gulf of Naples), with special attention to Astroides calycularis[END_REF] developing into intense activity from 2014 onwards. The abundance of A. calycularis in the cave has increased over time, with a low and patchy distribution between 1-2 m depth in the main chamber in the 1970's [START_REF] Cinelli | Distribution of benthic phyto-and zoocoenoses along a light gradient in a superficial marine cave[END_REF] to a high and continuous distribution in the 2000's [START_REF] Dappiano | New data on occurrence of thermophile Scleractinia (Cnidaria, Anthozoa) in the Phlaegrean Island (Ischia, Procida, Vivara, Gulf of Naples), with special attention to Astroides calycularis[END_REF]. The present study was performed in the main chamber of the cave. The reference sites with ambient pH were chosen following the criteria: i) A. calycularis naturally occurred there, ii) they hosted similar habitats and depths as the CO 2 vent site, and iii) and no venting activity was evident. Two reference sites were selected: Punta Vico (40°45'32.28"N, 13°52'55.38"E, another semi-submerged cave, with a main chamber 10 m wide x 30 m long, 5 m maximum depth, hereafter Ambient 1); and Sant'Angelo (40°41'33.78"N,13°53'38.88"E, an overhang, also a natural habitat of A. calycularis, located on a natural arch, with an opening of 10 m wide x 10 m height, 10 m maximum depth, hereafter Ambient 2). Initial investigations of the natural systems and environmental parameters started in June, 2016. These preliminary environmental data were used to plan subsequent field

The coral

A. calycularis (Pallas, 1766) is an azooxanthellate scleractinian colonial coral endemic to the Mediterranean, characterized by the bright orange color of its coenosarc and polyps [START_REF] Zibrowius | The "southern" Astroides calycularis in the Pleistocene of the northern Mediterranean-An indicator of climatic changes (Cnidaria, Scleractinia)[END_REF]. It is considered a long-lived species (e.g large colonies may have a life span of several decades) and commonly found in dimly lit, shallow rocky habitats (vertical walls, cave entrances, overhangs, from the intertidal fringe to 50 m depths) [START_REF] Zibrowius | The "southern" Astroides calycularis in the Pleistocene of the northern Mediterranean-An indicator of climatic changes (Cnidaria, Scleractinia)[END_REF]. It can be highly abundant covering more than 90% of local reefs. It has a limited geographic distribution, with a southwestern distribution in the Mediterranean Sea [START_REF] Zibrowius | The "southern" Astroides calycularis in the Pleistocene of the northern Mediterranean-An indicator of climatic changes (Cnidaria, Scleractinia)[END_REF]. This coral is considered a warm-water species with a narrow temperature tolerance confined to 14°C during the winter [START_REF] Bianchi | Biodiversity issues for the forthcoming tropical Mediterranean Sea[END_REF].

Fossil records reveal this species lived in the northwestern Mediterranean during part of the Pleistocene, where climatic fluctuations occurred leading to a reduction of the species [START_REF] Zibrowius | The "southern" Astroides calycularis in the Pleistocene of the northern Mediterranean-An indicator of climatic changes (Cnidaria, Scleractinia)[END_REF]. Interestingly, observed records north of its known distribution range in Italy and Croatia suggest that it is currently expanding northward [START_REF] Bianchi | Biodiversity issues for the forthcoming tropical Mediterranean Sea[END_REF]. Currently, A. calycularis is assessed as vulnerable on the IUCN Red List due to its limited geographic distribution and the historical and current regression caused by human activities in the littoral zone. A. calycularis broods its larvae and has relatively low dispersal capacity [START_REF] Casado-Amezúa | Genetic assessment of population structure and connectivity in the threatened Mediterranean coral Astroides calycularis (Scleractinia, Dendrophylliidae) at different spatial scales[END_REF].

Gas and temperature

Gas samples were collected in 200 ml glass bottles and analyzed using gas chromatography (Agilent 7890B combined with a Micro GC analyzer-INFICON, held at a constant temperature of 80 °C). The mean composition of the bubbling gas was predominantly CO 2 (92-95%, with undetectable levels of sulfur gas <0.0002 %) and did not elevate the temperature (see Supporting S1), subsequently resulting in water acidification. Vent activity was sampled by counting the number of vents in randomly placed 1 m 2 quadrats (n= 11) in the main chamber, with approximately 5 vents m -2 (mean ± SE= 4.9 ± 2.7 vents m -2 , min= 2 vents m -2 ; maximum = 11 vents m -2 ). Temperature was registered every hour by in situ temperature data loggers (Hobo TidbiT v2, Onset) in the cave and the reference areas and followed ambient seasonal fluctuations, from 14.7 to 15.2 °C in winter (n=16,754), and from 25.5 to 26.5 °C in summer (n= 19,011) over a 3-year period from 2016 to 2019 at 2 m depth (Figure S1, Table S1).

F o r R e v i e w O n l y 10 Note 1, Figure

pH T time series, pH T variability and pH sensor calibration

SeaFET TM Ocean pH sensors (Satlantic) were deployed to quantify variation in pH inside the cave at 2, 3 and 4 m depth. They were deployed in May-June (before summer) and in September (after summer) to assess whether differences in water temperature stratification could influence pH across depths. Dates of deployment were from September 8 to September 24, 2018 and from May 30 to June 18, 2019. Two sensors were deployed in the reference areas during the same period (Ambient 1 in September, 2018 and Ambient 2 in June, 2019). Before deployment, the SeaFETs were calibrated with ambient pH water in the aquarium facilities at the Center Villa Dohrn (Ischia, Italy) (for full details of pH sensor calibration, see Supporting Methods). The mean offset between calibration samples and calibrated SeaFET pH was ± 0.002 units, indicating high quality pH dataset (Figure S2).

Carbonate Chemistry and Nutrients

Discrete water samples were collected using Niskin bottles at the vent and reference areas with ambient pH to measure: i) the carbonate system parameters during the pH sensor deployment, and ii) dissolved inorganic nutrients. Salinity was measured using a CTD (CTD Sea Bird Electronics SBE 19 Plus Seacat). Samples for total alkalinity (A T ) were collected using standard operating #171, and #177). Precision of the A T measurements of CRMs was < 2.0 μmol kg -1 from nominal values. Means were reported as (mean ± SD): A T = 2562.41 ± 7.8 μmol kg -1 , n = 27 in September 2018; and A T = 2543.57 ± 21.78 μmol kg -1 , n = 21 in June 2019. A T and pH T were used to determine the remaining carbonate system parameters at in situ temperature and depth of each sampling period in the R package seacarb v3.2.12 (for constant details, see Supporting Methods). Dissolved inorganic nutrients (nitrite NO 2 , nitrate NO 3 , ammonium NH4 + , phosphate PO 4 and silicate Si(OH) 4 ) were determined using a colorimetric method (Supporting Methods) (Table S2).

Coral field surveys: cover, population structure and morphology

The A. calycularis cover was quantified using 24 photoquadrats (25 x 25 cm) positioned along six transects at four depths: 1, 2, 3, and 4 m in the three study sites (Vent, A1, A2). Percentage cover was analyzed with ImageJ image software (National Institutes of Health, https://imagej.net/ImageJ). Size frequency-distribution was calculated at 1 and 3 m depths by counting the number of polyps of each colony and each colony was then pooled into one of five size classes (I: 1-5 polyps: II: 6-10 polyps; III: 11-15 polyps; IV: 16-20 polyps; V: > 20 polyps).

These size classes were selected to span the range of colony sizes encountered in the field. We also assessed necrosis as the percentage of the colony exhibiting dead tissue or denuded skeleton, from white-grey to unpigmented or denuded skeleton. Finally, visual assessments were used to classify the colonies into two morphological categories: encrusting (flat growth form) and massive (extensive vertical and lateral growth). Encrusting colonies extended laterally over the surrounding substrate, whereas massive presented a greater vertical accretion which resulted in semi-spherical

F o r R e v i e w O n l y 12
shapes. This categorical criterion allowed us to obtain two simple morphological variables to capture biologically relevant axes of variation.

Sample collection for presence of coenosarc, skeletal characteristics and growth

Sixty-six colonies of A. calycularis were sampled haphazardly for coenosarc, biometric, growth, and skeletal parameters. Thirty-four colonies were collected at the vent site: 16 colonies were obtained from the vicinity of the CO 2 vents at 3 m depth (vent system deep, Vd) and 18 colonies at 1-2m depth (vent system shallow, Vs). Thirty-two colonies were collected from areas with ambient pH conditions: 17 colonies in Punta Vico, Ambient 1, 1 -2 m depth; and 15 colonies in Sant'Angelo, Ambient 2, 1 -2 m depth. The 66 colonies were photographed and the percentage of coenosarc (i.e., the living tissue connecting the polyps) was determined. The % of coenosarc was determined from the edges of the polyp tissue. The % of coenosarc was classified into ten classes at every 10% interval, from 100% to 0%. Loss of coenosarc in A. calycularis may occur mainly by two mechanisms: 1) loss of tissue due to necrosis (when colony exhibits dead tissue, from white-grey to unpigmented or denuded skeleton), or 2) the coenosarc is already absent due to physiological and morphological characteristic of the colonies.

Biometric parameters

Coral skeletons were rinsed in a solution of 10% commercial chlorine bleach for 3-4 days to dissolve polyp tissue, then they were dried at room temperature for 3 days. Colony was defined as the whole calcareous skeleton, which included the polyps (corallites) and the coenosteum. The following parameters were measured: colony length (L c , major axis of the colony) and colony width (W c , minor axis of the colony); number of polyps in each colony (NP c ), corallite length (L p , maximum axis of the oral disc) and corallite width (W p , minimum axis of the oral disc) (for full details, see Supporting Methods, Figures S3-S4, and Tables S3-S4).

Growth and age estimations

The age of each corallite skeleton was determined by counting the growth bands of 49-70 randomly selected corallites per site, by means of computerized tomography (CT). Growth bands are distinguished by a high-density band in winter and a low-density band in summer in temperate corals (see Supporting Methods). The age of all collected corallites was estimated using the von Bertalanffy's length-age growth function derived from the CT growth bands analysis. Coral growth is described by three parameters: linear extension rate (linear growth), net calcification rate (net mass deposited) and bulk skeletal density (mass per volume unit) [START_REF] Goffredo | Inferred level of calcification decreases along an increasing temperature gradient in a Mediterranean endemic coral[END_REF]. The measurement of all three components is fundamental when assessing the effect of the environment on coral growth, since none of the three parameters is a perfect predictor for the other two and each species can respond differently to environmental conditions. Then, the following three coral growth parameters were calculated for both polyp and colony levels: 1) linear extension rate; 2) net calcification rate and 3) bulk skeletal density (see below for bulk skeletal density measurements) (for full details, see Supporting Methods).

Skeletal parameters

Skeletal parameters of colonies were calculated by applying the buoyant weight technique through the density determination kit of the Ohaus Explorer Pro balance (± 0.0001 g; for further details, see Supporting Methods). This method is based on the Archimedes principle applied to a specimen after full saturation with the same fluid in which it was submerged. The measurements required to calculate the skeletal parameters were: density of the fluid medium (ρ); dry mass (DW), buoyant weight of the skeleton (BW= weight of the skeleton minus weight of the water displaced by it), SW (saturated weight of the coral = weight of the skeleton plus weight of the water enclosed in its). Measurements were repeated three times to get an average for BW and SW. Based on these

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14 measurements, the following parameters were calculated: V MATRIX (matrix volume = volume of the skeleton, excluding the volume of its pores); V PORES (pore volume=volume of the pores in the skeleton), V TOT (bulk volume = total volume of the skeleton including its pores). Finally, the skeletal parameters of colonies were calculated: the micro-density (ratio of DW to V MATRIX ); the bulk density (ratio DW to V TOT ); and the porosity (ratio V PORES to V TOT ).

Sample collection for genetics and transcriptome assembly

A. calycularis colonies were haphazardly collected between 1-2 m depth to compare gene flow among populations at the study sites. Nineteen colonies from the Vent, 14 colonies from Ambient 1 and 8 colonies from Ambient 2 were sampled for genetic analysis. RNA was extracted from a single polyp of each colony using a RNeasy kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions (for full details, see Supporting Methods). Approximately 1g of RNA was used to construct a cDNA library for each sample using the Illumina TruSeq RNA v2 Kit (Illumina, San Diego, CA, USA) (see Supporting Methods).

Libraries were sent to the Genomics Core Facility of the Health Sciences Cores at the University of Utah (Salt Lake City, Utah, USA) and samples were quantified using a Bioanalyzer (Agilent, Santa Clara, CA, USA) (see Supporting Methods). We assembled the first de novo transcriptome of A. calycularis with samples collected from Ischia. The following programs and scripts were run on Stanford University's Sherlock cluster and all scripts used in this pipeline (https://github.com/bethsheets/Astroides_transcriptomics) along with a general guide are available on GitHub (doi:10.5281/zenodo.2580291). Four population-specific de novo assemblies were generated using three individuals per population for each population in the program Trinity-2.8.4 (see Supporting Methods). Prior trials mapping to available corallimorpharian genomes produced incomplete assemblies. Therefore, assembled contigs were validated to be included in the

F o r R e v i e w O n l y 15
assembly by filtering for only metazoan matches found in the combined UniProt's Swiss-Prot and TrEMBL databases using BLASTX in the BLAST+ toolkit (see Supporting Methods). Matches were considered significant at values of  1 x 10 -3 and the top hit for each contig was kept for assembly filtering and annotation. Transcriptome completeness was assessed using BUSCO v3 (see Supporting Methods) against the metazoan (obd9) set. BUSCO analyses revealed that the final combined transcriptome was 97% complete (949 complete BUSCOs out of 978 searched: 366 complete single-copy, 583 complete and duplicated; 12 fragmented, and 17 missing). For the 41 individuals used for population analyses, the average overall mapping rates for each population were as follows: Vent-Grotta Mago 79% (range 71.91 -85.28), Ambient 1-Punta Vico 80% (range 65 -84.74), Ambient 2-Sant' Angelo-80.26% (range 71.91 -85.28). After filtering, we detected 46,784 biallelic SNPs among the vent and two ambient populations.

Mapping and SNP detection

For all 41 samples, raw paired-and single-end sequence files were mapped to the de novo assembly using HISAT2 (see Supporting Methods) with the very-sensitive setting. Duplicate reads due to PCR were removed with Picard tools (http://broadinstitute.github.io/picard/) MarkDuplicates using the lenient validation stringency. Overall mapping rates were compared among populations to assess whether certain populations were preferentially mapping to the de novo assembly.

Transcriptome-derived single nucleotide polymorphisms (SNPs) were called on all individuals using SAMtools mpileup and BCFtools (for filter settings, see Supporting Methods).

Identifying SNP candidates for environmental selection in high CO 2 conditions and enrichment analysis

We identified potential outlier SNPs related to the CO 2 vent location. We calculated pairwise Fst estimates (Vent -Ambient 1, Vent -Ambient 2, Ambient 1 -Ambient 2) per locus using the basic

Statistical analyses and data visualization

Environmental data analyses: Temperature, pH T , SeaFET sensor calibration, carbonate chemistry and figures were performed using the R packages: seacarb v3.2.12 and ggplot2 v3.

Carbonate Chemistry Associated with the CO 2 vent system in the Grotta del Mago' cave

The CO 2 vent system occurs at a 5 m depth inside the main chamber of the cave Grotta del Mago with approximately 5 vents m -2 (Figure 1) and do not elevate temperature (Table1, Figure S1, Table S1). The carbonate chemistry derived from discrete water samples and in situ monitoring of seawater pH T (pH on the total scale) delineated a pH T gradient from 4 m to 2 m depth caused by the distance from the bubbling of CO 2 gas from the seafloor (92-95% CO 2 , with undetectable levels of sulfur gas < 0.0002 %, see Supporting Note 1) (Figure 1, Table 1). pH sensors revealed reductions in mean pH T at each depth associated with increased temporal variability in pH T (Figure 1, Table 1, Figure S5, Table S5). Mean pH T were: 7.65 to 7.88 at 2 m; 7.62 to 7.74 at 3 m, and 7.60 to 7.60 at 4 m, respectively (see Table 1 and Table S5 for detailed pH statistics). At 2 m depth, 14% and 56% of the pH T measurements were below 7.8 (projected average global sea surface pH value for the year 2100 with the high emission scenarios RCP8.5) [START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO 2 emissions scenarios[END_REF] in June and September, respectively (Table S5). The percentage rose to 34% and 61% at 3 m depth, and 55% and 66% at 4 m depth, in June and September, respectively (Table S5). This pattern of depthdependent low pH T was also manifested as extreme pH events (defined as the pH value of 0.4 units less than the mean pH for each depth) that increased in number and duration with depth (Table S6). Mean pH T and variability were influenced by temperature stratification in June and September (Figure 1, Figures S5-S6). This is because during periods of stratification, and hence reduced vertical mixing [START_REF] Turner | Buoyancy Effects in Fluids[END_REF], the input of CO 2 is likely to be primarily confined to the lower part of the water column, leading to lower pH values near bed than when the water column is well mixed. The mean temperature difference between 2 m and 4 m in June was 0.25 °C, whereas the mean temperature difference was only 0.02 °C in September (Figure S6). In September, reductions

F o r R e v i e w O n l y 19
in seawater pH T were driven by increased dissolved inorganic carbon concentrations (C T ) and higher pCO 2 concentrations at relatively constant total alkalinities (A T ) and temperatures across depths (Table 1, Figure S5). Mean pCO 2 ranged from 2905 ± 1664 μatm at 2 m, to 3146 ± 1928 μatm at 3 m, to 3192 ± 1806 μatm at 4m depth, and aragonite saturation state (Ωa) ranged from 1.10 ± 0.4 at 2m, to 1.05 ± 0.4 at 3 m, and to 1.02 ± 0.4 at 4m depth (Table 1). In contrast, in June, the vent site was characterized by an increase in temperature along the water column (from ~ 18.5 °C to ~ 25°C), which created greater difference across the three depths in terms of pH T and associated carbonate chemistry parameters, particularly for the pCO 2 concentrations (from 1531 ± 627 μatm at 2 m, to 2082 ± 1502 μatm at 3 m, and 2812 ± 2310 μatm at 4 m depth) and Ωa (from 1.44 ± 0.27 at 2 m, to 1.23 ± 0.35 at 3 m, to 1.05 ± 0.42 at 4 m depth) (Table 1, Figure S5). At the two ambient pH sites, the mean pH T ranged from 7.97 to 8.05 units, pCO 2 from 322 ± 34 to 586 ± 76 μatm, and Ωa from 3.54 ± 0.23 to 3.86 ± 0.23 (Table 1).

Cover, population structure, and morphology of A. calycularis

The CO 2 vent population was characterized by small colonies (90% colonies had up to 10 polyps) and no large colonies of more than 20 polyps (class V) with massive morphology were found (Figure 2, Figure S7). Larger colonies (i.e. > 16 polyps, size classes IV and V) were only found in the two reference areas (percentage of larger size classes ranged from 13% in A1 to 16 % in A2), which differed significantly among the CO 2 vent site (χ 2 = 91.9, df= 8, p<0.0001). Additionally, necrosis was significantly higher at the CO 2 vent site (13 ± 4%) than the reference areas (both <0.5%, D= 0.56, p<0.0001). A. calycularis' cover at the CO 2 vent site decreased from 50% at 1 m depth, to 30% at 2m, 9% at 3 m, and 1% at 4m depth, as seawater pH T also declined (Figure S7).

This decline in coral cover was also observed in Ambient 1 (also a cave, from 69% at 1 m to 14%

F o r R e v i e w O n
l y 20 at 3 m) but not in Ambient 2 (overhang, from 72% at 1 m to 62% at 3 m; F= 14.1, df=11,55; p<0.001) (Figure S7).

Coenosarc, skeletal parameters and growth

Percentage of coenosarc (the living tissue connecting the polyps) significantly decreased between ambient pH sites (87 and 70%) and the CO 2 vent (28 and 14%) (Figure 2 Bulk density (ratio dry mass to bulk volume) and micro-density (ratio of dry mass to matrix volume) increased at low pH, while porosity (ratio pore volume to bulk volume; see Methods) decreased at low pH (p < 0.001; Figure 3A-B). Colonies at the CO 2 vent deep presented higher bulk density (~ + 27 %) and micro-density (~ + 7 %) and lower porosity (~ -28 %) compared to colonies from the ambient pH sites (p < 0.001) (Figure 3A-B, Figure S9).

Growth parameters of A. calycularis differed significantly between the CO 2 vent site and ambient pH sites (Figure 3B, Figure 4, Figure S10, Table S7). Mean polyp growth rate decreased exponentially with age at all sites (Figure S10). Young individuals (1-3 years old) grew relatively rapidly (> 2 mm yr -1 ), but, as they aged, their skeletal growth rate decreased (< 1.3 mm yr -1 at 8-10 years old) (Figure 4, Tables S7-S8). A trend toward higher linear extension and net calcification rate was observed at low pH at the polyp level (Figure 3B, Table S7). Polyp net calcification rate ranged from 3.95 mg mm -2 yr -1 at Vent deep, to 3.04 mg mm -2 yr -1 at Vent shallow, to 2.39 mg mm -2 yr -1 and 2.06 mg mm -2 yr -1 at ambient pH sites (Table S7). This indicates that net calcification rates increased approximately by 48%-93% from the more acidified (Vent deep) to the less acidified (Vent shallow) to the non-acidified (ambient pH) locations at polyp level. Linear S7).

Transcriptome assembly and population genomics

The A. calycularis transcriptome composed of 12 colonies contained 113,351 contigs with an N50 of 2,285 (range 501 -38,179). Based on 46,784 SNPs in 41 individuals, PCA analysis revealed strong clustering by population (Figure 5). The vent population in Grotta Mago was most distinct along PCA axis 1, but overlapped with Ambient1 (PV) along axis 2. Pairwise F ST measurements also support strong population structure within each of the three locations: CO 2 vent (Grotta Mago)

-Ambient 1 (Punta Vico) = 0.034, CO 2 vent (Grotta Mago) -Ambient 2 (Sant'Angelo) = 0.026, Ambient 1 (Punta Vico) -Ambient 2 (Sant'Angelo) = 0.024. SNPs with values over 2 show an excess of genetic differentiation in GM compared to the other populations. There were 334 loci with an excess F ST ratio of 10 or more, out of 2246 loci in our data set with known molecular

function. An analysis of the 402 unique molecular function Gene Ontology (GO) terms associated with these loci showed there to be three significant enrichment classes (Table S9): calcium ion binding (12 loci, padj = 7x10 -23 ), catalytic activity (4 loci, padj = 2x10 -9 ), and oxidoreductase action (12 loci, padj = 0.05).

Calcium ion loci include calmodulin, calcineurin, calnexin, calcium-binding proteins in the sarcoplasmic reticulum, and a set of two poorly characterized proteins with calcium binding motifs SNPs from the thirteen calcium-related loci for patterns across populations (Figure 6). As expected, the Grotta Mago population had highly divergent SNP genotypes at these loci (average minor allele frequency difference of 0.24), but these genes also showed a strong degree of linkage among SNP genotypes within a single gene often across 1000s of base pairs (Figure 6).

Such multi-SNP haplotypes are rare in our data set yet occur in 8 of 10 high excess, calcium ion loci with multiple SNPs.

Given the strong differences in calcium management suggested by excess differentiation of calcium ion genes in Grotta Mago, we queried our transcriptome SNP data base for other genes potentially involved in calcification. In corals, calcification occurs in the calicoblastic space through a combination of high calcium concentration and high pH (reviewed in [START_REF] Drake | How corals made rocks through the ages[END_REF]. High pH is achieved through proton transport by specific calcium/proton pumps, including the plasma membrane calcium ATPase (PMCA). There were no PMCA polymorphisms in our data set, but the V-type ATPase proton pump (contig DN1551, SNPs 1701-1805) showed six of seven SNPs with strong differentiation in Grotta Mago (excess 0.03 -28.8, average 11.4, Figure 6). Minor allele frequencies differed by about a factor of 2 in Grotta Mago compared to the other locations, and SNPs show strong linkage.

In addition to genes involved in calcium and pH regulation, we explored possible adaptation in Carbonic anhydrases convert highly diffusive CO 2 into charged carbonate ions, localizing them in the calicoblastic layer and regulating calcification [START_REF] Chen | Carbonic anhydrase, coral calcification and a new model of stable isotope vital effects[END_REF], though they also play a role in pH regulation of non-calcifying coral cells [START_REF] Bertucci | Carbonic anhydrases in anthozoan corals -A review[END_REF].

DISCUSSION

This study contributes to increasing our understanding of how coral populations persist under naturally high pCO 2 environments -and therefore how they might cope under future ocean acidification scenarios -and links trait-shifts with local variation in environmental parameters found in this new CO 2 vent system. Our results expand upon previous research on populations of corals exposed to naturally elevated pCO 2 [START_REF] Crook | Reduced calcification and lack of acclimatization by coral colonies growing in areas of persistent natural acidification[END_REF][START_REF] Enochs | Shift from coral to macroalgae dominance on a volcanically acidified reef[END_REF][START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF][START_REF] Fantazzini | Gains and losses of coral skeletal porosity changes with ocean acidification acclimation[END_REF], demonstrating unexpected shifts in patterns of skeleton and growth of the azooxanthellate coral A. calycularis. Specifically, colonies shift to a skeletal phenotype characterized by encrusting morphology, smaller size, reduced coenosarc tissue, fewer polyps, and less porous and denser skeletons at low pH. However, while individual polyps calcified more (greater net calcification rates), calcification rate of whole colonies were similar across sites. The resulting colony skeletons showed equal linear extension at low and ambient pH conditions, while their polyp skeletons extended faster in acidified conditions (Figure 7). Transcriptomic data revealed strong genetic differentiation among local populations of this low-dispersal species. We found excess differentiation in the Grotta Mago population for genes central to calcification, including genes for calcium management

F o r R e v i e w O n l y 24
(calmodulin, calcium-binding proteins), pH regulation (V-type proton ATPase), and carbonate localization (carbonic anhydrase).

Environmental variability in the CO 2 vent system

The vent system exhibits high temporal variability in seawater pH due to varying CO 2 venting intensity from the seabed, mixing due to variations in stratification, and fundamental thermodynamics processes fundamental to the carbonate system [START_REF] Takeshita | Including high frequency variability in coastal ocean acidification projections[END_REF]. The carbonate chemistry and in situ monitoring of seawater pH delineated a pH gradient (from 4 to 2 m depth) caused by the distance from the venting. This acidification gradient is important for the colonies exposed to more (deep) or less (shallow) acidified conditions, as reflected by the biological response of Astroides. The conditions in these zones are comparable with IPCC projections for near future acidification scenarios (RCP2.6 and RCP8.5), which project a decrease in surface pH from -0.14 to -0.4 pH units by 2100 relative to 1870 [START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO 2 emissions scenarios[END_REF]. pH and its variability found in this study are comparable with the range of natural variation observed in other CO 2 vent systems, with fluctuations of 0.6, 0.7 and 0.5 pH units in coral reefs [START_REF] Agostini | Ocean acidification drives community shifts towards simplified non-calcified habitats in a subtropical -temperate transition zone[END_REF], temperate reefs in Panarea [START_REF] Prada | Ocean warming and acidification synergistically increase coral mortality[END_REF] and in other CO 2 vents in Ischia [START_REF] Teixidó | Functional biodiversity loss along natural CO 2 gradients[END_REF], respectively. Likewise, [START_REF] Hofmann | High-frequency dynamics of ocean pH: A multi-ecosystem comparison[END_REF] reported pH fluctuations between 0.024 to 1.430 pH units, in which pH measurements were taken from different locations, ranging from polar to tropical, and open-ocean to coastal areas. Interestingly, as mean pH was reduced, its variability and the percentage of pH T measurements registered below 7.8 units increased, when seawater was uniformly warm. In contrast, in June when the water column was stratified, pH and its variability near the bottom was similar to what was observed in September whereas, farther

F o r R e v i e w O n l y
25 from the bottom, pH was higher and less variable. These results indicate that seawater stratification may play a key role in controlling the temporal and depth patterns of pH/pCO 2.

Shifts in coral skeleton, growth, and coenosarc

Each scleractinian coral species may adopt different growth strategies in response to ocean acidification. For example, investing calcification resources in bulk skeletal density by sacrificing the rate of linear extension has been observed in Orbicella annularis [START_REF] Carricart-Gavinet | Annual density banding in massive coral skeletons: result of growth strategies to inhabit reefs with high microborers' activity?[END_REF]. In contrast, investing calcification resources in linear extension rate at the expense of bulk density has been reported for some Porites [START_REF] Lough | Environmental controls on growth of the massive coral Porites[END_REF] and Dendrophyllidae species [START_REF] Goffredo | Inferred level of calcification decreases along an increasing temperature gradient in a Mediterranean endemic coral[END_REF]. Both strategies may imply different ecological trade-offs for the coral:

investing in a denser skeleton results in greater resistance to mechanical stress, while increasing linear extension rate may be advantageous for space competition and earlier sexual maturity [START_REF] Goffredo | Inferred level of calcification decreases along an increasing temperature gradient in a Mediterranean endemic coral[END_REF]. Unexpectedly, A. calycularis revealed unusual patterns in the calcification response to ocean acidification, such as higher bulk skeletal density and lower porosity while maintaining colony linear extension rates and net calcification. This response is different to what was previously shown in solitary corals (e.g. Balanophyllia europaea) growing in natural low pH conditions, where a decrease of net calcification resulted from preserving linear extension (to reach the polyp size of sexual maturity) at the expenses of lower bulk density (e.g. increased in skeletal porosity resulting in more fragile skeletons) [START_REF] Fantazzini | Gains and losses of coral skeletal porosity changes with ocean acidification acclimation[END_REF]. The authors suggested that under low-pH conditions, the increase in linear extension reflects the elongation of calcium carbonate crystals at the site of calcification, while the increase in skeletal density reflects the lateral thickening of calcified elements of coral skeleton [START_REF] Mollica | Ocean acidification affects coral growth by reducing skeletal density[END_REF].

The unusual response of A. calycularis to acidification may reflect an overall maintenance of energetic resources allocated to calcification at the level of the colonies, which extended at the same rate but were composed by fewer polyps, thus partitioning the available energy for calcification among fewer polyps [START_REF] Swain | Physiological integration of coral colonies is correlated with bleaching resistance[END_REF]. We can therefore reasonably assume that nutrients levels, and potentially the zooplankton, did not differ among study sites (Supporting Material Table S2). As a result, a single polyp would have more energetic resources available for calcification than in ambient pH conditions, as indicated by their greater skeletal growth parameters. This particular response may be possible in A. calycularis due to its colonial nature.

We hypothesize that in order to maintain the calcification rate at the colony level, colonies tend to decrease their number of polyps, but in turn, their few polyps extend their skeleton faster to reach the size of sexual maturity. No asexual division (fragmentation) has been observed over 5 years in A. calycularis in the three study sites, suggesting that this strategy of reproduction is not common.

A. calycularis exhibited at the vent site a morphological shift to encrusting and smaller colonies, with less porous, and potentially more robust corallite skeletal architecture.

While individuals from the vents were composed of corallites with higher skeletal density, this was less evident at the colony level. While skeletal integrity was not strictly quantified, observations at the vent site showed that the colonies were more fragile and lost their integrity more readily, suggesting that the section of the skeleton located between the polyps (coenosteum)

was either less calcified and/or more prone to dissolution. This could be the result of thinner or Contrasting responses between skeletal parts that either are or are not protected by living tissues has already been reported for corals [START_REF] Hennige | Hidden impacts of ocean acidification to live and dead coral framework[END_REF]Ries, 2011;[START_REF] Rodolfo-Metalpa | Coral and mollusc resistance to ocean acidification adversely affected by warming[END_REF]. This loss of coenosarc could indicate the beginning of a further shift from colonial forms towards solitary polyps to ensure survivorship, as has occurred throughout the history of scleractinian coral evolution and in laboratory conditions [START_REF] Fine | Scleractinian coral species survive and recover from decalcification[END_REF][START_REF] Kvitt | Breakdown of coral colonial form under reduced pH conditions is initiated in polyps and mediated through apoptosis[END_REF]. Interestingly, it was recently shown that following a heatwave and a bleaching event, the Mediterranean coral Cladocora caespitosa suffered from apparent mortality but its tissues actually retracted inside the individual corallites before rejuvenescence occurred a few years later [START_REF] Kersting | Living evidence of a fossil survival strategy raises hope for warming-affected corals[END_REF]. Here, we hypothesize that a similar, but less extreme phenomenon occurred with the corals reducing their coenosarc in response to low pH. A reduction in tissue thickness can have implications for calcification because the precipitation of calcium carbonate occurs in the calcifying fluid that is a medium semi-isolated from the external seawater by the overlying coral tissues. To promote calcification, corals have the ability to upregulate pH and C T (dissolved inorganic carbon) concentrations in the calcifying fluid [START_REF] Comeau | Decoupling between the response of coral calcifying fluid pH and calcification to ocean acidification[END_REF], a capacity that is reduced under ocean acidification [START_REF] Mcculloch | Coral resilience to ocean acidification and global warming through pH up-regulation[END_REF]. Here reduced calcification between the polyps was likely due to a reduction of coral ability to maintain optimal carbonate chemistry conditions in the calcifying fluid between the polyps. This could have been the result of natural spatial heterogeneity sensitivity of colonies to acidification [START_REF] Holcomb | Coral calcifying fluid pH dictates response to ocean acidification[END_REF]. In addition to reduced calcification, thinning or disappearing of the tissues likely led to local dissolution because exposed skeletons are more prone to dissolution (Ries, 2011). As a result of reduced calcification and dissolution of the coenosteum, colonies at the vent site were weaker and smaller despite heavily calcified corallites. calycularis using microsatellites also found significant genetic differentiation at km-scale distances, likely a reflection of this species limited dispersal capabilities [START_REF] Casado-Amezúa | Genetic assessment of population structure and connectivity in the threatened Mediterranean coral Astroides calycularis (Scleractinia, Dendrophylliidae) at different spatial scales[END_REF]. These data showed strong linkage among SNPs across 1000s of bp within genes and strong linkage across different genes. These patterns could be generated by selective sweeps acting on a small number of founder colonies, but because linkage among genes and SNPs occurs among ambient pH (Ambient 1 and Ambient 2) individuals as well as Vent corals. These linkages were probably not due to recent selection at the CO 2 vent site alone but reflect the underlying architecture of adaptation. A. calycularis is a warm-water coral whose distribution range is currently expanding northward [START_REF] Bianchi | Biodiversity issues for the forthcoming tropical Mediterranean Sea[END_REF], where Ischia belongs to the north-east margin of the confirmed distribution. As a result, it is also likely that selection is acting at the Ambient 1 and 2 sites compared to more southerly populations. Disentangling the ways in which selection at high CO 2 locations combines with selection at higher temperatures may be particularly important in future ocean conditions. The matrix of genetically distinct populations of A. calycularis experiencing a variety of selection regimes for heat and CO 2 may be a powerful setting for this future work.

The most highly differentiated genes in the vent site population, Grotta Mago, are annotated for calcium regulation, proton pumping, and inorganic carbon regulation. It is possible that they are differentiated in Grotta Mago for reasons other than selection on Two loci of calmodulin were highly differentiated in Grotta Mago, with linked SNPs in our data set. Calcium transporter genes are thought to be important in delivering calcium to the calicoblastic space [START_REF] Allemand | Coral calcification, cells to reefs[END_REF]. Though the precise mechanism is not known, calmodulin along with calbindin and calreticulin are hypothesized to play a role in managing calcium levels and can be sensitive to pH [START_REF] Allemand | Coral calcification, cells to reefs[END_REF]. For example, [START_REF] Kaniewska | Major cellular and physiological impacts of ocean acidification on a reef building coral[END_REF] showed 8-fold down regulation of calmodulin in a CO 2 -treated reef building coral. An increase in calcium at the site of calcification has been shown as a mean to alleviate the negative effect of low pH in some corals [START_REF] Decarlo | Coral resistance to ocean acidification linked to increased calcium at the site of calcification[END_REF]. Our data also showed excess differentiation in genes that depend on calcium concentrations for their function, such as calineurin, calnexin and the sarcoplasmic reticulum histidine-rich calcium-binding protein, which are thought to play a role in calcium homeostasis. proton pump is localized in the symbiosomes of corals that contain intracellular symbionts [START_REF] Tresguerres | Novel and potential physiological roles of vacuolar-type H + -ATPase in marine organisms[END_REF], but is also highly expressed in non-symbiotic gastroderm of symbiont-free tips of quickly calcifying corals, suggesting a role in calcification in the absence of symbionts [START_REF] Perez | Characterization of sodium potassium -ATPase and vacuolar proton -ATPase in three coral species from two different clades[END_REF]. In particular, the protein VHA (V-type proton ATPase) is differentially regulated in corals exposed to low pH, being downregulated more than four fold in experiments on the reef building coral Acropora millepora [START_REF] Kaniewska | Major cellular and physiological impacts of ocean acidification on a reef building coral[END_REF]. The population of A. calicularis in Grotta Mago showed strong differentiation at 6 SNPs across a 100 bp region of this gene.

CO 2 diffuses very quickly through cells, and is hard to localize in cell regions that need it for photosynthesis or calcification. Carbonic anhydrase catalyzes the reaction to convert CO 2 to carbonate ions that diffuse much less quickly. As a consequence, carbonic anhydrase is central to calcification in many marine species. In fact, [START_REF] Zebral | Carbonic anhydrase as a biomarker of global and local impacts: Insights from calcifying animals[END_REF] suggest use of it as a biomarker to monitor effects of acidification. In corals, carbonic anhydrase is thought to favor carbonate ion concentration in the calicoblastic space and in symbiosomes through conversion of CO 2 [START_REF] Chen | Carbonic anhydrase, coral calcification and a new model of stable isotope vital effects[END_REF][START_REF] Zoccola | Coral carbonic anhydrases: Regulation by ocean acidification[END_REF].

However, low-pH experiments on a deep water coral (Lophelia pertusa) did not find strong shifts in carbonic anhydrase activity, nor did an examination of carbonic anhydrase in polychaete worms from the Ischia CO 2 vents (Del Pasqua, Gambi, Caricato, Lionetto, & Giangrande, 2019). Experiments on reef building corals have shown mixed results [START_REF] Kurihara | Intraspecific variation in the response of the scleractinian coral Acropora digitifera to ocean acidification[END_REF]. We found strong differentiation of one carbonic anhydrase locus in Grotta Mago. An average shift in minor allele frequency from 0.14 to 0.50 in six linked SNPs may signal differential activity of this gene in some functionally important way. These SNPs appear to be downstream of the carbonic anhydrase coding region and if they play a role it may be as allele-specific regulators of expression. These allele differences may be a good tool to understand the role of carbonic anhydrase in reaction to high CO 2 .

In conclusion, our study demonstrates that the natural population of the azooxanthellate coral A.

calycularis living near the CO 2 vent system shows variable responses in terms of skeleton and growth patterns that result in a shift in phenotypic and ecological traits. We have shown that these

  local adaptation of organisms to global environmental change.

  of acclimatization and adaptation to climate variability and extremes by studying populations in naturally variable environments. Such studies are critical for predicting future biological responses to rapid global environmental change.

  carbonate chemistry associated with the CO 2 vent system and reference sites inSeptember, 2018 andJune, 2019.

  full details, see Supporting Methods). The HCl (0.1 M) titrant solution was calibrated against certified reference materials distributed by A.G. Dickson (CRM, Batches #153,

  HIERFSTAT package in R (see Statistical analyses). We used these estimates to compare the genetic distance for each SNP between the three populations [F ST (Vent-A1) + F ST (Vent-A2))/F ST (A1-A2)]. To identify potential outlier SNPs related to the CO 2 vent location, we compared the genetic distance for each SNP for the population comparisons including Grotta Mago to the genetic distance between the ambient populations A1 and A2 [F ST (Vent-A1) + F ST (Vent-A2))/F ST (A1-A2)]. SNPs with values over 2 showed an excess of genetic differentiation in the CO 2 Vent compared to the other ambient pH populations. Using the transcriptome assembly annotations, we searched for enrichment patterns in the contigs holding these candidate SNPs by using their UniProt identifiers (https://www.uniprot.org///geneontology.org/).

  , p<0.0001). Mean colony area decreased by ~ 80 -71% and mean polyp number by ~ 27 -18% at the Vent deep compared to ambient pH sites (p < 0.001) (Figure 3A). The skeletal parameters that characterize the architecture of colonies showed different patterns in relation to pH (Figure 3A-B, Figures S8-S9).

  calcification rates at colony level were homogenous in ambient pH and acidified conditions (Figure3B, Table

  [START_REF] Tambutté | Morphological plasticity of the coral skeleton under CO 2 -driven seawater acidification[END_REF] found the same response of decreasing calcification and bulk skeletal density while linear extension of skeleton remained unchanged in the tropical coral Stylophora pistillata subjected to low pH conditions in laboratory.[START_REF] Mollica | Ocean acidification affects coral growth by reducing skeletal density[END_REF] modelled the skeletal growth to changes in seawater chemistry and predicted declines in Porites skeletal density but no linear extension across global the large variability in the response of coral calcification to ocean acidification.

  the tissue covering the coenosteum) found in the colonies at the CO 2 vent.

  of corals at the CO 2 vent system Our results show strong genetic differentiation of all three A. calycularis populations (F ST averaging 0.024 -0.034), with over 5000 SNPs showing F ST >0.10. A previous genetic study of A.

  presence of high CO 2 . However, the strong shift in alleles at these loci and the linkage among those differentiated SNPs provides a strong set of hypotheses for the way selection might act to favor coral calcification at low pH.

  H+ concentrations are central to coral calcification by controlling pH of the calcifying fluid and the calicoblastic and gastrodermal cells. The V-type ATPase

FIGURE 1 .FIGURE 3 .

 13 FIGURES AND TABLES

FIGURE 4 .

 4 FIGURE 4. Relationships between age-length growth curves of A. calycularis. A) Age-length

FIGURE 5 .

 5 FIGURE 5. Population genetic structure of A. calycularis based on 46,784 single nucleotide

FIGURE 6 .

 6 FIGURE 6. Upper: SNP genotypes for 13 calcium ion related loci showing high levels of

FIGURE 7 .

 7 FIGURE 7. Schematic summary of the responses on skeletal and growth parameters to ocean

TABLE 1 .

 1 Measured and estimated seawater physiochemical parameters at the CO 2 vent site and 1039 reference areas with ambient pH for salinity (S), temperature (T), total alkalinity (A T ), dissolved 1040 inorganic carbon (C T ), pH T , pCO 2 , calcite (Ωc) and aragonite(Ωa) saturation. Values are means, ± 1041 SD with 25th and 75th percentiles. Calculated concentrations of C T , pCO 2 , Ωc and Ωa are shown.

1042 1 :

 1 Parameters measured from discrete water samples; 2: parameters measured with in situ sensors.1043For detailed SeaFET pH sensor statistics and the carbonate system parameters, see FigureS5and 1044

  Chi-square contingency tables were used to compare the size-frequency distributions among sites, as well as the frequency of encrusting and massive forms. Kolmogorov-Smirnov twosample tests were used to determine whether there were significant differences in necrosis between the CO 2 vent and ambient pH sites. These analyses were computed using lme4 v1.1.21 package implemented in R. Skeletal characteristics and growth: Relationships between biometrical and skeletal parameters were calculated using the power function model. Pearson's correlation coefficients were calculated for the relationships among biometric and skeletal parameters at both colony and polyp levels. Spearman's rank correlation coefficient was used to calculate the significance of the correlations between colony biometric and skeletal parameters and pH T .

	F o r R e v i e w O n l y effect. Supporting Methods). These analyses were carried out in the software MATLAB R2012a
	1.1 (see (MathWorks, Natick, USA). Population genetics: Population genetic analyses of SNPs, Weir and
	Supporting Methods for R package references). Carbonate system parameter figures in the vent Cockerham's pairwise F ST estimates among populations, and the heatmap of divergent SNP
	system were created with Ocean Data View software (version 5.1.2, http://odv.awi.de). Biological genotypes were conducted in the R-based program HIERFSTAT v4.22 and ComplexHeatmap
	v2.4.2, respectively.
	17

surveys: A linear mixed model was used to test for differences in % cover (logit transformation) as a function of site (fixed factor, 3 levels), depth (fixed factor, 4 levels) and quadrat as a random ANOVA was used to test % cenosarc (with arcsin transformation), mean mass, polyp number, bulk density, linear extension rate, calcification rate, and porosity of the colonies and mean length of corallites among sites. We used the non-parametric Kruskal-Wallis test for differences in means for data that did not meet the assumption for normality and equal variance. Kruskal-Wallis tests were applied to mean area, length, width, micro-density of colonies, corallite mean age, polyp linear extension rate, net calcification rate, and length of central and all corallites. These analyses were computed using IBM SPSS Statistics 12.0 (IBM Corporation). The Von Bertalanffy growth model and confidence intervals (CI) were estimated through a regression analysis by least squares procedure using raw data of corallite length and age (measured by computerized tomography) (see

  genes managing intracellular carbonate chemistry. Coral calcification depends on the delivery of CO 2 to the calicoblastic layer where it is converted to carbonate ions(Cohen & McConnaughey, In A. calycularis, a plethora of carbonate related genes showed high differentiation and high linkage in Grotta Mago, including carboxylases and transporters, but the most interesting is a carbonic anhydrase (Contig 15508, 5 of 12 SNPs with excess Fst above 10, average=10.3).

	2003).
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  variable responses at the polyp and colony level allow this coral to cope with low and variable pH in the long term by re-allocating energy investments between individual and colony growth as well

	as mineralogical characteristics. Transcriptomic data revealed strong genetic differentiation among
	local populations with several candidate loci and linkage blocks under selection. In addition, we
	found excess differentiation in the CO 2 vent population for genes central to calcification, including
	genes for calcium management (calmodulin, calcium-binding proteins), pH regulation (V-type
	proton ATPase), and inorganic carbon regulation (carbonic anhydrase). These patterns highlight
	both the CO 2 vents as well as the fringes of this species' expansion as potential drivers of
	adaptation.
	F o F o F o F o F o F o F o F o F o F o F o F o F o
	r r r r r r r r r r r r r
	R R R R R R R R R R R R R
	e e e e e e e e e e e e e
	v i v i v i v i v i v i v i v i v i v i v i v i v i
	e e e e e e e e e e e e e
	w w w w w w w w w w w w w
	O n O n O n O n O n O n O n O n O n O n O n O n O n
	l l l l l l l l l l l l l
	y y y y y y y y y y y y y
	32 44

Table S5 ,

 S5 respectively.

			Vent site (GM)		A1-PV	A2-SA
	Month/year	2m	3 m	4 m	2 m	2 m
	September 2018
	S	37.3 1 ± 0.2
		(37.2, 37.5), n=9

ACKNOWLEDGEMENTS

We thank P. Sorvino (ANS Diving, Ischia), A. Passaretti and E. Di Meglio for their field assistance.

We also thank S. Durante (Rizzoli Orthopaedic Institute of Bologna) for assistance in performing computerized tomography scans and F. Italiano (National Institute of Geophysics and Volcanology) for the gas data. N.T. thanks M. Khamla for assistance in Figure 7 andE. Ferrer for English grammar reviewing. Funding: This research was supported by the Total Foundation (High-CO 2 Seas grant, Grant No. BIO-2016-081-4), the French National Research Agency (4Oceans-MOPGA grant, ANR-17-MPGA-0001), and the ALMA IDEA (STRAMICRO grant, University of Bologna). N.T. was supported by a Maire Curie-Cofund (FP7-PEOPLE-Marie Curie Bandiera-Cofund, GA No. 600407) and by a Marie Sklodowska-Curie Global Fellowship under the European Union's Horizon 2020 research and innovation programme (H2020-MSCA-IF-2015, GA No. 702628).

DATA AVAILABILITY STATEMENT

RNASeq FASTQ files for all 41 samples sequenced in this study were deposited in the NCBI Short Read Archive (SRA) under BioProject PRJNA643775 (Accession numbers SRR12135922 -SRR12135962), https://dataview.ncbi.nlm.nih.gov/object/PRJNA643775?reviewer=5j14na61906tr4dne0bvg9kbhq.

The de novo transcriptome assembly generated in this study and used for mapping the samples has been deposited in the NCBI Transcriptome Shotgun Assembly (TSA) database at DDBJ/ENA/GenBank under accession number GIRZ01000000

(https://www.ncbi.nlm.nih.gov/nuccore/GIRZ00000000). The version described in this paper is the first version, GIRZ01000000. The bioinformatics scripts used for assembly, mapping, and SNP-calling are available on Github at DOI: 10.5281/zenodo.3934433.

AUTHOR CONTRIBUTIONS

N.T., E.C., S.P., E.S., S.G., M.C.G designed the study. N.T., E.C., S.A., S.C., F.M., A.M., M.M., S.P., E.S., L.U., C.d.V, M.C.G were involved with fieldwork. N.T., S.A., J.P.G., A.M., S.G.M., L.U., C.d.V. analyzed the environmental data; E.C., C.C., P.F., S.G. analyzed the skeletal data; E. S., S. P., and N.T analyzed the genomic data. N.T. drafted the initial manuscript and all authors contributed discussion, writing and interpretation.

COMPETING INTERESTS

The authors declare that they have no competing interests.

ETHICAL STATEMENT

All work undertaken in this study complied with current laws of Italy and United States of America for collecting and importing/exporting coral specimens. Cites permits IT/EX/2018/MCE/00170, IT/EX/2017/MCE/00214, IT/EX/2017/MCE/00325.


Eve-Isabelle Pecheur
email: eve-isabelle.pecheur@inserm.fr

Eve-Isabelle Pécheur

Circulating miRNAs as biomarkers of liver fibrosis progression in HCV/HIV-1 coinfected patients : a « miR »-velous opportunity of early diagnosis ?

Keywords: HCV-HIV-1 coinfections, miRNA, noninvasive biomarker, liver fibrosis, prediction

Circulating miRNAs as biomarkers of liver fibrosis progression in HCV/HIV-1 coinfected patients: a « miR »-velous opportunity of early diagnosis ?

The main causes of liver fibrosis are infections with the hepatitis B or C viruses (HBV and HCV, respectively), alcohol abuse, and non-alcoholic fatty liver disease. Fibrosis is a reversible exuberant wound healing and scarring process in which excessive connective tissue builds up in the organ [START_REF] Lee | Pathobiology of liver fibrosis: a translational success story[END_REF], with overly abundant deposits of collagen and elastin fibrils. This dynamic phenomenon is triggered by a chronic liver injury, which causes an imbalance between excessive production (fibrogenesis) and deficient degradation (fibrolysis) of extracellular matrix (ECM), and during which several cell types are recruited onsite to help « seal off » the injury [START_REF] Schuppan | Liver fibrosis: Direct antifibrotic agents and targeted therapies[END_REF]. Whatever the etiology, this injury is linked to impaired hepatocyte replicative capacities and hepatocyte death. It also activates hepatic progenitor cells, triggering their proliferation and differentiation [START_REF] Jung | Signals from dying hepatocytes trigger growth of liver progenitors[END_REF], which helps provide new hepatocytes and maintain the organ's functional integrity, and is accompanied by liver inflammation. In the context of virus-induced liver fibrosis, HCV coinfection with the human immunodeficiency virus 1 (HIV-1) aggravates the clinical features of the disease. Indeed, the prevalence of liver disease is 7 to 10 times greater in HIV-positive than in HIV-negative individuals, in close link with HCV coinfection [START_REF] Jeyarajan | Insights Into the Pathophysiology of Liver Disease in HCV/HIV: Does it End With HCV Cure?[END_REF]. Furthermore, even though HIV-1 does not infect hepatocytes but immune cells, it contributes to liver damages by fueling oxidative stress, activating the release of pro-fibrogenic cytokines such as TGF-, and directly stimulating the production of ECM components by hepatic stellate cells. Therefore, HCV/HIV-1 coinfected individuals are at higher risks of developing liver fibrosis and of progressing more quickly to severe liver dysfunctions.

A major pitfall in the diagnosis of liver fibrosis is that initial stages are asymptomatic, delaying the implementation of any appropriate therapy. Indeed, a successful resolution of fibrosis largely depends upon the stage and extent of scarred tissue, and treatments involve correcting the underlying condition when possible, i.e., in the case of coinfected patients, administering appropriate antiviral therapies. Aside from liver biopsies, non-invasive strategies have been developed to evaluate the presence and extent of the liver disease, such as ultrasound imaging techniques and analyses of serum biomarkers [START_REF] Martínez | Noninvasive assessment of liver fibrosis[END_REF]. These markers can be classified as indirect (a combination of routine liver biochemistry and general features) or direct (a reflection of liver ECM turnover and accumulation). Direct markers include soluble components of the ECM (proteolytic or inhibitory enzymes, collagen by-products, hyaluronic acid). Also algorithms combining direct and indirect biomarkers have been proposed [START_REF] Martínez | Noninvasive assessment of liver fibrosis[END_REF].

In an attempt to identify in HCV/HIV-1 coinfected patients serum biomarkers : (i) indicative of a liver-specific disease, and (ii) predictive of disease progression before signs of fibrosis, Martinez and coworkers focused on circulating micro-RNAs (miRNAs) (this issue, [START_REF] Franco | Circulating microRNA signatures that predict liver fibrosis progression in patients with HIV-1/HCV co-infections[END_REF]). MiRNAs are small (< 25 nucleotides) non-coding RNAs, which act as regulators of transcription by targeting messenger RNA transcripts. This results in the degradation or the inhibition of expression of these transcripts. Using miRNAs as disease biomarkers recently raised increasing interest, since these molecules are highly stable, easily detected in the circulation and some of them are organ-specific or, within a given organ, cell-type specific. In the liver, miR-122 is hepatocyte-specific, miR-192/194 are highly enriched in hepatocytes, and miR-29 is specific of hepatic stellate cells [START_REF] Wang | MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases[END_REF]. Elevated amounts in the circulation are often a bona fide reflection of reduced levels in the organ of origin, in link with organ dysfunction/failure. In their study, Martinez and coworkers used large-scale deep sequencing to detect miRNAs in the plasma of 46 HCV/HIV-1 coinfected but non fibrotic patients. These patients were followed over a 10 year-period, during which some developed liver fibrosis, others did not [START_REF] Franco | Circulating microRNA signatures that predict liver fibrosis progression in patients with HIV-1/HCV co-infections[END_REF]. First, when comparing coinfected patients with healthy individuals, a set of sixteen miRNAs was found upregulated in infected patients. Among these miRNAs, miR-122, miR-192, and miR-375 had previously been associated with liver disease [START_REF] Wang | MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases[END_REF], and miR-100, miR-99a, miR-30a, and miRNA-96-5p associated with HIV-1 infections. Second, when comparing the groups of HCV/HIV-1 coinfected patients progressing toward liver fibrosis or not, a signature of seven miRNAs was unraveled, which strongly correlated with the progression toward liver fibrosis. These miRNAs (miR-99a, miR-100, miR-122, miR-125, miR-192, miR-194 and miR-1246) detected liver fibrosis progression with high sensitivity (88%) and specificity (85%) yields. Therefore, their plasma baseline levels could serve as a robust prediction of the advancement stage of liver fibrosis, upstream to the appearance of signs of hepatic damages. This exploratory study still needs to be validated in larger patients cohorts, but it already opens bright perspectives to propose such a non-invasive signature for the prediction of liver disease progression in HIV-1 mono-infected patients, and in HCV/HIV-1 coinfected patients cured of HCV infection after treatment with direct-acting antivirals. This circulating miRNA signature, or some miRNAs therein, could also be candidates to design new clinical algorithms of prediction of liver fibrosis progression in relevant patients cohorts.

Eve-Isabelle Pécheur discloses no conflict of interest. This work was funded by the Ligue Contre le Cancer -Comité de la Loire.