Aurélie Vande Put
email: aurelie.vandeput@ensiacet.fr

Djar Oquab
email: djar.oquab@ensiacet.fr

Daniel Monceau
email: daniel.monceau@ensiacet.fr

Characterization of TBC systems with NiPtAl or NiCoCrAlYTa bond coatings after thermal cycling at 1100°C: a comparative study of failure mechanisms

Keywords: TBC, MCrAlY, NiPtAl, spallation, thermal cycling, thermogravimetry, failure mechanism, rumpling

During service, TBC can suffer degradation by CMAS, FOD, erosion or spallation. Whereas the first three are due to foreign particles, the last one is related to thermal cycling. When subjected to high temperature exposures followed by rapid coolings under oxidizing conditions, a TBC system undergoes morphological changes and stress development. This will initiate cracks which propagate and finally lead to failure by spallation. Consequently, the aim of the present study is to understand better the mechanisms responsible for such spallation events. Two kinds of TBC systems with different bond coatings (NiCoCrAlYTa or Pt-modified nickel aluminide bond coatings) are thermally cycled. Subsequently, SEM investigations on TBC systems after spallation concentrate on failure path, defect, morphological and microstructural changes to propose way for improving TBC system lifetime.

Introduction

Thermal barrier coatings are widely used in gas turbine engines. They are composed of a superalloy substrate with good mechanical properties, an Al rich bond coating (BC) which will form a protective alumina scale by reaction with oxygen and a top coat (TC) which should be strain tolerant and which is used to reduce the temperature seen by the entire system. If only thermal cycling conditions are considered, two failure categories can be distinguished for a TBC system [1]: intrinsic or extrinsic mechanisms. Intrinsic mechanisms are due to strain misfits between the different layers which compose a TBC system. They refer to rumpling and interface delamination. The other category is related to foreign particles. When these particles impact the top coat, this can be eroded or damaged (FOD). In the case where such particles would accumulate on the top coat surface and melt, top coat spallation will occur. In all cases, the top coat will be damaged and its beneficial effect on the TBC system will be lost. In the present study, the focus is on the intrinsic mechanisms which are rumpling and interface delamination. Thermal cycling tests of different TBC systems are then conducted, followed by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS).

Experimental Procedures

Materials. Two kinds of thermal barrier coating (TBC) systems were investigated. AM3 single crystal nickel-based superalloy was used as the substrate. The bond coating was either NiCoCrAlYTa or NiPtAl. The NiCoCrAlYTa bond coating was vacuum plasma sprayed (VPS).

After heat treatment, the microstructure is composed of β, γ', γ and TaC phases. The NiPtAl BC was formed by platinum deposition via an electrolytic process followed by vapour phase aluminizing (APVS). After heat treatment, the bond coating is single-phase β-NiPtAl. The top coat, composed of ZrO 2 -7wt%Y 2 O 3 , was deposited by electron beam physical vapour deposition (EB-PVD) on grit blasted (NiPtAl) or partially machined (NiCoCrAlYTa) bond coatings. Bond coatings were preoxidized 3 h at 1000°C in air under 0.5 mbar before EB-PVD. Because of process requirement, a Hastelloy X stem was welded to the AM3 disc. The entire surface of the stem was also covered by the bond coating and the top coat except its extremity which was only covered by a NiCoCrAlYTa bond coating. More details about these systems can be found in [2,3].

Thermal cycling. Before thermal cycling, specimens were oxidized 20 h at 1100°C or 900°C under flowing dry O 2 in a thermobalance in order to measure the initial oxidation rates with precision. Then cyclic oxidation was performed using two devices: a SETARAM TAG24S thermobalance and a CTGA thermobalance. Due to its symmetrical furnace arrangement, the SETARAM TAG24S offers an excellent accuracy (about 1 µg) with limited drift and buoyancy effects. In one compartment, an inert alumina reference sample is placed, in the other one the TBC system under investigation. Then, the apparatus records the mass difference between the two specimens during thermal cycling, as detailed in [4]. The second apparatus was a CTGA thermobalance which was developed and patented in the laboratory [5]. Ten halogen lamps heat 5 independent samples whose weight is measured continuously. As a consequence, the initial heating and cooling of CTGA thermobalance are faster than those of the SETARAM TAG24S thermobalance. Nevertheless, both thermal cycles consisted of a high temperature dwell of 1 h at 1100°C and a 15 min cold dwell at 200°C (more details on thermal cycles are given in [3]). Thermal cycling was realized under flowing synthetic air (0.2-1 l/h for each sample). Both kinds of TBC systems were tested in both thermobalances. TBC29 and TBC30 samples have a NiPtAl bond coating, TBC47 and TBC48 samples a NiCoCrAlYTa bond coating. Specimens cycled in the SETARAM TAG24S thermobalance, TBC29 and TBC47, were oxidized for 20 h at 900°C before cycling. Samples cycled in the CTGA thermobalance, TBC30 and TBC48, were oxidized for 20 h at 1100°C before cycling. Thermal cycling is stopped at a certain number of cycles, depending on the system, before complete spallation determined by critical mass change.

Results and Discussion

Cyclic Oxidation Kinetics. The thermobalances record net mass change. Oxidation kinetics and mass losses due to spallation can then be determined. Oxidation kinetics values were found to be characteristic of α-alumina formation [3]. Fig. 1 shows the net mass change of each TBC system during thermal cycling. For easier interpretation of the results, only the net mass change at the end of each high temperature dwell is plotted. TBC48 spallation was severe and occurred very rapidly. For the other TBC systems, although oxidation kinetics are similar, spallation is more pronounced for TBC systems with a NiCoCrAlYTa bond coating.

Systems degradation. Samples were examined by SEM after thermal cycling (Fig. 2). White areas represent the bond coating while black domains correspond to TGO. Bright regions on the top of TGO layer are top coat fragments. Whatever the bond coating and whatever the cycling duration, the bond coating, TGO and top coat are visible, as can be seen in Fig. 2a andb. The bond coating shows alumina grain imprints which are characteristic of an inward growing oxide. The main difference is that the bond coating surface, which is highly convoluted in the case of a NiPtAl bond coating does not appear rumpled for a NiCoCrAlYTa bond coating, Fig. 2c andd. O means that the thermal cycling was stopped and that the furnace was open. For both BCs, a major part of TGO layer has been lost during spallation. Nevertheless, dispersed remnant pieces of TGO are observed at the bond coating surface, Fig. 2. In the case of a Pt-modified β-NiAl bond coating, the bond coating surface is severely rumpled. Then, by propagating along the BC/TGO interface, cracks can go through alumina layer in concave areas, Fig. 3a. Such TGO remnants refer to ruptured ligaments [6]. In the case of a NiCoCrAlYTa bond coating, Y-rich oxides, Y, Ta-rich oxides and Ta,Ti-rich oxides are identified within TGO. Formation of reactive element compounds at the BC/TGO interface through which oxygen diffuses faster leads to enhanced oxidation, Fig. 3b. Therefore, TGO is thicker in these regions which are usually called pegs, assuming that they mechanically peg the interface [7].

After spallation, zirconia appears showing two different shapes according to the bond coating. For NiPtAl, only remnants of zirconia base columns are still adherent to the top of ruptured ligaments, Fig. 3c. For a NiCoCrAlYTa BC, zirconia is usually present as "corn kernel defect" [8] on the top of TGO, Fig. 2d and3d.

Cross section observations also highlight BC phase transformation, Fig. 4. NiCoCrAlYTa bond coating is composed initially of β, γ', γ phases with some TaC precipitates. After 80 cycles, it is still composed of β, γ' and γ but it presents an external layer of γ, Fig. 4a. After 456 cycles, the entire β phase disappeared and only γ and γ' are detected, Fig. 4b. For Pt-modified β-NiAl, the coating was initially single-phased β. After 480 and 1007 cycles, β, γ' and γ are present, Fig. 4c andd. In both cases, γ is localized near BC/AM3 interface, a γ' layer develops under the TGO but β fraction decreases with oxidation time. Surface undulations have been observed for MCrAlY BC without top coat only in a few studies [9,[START_REF] Peichl | Proc. Materials for Advanced Power Engineering Conf[END_REF], rumpling has often been observed in the case of NiPtAl BC with and without top coat, e.g. [START_REF] Tolpygo | [END_REF][12][13]. Different explanations have been given to explain such phenomenon. Compressive stress development within TGO is one of those and is mainly due to CTE mismatch. Besides, Pennefather and Boone [9] suggest that the mechanical strength of the coating could also play a role in rumpling whereas Evans et al. [14] assume that stresses due to TGO growth add to CTE mismatch in order to favor rumpling. Haynes et al. suggest that the CTE mismatch between the substrate and the BC has also to be taken into account [15]. But rumpling can also be related to phase transformation. Chen et al. [16] assume that the martensitic phase transformation is responsible for nickel aluminide coating rumpling. Tolpygo and Clarke claim that the β→γ' transformation, which is associated with a volume change, can also explain the rumpling mechanism [13]. Vialas et al. [17] showed that this volume change can be either an increase or a decrease, and that the β/γ' phase transformation boundary can be either parallel to the surface or localized at BC grain boundaries. In the case of the present study, the attention is focused on this last explanation. The β→γ' transformation can occur in such a way that a γ' layer forms continuously along the interface. Its penetration front gradually moves inward as the transformation continues and BC thickness remains uniform on the entire surface. If only a certain portion of β transforms locally to γ' and this is associated to a volume reduction, the BC surface can rumple or voids can form [13]. In the case of the NiCoCrAlYTa BC, consumption leads to the β→γ instead of the β→γ' transformation [18]. Given that such transformation occurs homogeneously, a continuous γ phase develops under the TGO/BC interface, Fig. 4a andb. Besides, the γ/(β+γ'+γ) plane remains parallel to the interface as the transformation goes on. The formation of a continuous γ layer with a plane γ/(β+γ) was also observed in the case of a MCrAlY BC, initially biphased β+γ [19]. Then rumpling does not occur and TGO/TBC interface remains planar, Fig. 5a. The only TGO layer deformation observed is related to the presence of corn kernel defects. These are conical defects within YSZ which are no longer bonded to the top coat. Consequently, bond coating deformation is not prevented at these specific locations [12] and TGO imperfections due to undulations develop [14]. Such BC deformation due to corn kernel defects has already been observed by Sohn et al. [19] in the case of MCrAlY bond coating TBC systems. Concerning NiPtAl BC, the rumpling of the surface is the result of the volume reduction associated with the localized transformation, Fig. 4c andd. When TGO is adherent on the BC, it follows the bond coating surface undulations and imperfections due to a convoluted TGO are then created [14]. Out of plane stresses develop and cracks nucleate around concave and convex areas, Fig. 5b. The large fraction of BC surface observed suggests that these cracks go through the TGO in order to reach and propagate at the TGO/BC interface the release of stored energy is more important [8]. Rumpling is then responsible for the failure of TBC systems with a NiPtAl BC even though this allows a decrease in the TGO elastic strain, as assumed by Tolpygo et al. [12]. Such failure path (cracks initiated at TGO/TC interface which propagate at TGO/BC interface) has already been observed by Tolpygo and Clarke on a TBC system composed of a René superalloy and a Ptmodified nickel aluminide BC after thermal cycling [13].

NiCoCrAlYTa NiPtAl (a) TBC47, 456 cycles (BSE). (b) TBC30, 480 cycles (BSE). Fig. 5: Interface between TGO/top coat for both bond coatings.

It is worth mentioning that such a failure mechanism occurs for the entire surface of TBC30 but for only 70% of the TBC29 surface (after 1007 thermal cycles). Actually, on the other 30% of the surface of TBC29, the TGO layer was still adherent to the BC and appeared much more convoluted than the BC surface without TGO. Fig. 6a is a top view of the TGO layer where it is still adherent to the BC. This strong rumpling increases TGO length and consequently Al consumption. Therefore, it is not surprising to discover γ phase just below the TGO/BC interface in convex areas, Fig. 6b. On the top of this TGO layer, the base of zirconia columns is observed but only at few places. This suggests that cracks coalesce at the TGO/TC interface but cannot propagate along the complete interface because top coat is still adherent to the TGO where the base of zirconia columns is observed. Given that top coat cannot constrain TGO undulations anymore, the TGO is free to rumple. This explains why rumpling is much more important here than in the areas where TGO is no longer adherent to the bond coating. Finally, if no large mass loss is detected during TGA measurement, it is because TGO/TC interface is still adherent at few points, preventing it from spalling.

Rumpling for TBC systems with a NiPtAl BC, and corn kernel defects for TBC systems with a NiCoCrAlYTa BC, are both responsible, in different ways, for TGO undulations. In the case of a NiCoCrAlYTa BC, another important feature with regard to the TGO microstructure and morphology is the important formation of pegs. As mentioned previously, pegs develop because of the presence of yttrium oxides which locally increase oxidation kinetics [20,[START_REF] Toscano | Materials and Corrosion[END_REF]. Like other reactive elements (Hf, Zr) [START_REF] Toscano | Materials and Corrosion[END_REF][START_REF] Mendis | [END_REF], yttrium is known to have a beneficial effect on the TGO adherence. This effect is attributed to many different mechanisms, the most important of which is the impurity gettering [START_REF] Mendis | [END_REF]. But the beneficial effect of reactive elements is highly linked to their concentration.

A study of Haynes et al. [15] on cyclic oxidation of various MCrAl BC clearly reveals that lifetime and TGO adherence are strongly dependent on reactive element concentration within the coating, as shown also by Lau et al. [23]. According to Toscano et al., the Y reservoir has to be sufficient in order to favor TGO adherence but it must not be too high in order to avoid large oxidation kinetics [20]. Two alternative explanations have been offered for the pegs effect. Nijdam et al. [24,25] consider that the pegs density has to be important to prevent crack propagation whereas in a study on the effect of Y or Hf implantation in nickel aluminide coatings, Fisher et al. conclude that spallation was due to excessive Hf-rich peg formation [26]. Besides, pegs create TGO thickness imperfections [14]. As mentioned by Yanar et al., defects in TBC systems are initiation sites and the stored energy in the TGO is the driving force for the failure propagation [8]. Consequently, a and non-uniform TGO thickness is not favorable given that the strain energy will be more important and the failure probability increased. If we refer to the statement of Yanar et al., defects and TGO thickness have to be limited to improve TBC system lifetime. In the present study, TBC systems with NiCoCrAlYTa BC present many defects: corn kernel defects which cause a localized BC deformation and many TGO imperfections. All these defects, associated with a high strain energy due to pegs, are responsible for the short TBC system lifetimes. Corn kernel defects result from initial BC surface roughness [8]. In the present case, rumpling is likely to be due to BC composition and microstructure. The formation of pegs is caused by BC surface roughness and composition. The fact that peg formation is caused by BC composition has already been explained previously. But other works have shown that the surface preparation also plays an important role on the lifetime of TBC systems. Indeed, two studies on MCrAlY, one by Naumenko et al. [27] and the other one by Gil et al. [28], show the importance of surface preparation on TGO morphology and composition. A rough surface will favor peg formation and development of a non-uniform TGO while a uniform TGO, in composition and microstructure, forms on an almost planar surface. Then, two ways to improve TBC systems durability can be undertaken. The first one is based on an accurate control of BC composition and microstructure. In the case of a nickel aluminide BC, efforts are to be made to avoid rumpling. Using a γ+γ' bond coating [29] can offer the advantages of having a CTE and a composition closer to those of the superalloy. Besides, the two-phased composition of the bond coating might minimize rumpling. In the case of MCrAlY BC, reactive elements have to be added for their beneficial effect on scale adherence without leading to excessive peg formation. The second way to improve the TBC systems lifetime is to minimize defects by controlling the surface roughness and consequently by using the appropriate surface preparation, as already shown by Yanar et al. [8]. However, BC surface must not be too smooth otherwise nothing can prevent fast crack propagation. Thus, the formation of a limited number of pegs could be a good compromise with regards to crack propagation given that pegs roughen the interface.

Conclusion

The composition of the bond coating plays a major role in determining the failure mechanism of a TBC system. In the case of a TBC system with a Pt-modified bond coating, failure is mainly caused by rumpling. For a TBC system with a NiCoCrAlYTa bond coating, the high density of defects and TGO heterogeneities are found to be responsible for top coat spallation with shorter lifetimes than the NiPtAl-base system. Such defects result from bond coating surface roughness before EB-PVD. TGO heterogeneities come from reactive element rich oxide formation. Consequently, bond coating surface preparation and also the reactive element reservoir within the bond coating have to be carefully controlled in order to increase TBC system lifetime.

Fig. 1 .

 1 Fig.1. Net Mass Change versus number of cycles. TBC29 and TBC30: NiPtAl BC, TBC47 and TBC48: NiCoCrAlYTa BC. S means that the thermal cycling was stopped. O means that the thermal cycling was stopped and that the furnace was open. For both BCs, a major part of TGO layer has been lost during spallation. Nevertheless, dispersed remnant pieces of TGO are observed at the bond coating surface, Fig.2. In the case of a Pt-modified β-NiAl bond coating, the bond coating surface is severely rumpled. Then, by propagating along the BC/TGO interface, cracks can go through alumina layer in concave areas, Fig.3a. Such TGO remnants refer to ruptured ligaments[6]. In the case of a NiCoCrAlYTa bond coating, Y-rich oxides, Y, Ta-rich oxides and Ta,Ti-rich oxides are identified within TGO. Formation of reactive element compounds at the BC/TGO interface through which oxygen diffuses faster leads to enhanced oxidation, Fig.3b. Therefore, TGO is thicker in these regions which are usually called pegs, assuming that they mechanically peg the interface[7].

  TBC30 (BSE). (b) TBC47 (BSE) (c) TBC30, tilted image (BSE).(d) TBC47, tilted image (BSE). Fig.2. Bond coating surface after thermal cycling (480 cycles for TBC30, 456 cycles for TBC47). Few parts of TGO and top coat remain on BC surface after spallation. Bond coating rumpling for TBC systems with Pt-modified β-NiAl BC (c), no rumpling for NiCoCrAlYTa BC (d).It has been seen that whatever the bond coating of TBC systems is, failure mainly occurred at TGO/BC interface. Nevertheless, many differences were noticed between the two BC: rumpling vs. unconvoluted surface, pegs vs. anchorage points, corn kernel defects vs. base of zirconia columns, localised vs. homogeneous β transformation. All of these characteristics indicate that failure results different mechanisms depending on the bond coating. TBC30 (BSE). (b) TBC47 (BSE). (c) TBC30 (BSE). (d) TBC48 (BSE). Fig. 3. a) Ruptured ligament, b) Peg. c) Base of ZrO 2 columns and d) "corn kernel defect" on TGO. NiCoCrAlYTa BC NiPtAl BC (a) TBC48, 80 cycles. (b) TBC47, 456 cycles. (c) TBC30, 480 cycles. (d) TBC29, 1007 cycles. Fig. 4. Phases composition of bond coatings after thermal cycling, (BSE).

  (a) (SE).(b) (BSE).

Fig. 6 :

 6 Fig. 6: TBC29 after 1007 cycles (NiPtAl bond coating). (a) TGO remains adherent to the bond coating in some areas and is highly convoluted. (b) This leads to the γ' → γ transformation.

Acknowlegments

Turboméca-SAFRAN (F. Crabos and A. Raffaitin) and Snecma Services-SAFRAN (A. Malié) companies are gratefully acknowledged for providing TBC samples, Turboméca and CNRS for financial support of A. Vande Put PhD.


Sophie Martin
email: sophie.martin@sb-roscoff.fr

Virgile Calvert

Anne Chenuil

PHYSIOLOGICAL DIFFERENCES AMONG CRYPTIC SPECIES OF THE MEDITERRANEAN CRUSTOSE CORALLINE ALGA LITHOPHYLLUM

Keywords: Calcification, coralline algae, cryptic species, depth, light, Lithophyllum, photo-acclimation, photosynthesis, physiology

The crustose coralline alga Lithophyllum stictiforme is an important reef builder, forming coralligenous concretions in the Mediterranean Sea. This algal species complex is composed of eight cryptic species (or clades). The objective of our study was to compare light-dependent physiological processes within and between the two most abundant cryptic species (C1 and C4) of L. stictiforme from the Bay of Marseille (France). Physiological rates of respiration, photosynthesis and calcification were measured using incubation chambers under various irradiance levels and in the dark during the summer period. We compared the physiology of the C1 and C4 cryptic species of L. stictiforme among three algal groups: C1 (the most common in the Marseille area) from 28 m depth, C4 from the same site and depth, and C1 from a deeper site at 45 m depth. We found both interspecific (between C1 and C4) and intraspecific (within C1) physiological differences. Photosynthetic parameters showed acclimation to light (or depth) with higher values of initial slope of photosynthetic light response curve (α) and lower values of saturating irradiance (Ek) and compensating irradiance (Ec) for C1 from 45 m than for C1 and C4 from 28 m depth. At the shallower depth, significant physiological differences were observed between C1 and C4 with a higher value of maximum rate of gross photosynthesis (Pg max) in C4, suggesting better ability to grow under high irradiance. Light and dark calcification rates differed only between C4 from 28 m and C1 from 45 m depth, being intermediate in C1 from 28 m depth. On a 24 h basis, diel net calcification rates were significantly higher in specimens from the shallower waters than from the deeper waters. Physiological differences suggest physiological plasticity within the regionally dominant cryptic species C1 and speciesspecific physiological traits between the cryptic species C1 and C4.

HIGHLIGHTS

 Photosynthesis and calcification rates differ among Lithophyllum stictiforme cryptic species  The regionally dominant cryptic species exhibits light-dependent physiological plasticity  Physiological differences occur between cryptic species found in similar environments

INTRODUCTION

Crustose (nongeniculate) coralline algae (CCA) are marine red algae (Rhodophyta) of the family Corallinaceae. They are characterized by a calcareous thallus formed by the extensive crystalline calcium carbonate deposits found in the walls of their vegetative cells. CCA occur worldwide from polar regions to the tropics [START_REF] Johansen | Coralline Algae, a First Synthesis[END_REF][START_REF] Steneck | The ecology of coralline algal crusts: Convergent Patterns and Adaptative Strategies[END_REF] and occupy the entire depth range inhabited by photosynthetic organisms, from the surface to depths below 200 m [START_REF] Littler | Deep-water rhodolith distribution, productivity, and growth history at sites of formation and subsequent degradation[END_REF]; some grow under intense illumination, others grow in very dim light conditions. They are key ecosystem engineers that produce or consolidate biogenic benthic habitats [START_REF] Nelson | Calcified macroalgae -critical to coastal ecosystems and vulnerable to change: a review[END_REF]. In the Mediterranean Sea, CCA are the main builders of a typical habitat: coralligenous reefs, unique calcareous formations of biogenic origin [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. The complex structure of the calcareous coralligenous concretions fosters the development of high species diversity comprised of taxonomic groups as diverse as algae, sponges, gorgonian corals, molluscs, echinoderms, bryozoans, polychaetes, tunicates, crustaceans or fishes, which live on these concretions as epiflora and epifauna, in the small holes and crevices as cryptofauna, inside the concretions as borers, and even in the sediment deposited in cavities and holes as endofauna [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. Coralligenous reefs are considered the second most important Mediterranean benthic ecosystem in terms of biodiversity, after Posidonia oceanica meadows [START_REF] Boudouresque | Marine biodiversity in the Mediterranean: status of species, populations and communities[END_REF] and provide a wide range of ecosystem services including original resources for local fisheries and beautiful seascapes for recreational scuba diving (Thierry de Ville d' Avray et al., 2019).

Mediterranean coralligenous concretions develop under low-light conditions, at depths ranging from 20 to 120 m, where irradiance is reduced to 0.05-3% of the surface irradiance [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. In these environments of limited light, the main coralligenous reef-forming CCA are species of the genus Lithophyllum, previously reported as L. stictiforme (previously known as "stictaeforme") (Areschoug) [START_REF] Hauck | Beiträge zur Kenntnis der adriatischen Algen[END_REF] and L. cabiochiae (previously known as "cabiochae") (Boudouresque & Verlaque) [START_REF] Athanasiadis | The taxonomic status of Lithophyllum stictaeforme (Rhodophyta, Corallinales) and its generic position in light of phylogenetic considerations[END_REF] [START_REF] Sartoretto | Age of settlement and accumulation rate of submarine 'coralligene' (-10 to -60 m) of the northwestern Mediterranean Sea; Relation to Holocene rise in sea level[END_REF]. The recent molecular phylogenetic assessment of this group of algae suggests that L. cabiochiae is not a taxonomically distinct species and is part of the L. stictiforme species complex (De Jode et al 2019, [START_REF] Pezzolesi | Mediterranean Lithophyllum stictiforme (Corallinales, Rhodophyta) is a genetically diverse species complex: implications for species circumscription, biogeography and conservation of coralligenous habitats[END_REF]). These common CCA species are known to play a major role in the development and biodiversity of Mediterranean coralligenous assemblages. They are the main Lithophyllum species that contribute to coralligenous concretions [START_REF] Pezzolesi | Mediterranean Lithophyllum stictiforme (Corallinales, Rhodophyta) is a genetically diverse species complex: implications for species circumscription, biogeography and conservation of coralligenous habitats[END_REF] and they favour establishment of other coralligenous reef species, with the recruitment of algae and invertebrates on their thalli [START_REF] Piazzi | Crustose coralline algae and biodiversity enhancement: The role of Lithophyllum stictiforme in structuring Mediterranean coralligenous reefs[END_REF]. The L. stictiforme species complex is the single most important reef builder that is widespread across the whole Mediterranean Basin. The complex is made up of numerous genetically isolated, but morphologically similar species, named cryptic species (De Jode et al., 2019;[START_REF] Pezzolesi | Mediterranean Lithophyllum stictiforme (Corallinales, Rhodophyta) is a genetically diverse species complex: implications for species circumscription, biogeography and conservation of coralligenous habitats[END_REF]. Not accounting for these cryptic species -a frequent phenomenon among marine species -may lead to an overestimation of the distribution range size (or niche width) when distinct cryptic species have distinct distributions (or niches, respectively) [START_REF] Chenuil | Problems and Questions Posed by Cryptic Species. A Framework to Guide Future Studies[END_REF]. Such errors, particularly when they involve keystone species, may have serious consequences for ecosystem monitoring/management. In L. stictiforme, eight cryptic species (C1 to C8) are found in sympatry in the Bay of Marseille (north-western Mediterranean Sea;De Jode et al., 2019). Spatial distributions of these cryptic species overlap, but their relative abundances are significantly differentiated according to depth and irradiance, suggesting that their niches are differentiated (De Jode et al., 2019). Moreover, irradiance is the most important environmental factor shaping coralligenous communities [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. The distribution of the cryptic CCA engineering species according to irradiance can thus potentially have a great influence on the composition of these communities (De Jode et al., 2019). Although the cryptic species of the L. stictiforme complex are adapted to low irradiances, their light-dependent ecological and physiological traits are likely to vary among species.

Before the presence of cryptic species was revealed, Martin et al. (2013a,b) had studied physiological responses to irradiance of L. stictiforme (reported as L. cabiochae in that study) collected in the Bay of Villefranche (NW Mediterranean) at 25 m depth. They showed that both photosynthesis and calcification depend strongly on irradiance in this species. Although poorly documented, physiological differentiation has already been observed among cryptic marine red macroalgal species of the families Delesseriaceae and Rhodomelaceae [START_REF] Kamiya | Cryptic diversity in the euryhaline red alga Caloglossa ogasawaraensis (Delesseriaceae, Ceramiales)[END_REF]Muanmai et al., 2015). If this physiological differentiation is a response promoting adaptation to particular environments, then cryptic species may perform in different ways in response to specific environmental factors. As previously reported for other temperate coralline algae [START_REF] Martin | Primary production, respiration and calcification of the temperate free-living coralline alga Lithothamnion corallioides[END_REF][START_REF] Qui Minet | Physiology of maerl algae: Comparison of inter-and intraspecies variations[END_REF], respiration, photosynthesis and calcification processes in L. stictiforme are strongly influenced by seasonal variations in irradiance and temperature and are the highest in summer when temperature and irradiance levels are maximal (Martin et al., 2013b). In addition to their metabolism, both survival and reproduction have also been reported to depend on temperature and irradiance in L. stictiforme (as L. stictaeforme in that study) collected at 30-35 m depth in the NW Mediterranean (Rodríguez-Prieto, 2016). As those investigations were carried out without reference to cryptic species, they thus overlooked potential differences in the responses among cryptic species to these environmental factors.

In the present study, we measured the physiological rates of L. stictiforme in the summer period, probably under optimal conditions of temperature and irradiance. In their natural environment along Marseille's coastline, the various L. stictiforme cryptic species are exposed to comparable temperature values because they generally thrive below the summer thermocline, located at a depth of between 16 and 20 m (Harmelin, 2004;Haguenauer et al., 2013). Therefore, De Jode et al. (2019) considered irradiance as the main environmental factor differentiating between cryptic species. Here, we compared light-dependent physiological processes (i.e. photosynthesis and calcification) of two of the cryptic species of the L. stictiforme complex, one, clade C1, which is the most abundant along the Marseille coastline at depths ranging from 28 to 45 m and, the other, clade C4, which is less abundant and mainly found in the northern sector of the Bay of Marseille at relatively shallow depths (De Jode et al., 2019). Because photosynthetic and calcification processes depend on the availability of light (Martin et al., 2013a,b), we assessed the physiological performances of L. stictiforme cryptic species by measuring their specific photosynthetic parameters derived from photosynthesisirradiance curves and their specific calcification rates under representative in situ irradiances.

Photosynthetic and calcification responses under various irradiance levels allowed us (i) to identify potential physiological differences between the cryptic species and/or depths and (ii) to assess whether these reflect differences in the relative abundance of cryptic species across our study area. Two sites at two distinct depths were chosen to allow interspecific comparisons at similar depths and intraspecific comparisons at different depths.

MATERIALS AND METHODS

Studied area and biological material

Specimens of the crustose coralline algae Lithophyllum stictiforme species complex were collected in coralligenous habitats of the Bay of Marseille (France), a semi-opened embayment (about 40 km by 25 km, from the Gulf of Fos to the Riou Archipelago) located in the northwestern Mediterranean Sea. Two cryptic species (or clades, C1 and C4) were chosen among the eight cryptic species found along the Marseille coastline, based on (i) their relative abundance, to obtain sufficient individuals of each species for the experiment and (ii) their field distribution, to have cryptic species coexisting in the same environment in at least one site (in syntopy) and being present in contrasting environments (different sites and depths). C1 and C4 are the most abundant cryptic species in the Bay of Marseille (De Jode et al., 2019). On the one hand, they can be found at the same site. On the other hand, they have different environmental preferences: C1 is rare (~1% of sampled C1) in the shallowest waters (15-20 m depth) along the French Mediterranean coastline, but well represented in the deepest waters at depths ranging from 30 to 45 m (~80% at 28-31 m and ~20% at 37-46 m, Table S1). Conversely, C4 displays preference for the shallowest waters (~30% at 15-20 m and ~65% at 28-31 m), but is rare (~5%) below 30 m depth (Table S1). Individuals of the C1 and C4 cryptic species were sampled by scuba divers in two sites of the Bay of Marseille at distinct depths: (i) "La Couronne" (LC) in the northern sector of the Bay of Marseille at 28 m (43°19.668'N, 5°1.775'E) on 9 July 2018 and (ii) "Riou-Moyade" (RM) in the Riou archipelago in the southern sector of the Bay of Marseille at 45 m (43°10.574'N, 5°22.208'E) on 11 July 2018 (Fig. 1). According to De Jode et al. (2019), two cryptic species (C1 and C4) have been found at LC, while only one cryptic species (C1) was found in deep waters at RM. Light and depth were homogeneous for all collected specimens at each sampling site, even at LC, where C1 and C4 co-occur in similar light conditions. The slope of the substrate was similarly inclined at LC and RM, while light exposure of the substrate was to the south-west at LC and east at RM (De Jode, 2018). Photosynthetically available radiation (PAR; μmol photons m -2 s -1 ) at 28 and 45 m in the Bay of Marseille was calculated from surface PAR and vertical PAR attenuation coefficient (KPAR) for a typical month of July. KPAR was calculated according to [START_REF] Kirk | Light and Photosynthesis in Aquatic Ecosystems[END_REF] from vertical irradiance profiles obtained in the Bay of Marseille at the SOMLIT Frioul Station on four separate dates (11 and25 July 2018, and11 and23 July 2019). The mean KPAR were 0.094 and 0.085 m -1 at 28 m and 45 m depth, respectively. They were used in combination with the incident PAR at the surface to determine the daily cycle of irradiance at 28 and 45 m depth (Fig. 2). The incident PAR at the surface was measured on July 2007 using a flat quantum sensor (LI-COR, LI-192SA) at Saint-Jean-Cap-Ferrat located at similar latitude (43,7°), 150 km east of Marseille on the French Mediterranean coast (Fig. 2). In the Bay of Marseille in July, the mean maximal PAR at noon was 135 μmol photons m -2 s -1 at 28 m depth and 40 μmol photons m -2 s -1 at 45 m depth and the mean daily values were 70 and 20 μmol photons m -2 s -1 , respectively. Algae were transported to the Mediterranean Institute of Biodiversity and Ecology (IMBE) in seawater in a thermostated tank to maintain samples at their in situ temperature (c. 16°C). Algae were thoroughly cleaned of epiphytic organisms without causing any damage to the thalli. LC samples were divided in two groups of algae according to their morphology. The first group of algae from LC was characterized by a thinner and slightly more purple and rougher thallus. It was identified as clade C4 by genetic analysis (see below). The second group of algae from LC was characterized by a thicker and slightly pinker and smoother thallus; it was then identified as clade C1 (Fig. 3). RM samples were all morphologically similar with a pink, smooth thin thallus. Their identification as clade C1 at this location by De Jode et al.

(2019) was confirmed by genetic analysis. Differences in thickness, measured as surface-tovolume (S/V) and surface-to-weight (S/W) ratios were significant among algal groups (p ≤ 0.002, Table 1, Fig. S1). Both ratios were the lowest in the clade C1 from LC (28 m depth) and significantly (50%) higher in the clade C1 from RM (45 m depth). The S/V and S/W ratios of clade C4 (LC) were intermediate and did not differ significantly from C1 from LC and RM.

From each of the three groups of algae (LC-C4, LC-C1 and RM-C1), 12 specimens with a flat thallus of c. 20 cm 2 were selected for the experiments. At the end of the experiments, the algal volume was measured by displacement of water, considering that the algal volume submerged equals the volume of seawater displaced. It was determined on a balance to the nearest 0.01 g and converted to ml using the density of seawater. The algal fresh weight was determined to the nearest 0.01 g after drying with an absorbent paper to remove seawater. The algal surface area was determined from photographs using the software Image J Version 1.8.0.

Genetic analyses

To identify the cryptic species for each sample, a piece of thallus was cut from each individual

Experimental setup

Algae were maintained in aquaria continuously supplied with natural Mediterranean seawater pumped at 5 m depth in the Bay of Marseille to prevent any change in seawater composition.

Temperature in the aquaria was adjusted to 16.0°C and controlled in the aquaria to within ± 0.1°C using a temperature controller (Model Teco TK 150). Salinity was measured with a conductivity probe (Horiba U-5000) and remained constant (37.9) during the experiment. The pH on the total scale (pHT) and total alkalinity (AT) were measured according to the methods described in [START_REF] Qui Minet | Physiology of maerl algae: Comparison of inter-and intraspecies variations[END_REF]. Temperature, salinity and parameters of the seawater carbonate system (pHT-16°C = 8.11 ± 0.02, corresponding to pHT-25°C = 7.98 ± 0.02, and AT = 2560 ± 2 µEq kg -1 , n = 8) in the aquaria were in the range of those measured at the SOMLIT

Physiological measurements

Physiological measurements were performed after a one-day acclimation period under laboratory conditions. Algal physiology (photosynthesis, respiration and calcification) was assessed through two independent sets of light and dark incubation measurements.

Physiological rates were measured in 100 or 185 ml transparent acrylic respirometry chambers (Engineering & Design Plastics Ltd, Cambridge, UK) filled with filtered seawater and continuously stirred with a magnetic stirring bar. The chambers were placed in a thermostated bath at 16.0°C, above which was installed similar light sources (Blue and Cool White LEDs) as above the aquaria. The intensity of photosynthetically active radiation (PAR) was adjusted using an Apogee SQ-120 quantum sensor and a computer-controlled system (Alpheus Radiometrix EVO).

Control incubations without algae were performed to check for biological activity in seawater. No significant activity (no difference from 0; t-test, p > 0,05) was detected in the seawater, whatever the irradiance levels.

Two sets of complementary measurements were performed in each group of algae: (i) a first set of incubations to determine photosynthetic parameters derived from the response curves of photosynthesis to irradiance (P-E) under a range of irradiance levels from 0 to 200 µmol photons m -2 s -1 and (ii) a second set of incubations to determine calcification rates in the dark and under representative in situ irradiances at c. 30 m and 45 m depth (i.e. 100 and 50 µmol photons m -2 s -1 , respectively).

(i)

Photosynthetic parameters

The 12 selected specimens of each group of algae (LC-C4, LC-C1 and RM-C1) were incubated individually through independent dark and light incubations in 185 and 100 ml chambers, respectively. Respiration rates (Rd) were measured in the dark for 30 minutes after 1-hour exposure to the dark. Net primary production rates (Pn) were measured for 10 minutes at each irradiance level provides by the controlled LED-based light source in increasing orders (5, 10, 25, 50, 100, 150, and 200 µmol photons m -2 s -1 ). Oxygen concentrations were measured at the beginning and at the end of each irradiance level using a non-invasive optical fibre system (FIBOX 3, PreSens, Regensburg, Germany). Reactive oxygen spots in the chambers were calibrated with 0% and 100% oxygen buffers.

As recommended by [START_REF] Martin | Response of Mediterranean coralline algae to ocean acidification and elevated temperature[END_REF] and Martin et al. (2013a,b) for species of the L. stictiforme complex (as L. cabiochae), physiological rates were normalized to thallus surface area since photosynthetic tissues are present in the living upper thallus layer of the crusts. Indeed, CCA metabolic rates are usually expressed per unit of crust surface to avoid any artefact due to the thickness of skeletal carbonate below the living coralline layer [START_REF] Chisholm | Primary productivity of reef-building crustose coralline algae[END_REF]. This enables comparison of the physiological rates between algal groups of different surface-to-volume (or weight) ratios.

The rates of net photosynthesis (Pn) and dark respiration (consumption of O2, Rd) (in µmol O2 cm -2 thallus h -1 ) were calculated following Eq. (1):

Pn (or -Rd) = (ΔO2 × V) / (Δt × S) ( 1
)
where ΔO2 is the difference between the initial and final oxygen concentrations (µmol O2 l -1 ), V is the volume of seawater in the chamber (the volume of the chamber minus the volume of the alga, l), Δt is the incubation time (h) and S is the thallus planar surface area (cm 2 ).

The response of the photosynthetic oxygen-evolving rate to irradiance (E, μmol photons m -2 s -1 ) was fitted with the model of [START_REF] Chalker | Simulating light-saturation curves for photosynthesis and calcification by reef-building corals[END_REF]. Photosynthesis versus irradiance (Pn-E) curve parameters were obtained for each specimen according to Eq. ( 2):

Pn = Pg max × (1-Ek E - e ) -Rd (2)
where Pg max is the maximum rate of gross photosynthesis (µmol O2 cm -2 thallus h -1 ),

Ek (µmol photons m -2 s -1 ) is the saturating irradiance (irradiance at which the initial slope intercepts the horizontal asymptote, µmol photons m -2 s -1 ). The initial slope of the Pn-E curve (α) was calculated as α = Pg max / Ek.

The compensation irradiance (Ec, µmol photons m -2 s -1 ) is the irradiance at which Pn = 0 (or Pg = Rd).

(ii) Net calcification rates

The rates of net calcification (gross calcification -dissolution) were measured through longer incubation times (from 1 to 2 h, depending on the chamber volume) necessary to obtain a significant signal to quantify the amount of CaCO3 precipitated, based on measurements previously performed in this species at 16.0°C by Martin et al. (2013a). The first 6 of the 12 specimens of each group of algae (LC-C4, LC-C1 and RM-C1) were incubated individually in independent incubations in the dark and in the light at two irradiance levels of 50 and 100 µmol photons m -2 s -1 . Net calcification rates in the dark (Gd) were measured for 2 h in 185 mL chambers after 1-h exposure to the dark. Net calcification rates in the light (G) were measured at 50 µmol photons m -2 s -1 for 1 h in 100 mL chambers and at 100 µmol photons m -2 s -1 for 1.5-2 h in 185 mL chambers. G and Gd were estimated using the alkalinity anomaly technique [START_REF] Smith | Carbon dioxide and metabolism in marine environments[END_REF], which is a good estimator for short-term incubations. It is based on a decrease of 2 moles total alkalinity (AT) by two equivalents per molecule of CaCO3 precipitated [START_REF] Wolf-Gladrow | Total alkalinity: The explicit conservative expression and its application to biogeochemical processes[END_REF]. Seawater was sampled at the beginning and at the end of incubation, and AT analyses were performed as previously described.

Net calcification rates (G or Gd) were calculated as Eq. ( 3):

G (or Gd) = (ΔAT × V) / (2 × Δt × S) ( 3
)
where ΔAT is the difference between initial and final AT (μEq l -1 ), V is the volume of the chamber (l), Δt is the incubation time (h) and S is the thallus surface of the algae (cm 2 ).

Diel carbon and carbonate production

Diel (24 h) Pn (or G) was calculated by summing daily Pn (or G) during the daylight period and Rd (or Gd) during the night-time period. Daily Pn was calculated by integrating the Pn versus E curves against the incident irradiance at 28 m (LC) or 45 m (RM) depth in July in the Bay of Marseille during the daylight period (Fig. 1). The rates of Pn and Rd in µmol O2 cm -2 thallus h - 1 were converted to µmol C cm -2 thallus h -1 using the photosynthetic (PQ = Pn O2 / Pn CO2 = 1) and respiratory (RQ = Rd CO2 / Rd O2 = 1) quotients previously reported for species of the L.

stictiforme complex (as L. cabiochae) (Martin et al., 2013a). Daily G was estimated from linear relationship between 0 and mean maximal in situ incident irradiance (i.e. 100 µmol photons m - 2 s -1 at LC and 50 µmol photons m -2 s -1 at RM) since the rate of calcification increases linearly with light until saturating around maximal in situ light intensities (Martin et al., 2013a,b).

Statistical analyses

Results are expressed as mean ± standard error of the mean (SE).

Differences in morphological and physiological parameters between algal groups were tested by using one-way ANOVA when normal distribution (Shapiro-Wilk test) and equality of variance (Levene's test) were verified. When necessary, data were log transformed to meet ANOVA assumptions. Otherwise, Kruskal-Wallis tests were performed. ANOVAs were followed by Tukey's honestly significant difference (HSD) post hoc tests and Kruskal-Wallis tests were followed by Mann-Whitney U-tests to separate sets of homogeneous data.

RESULTS

Genetic analyses

Nucleotide sequences confirmed that all specimens belonged to the clades C1 or C4 and that the three algal groups morphologically identified a priori were homogeneous (containing only C1 or only C4 specimens) so that our sample sizes for physiological analyses were balanced accordingly.

Photosynthetic parameters

Dark respiration (Rd) did not differ significantly among the three algal groups and averaged 0.11 ± 0.01 µmol O2 cm -2 thallus h -1 in terms of O2 consumption (Table 1). The response of net photosynthesis in terms of O2 production to irradiance (Pn-E curves) is presented in Fig. 4 for each algal group. Maximal gross photosynthesis (Pg max) differed significantly between algal groups (Table 1). It was minimal in clade C1 from LC and RM, averaging 0.64 ± 0.03 µmol O2 cm -2 thallus h -1 and significantly higher in clade C4 from LC, reaching 0.93 ± 0.08 µmol O2 cm -2 thallus h -1 . The mean ratio of Pg max / Rd was 6 ± 1 both in clade C1 from LC and RM. It was 2-fold higher (12 ± 3) in clade C4 from LC (Table 1). The saturating irradiance (Ek) and the compensation irradiance (Ec) were significantly lower in RM (45 m depth) samples than in LC (28 m depth) algal groups. Mean Ek was 25 ± 2 µmol photons m -2 s -1 in specimens from RM and was 3-to 4-fold higher in specimens from LC. It did not differ between clades C1 and C4 from LC, in which Ek averaged 88 ± 11 µmol photons m -2 s -1 . Mean Ec was 2-fold lower in specimens from RM (5 ± 1 µmol photons m -2 s -1 ) than those from LC (12 ± 2 µmol photons m -

Net calcification rates

Net calcification rates in the dark (Gd) significantly differed among algal groups. Gd were lowest in clade C4 from LC, intermediate in clade C1 from LC and highest in clade C1 from RM, averaging -0.004 ± 0.008, 0.014 ± 0.006 and 0.022 ± 0.003 µmol CaCO3 cm -2 thallus h -1 , respectively (Fig. 5, Table 1). Net calcification rates in the light (G) followed the opposite trends as Gd with the lowest rates in clade C1 from RM, intermediate in clade C1 from LC and highest in clade C4 from LC, averaging 0.11 ± 0.01, 0.14 ± 0.01 and 0.17± 0.01 µmol CaCO3 cm -2 thallus h -1 , respectively, under 50 µmol photons m -2 s -1 and 0.13 ± 0.02, 0.27 ± 0.06 and 0.36 ± 0.08 µmol CaCO3 cm -2 thallus h -1 , respectively, under 100 µmol photons m -2 s -1 (Figs 6- 7, Table 1).

Diel carbon and carbonate production

Diel (24 h) net organic C production (Diel Pn) was estimated to be highest in clade C4 from LC, intermediate in clade C1 from LC and lowest in clade C1 from RM (Table 1). Diel Pn was 2-fold higher in clade C4 from LC (4.6 ± 0.6 µmol C cm -2 thallus d -1 ) than in clade C1 from RM (2.3 ± 0.3 µmol C cm -2 thallus d -1 ). Diel net inorganic C production (Diel G) did not differ significantly between clades C1 and C4 from LC and averaged 3.7 ± 0.5 µmol CaCO3 cm -2 thallus d -1 . Diel G was estimated to be 3-fold lower in specimens from RM than those from LC (Table 1).

DISCUSSION

In the present study, we identified differences in physiological performance between the two cryptic species C1 and C4 from the same depth and light environment, and within the dominant and ubiquitous cryptic species C1 from different depths and light environments. Light is indeed one of the most crucial factors that drive the distribution and physiological activities of macroalgae (Gattuso et al., 2006). Red coralline algae are the deepest known marine benthic macroalgae. They are found down to depths of more than 200 m, where irradiance levels are less than 0.001% of the surface irradiance (ca. 0.01 μmol photons m -2 s -1 ) [START_REF] Littler | Deepest known plant life discovered on an uncharted seamount[END_REF].

Their ability to acclimate/adapt to low light conditions is attributed to diverse physiological mechanisms. For example, algae living in deep habitats have low respiratory activity, a strategy to avoid excessive carbon losses [START_REF] Littler | Deep-water plant communities from an uncharted seamount off San Salvador Island, Bahamas: distribution, abundance, and primary productivity[END_REF]Gomez et al., 1997). Here, dark respiration rates (Rd) measured in L. stictiforme (0.10-0.13 µmol O2 cm -2 thallus h -1 ) collected at 28-45 m depths in the Bay of Marseille were in the low range of those reported for temperate CCA (see Martin et al., 2013a for a review) and consistent with Rd values reported for species of this complex (as L. cabiochae) collected at 25 m depth in the Bay of Villefranche (NW Mediterranean Sea) and measured in similar temperature conditions (0.1-0.2 µmol O2 cm -2 thallus h -1 ; Martin et al., 2013a,b).

Photosynthetic parameters derived from photosynthesis-irradiance curves indicate how well the three algal groups are adapted to their light environment. As a sublittoral species growing in deep water (28-45 m depth), L. stictiforme is typically adapted to chronic low light conditions, as indicated by low values of compensation irradiance (Ec). In the three groups of algae, the energy balance between gross photosynthesis (supply of energy) and respiration (energy used for maintenance and growth) was positive at very low irradiance levels (Ec < 15 µmol photons m -2 s -1 ). The photosynthetic parameters Ec, Ek and α of L. stictiforme were strongly related to water depth and concomitant changes in light availability. The compensation irradiance Ec averaged 12 µmol photons m -2 s -1 in C1 and C4 specimens from 28 m depth, whereas an irradiance of 5 µmol photons m -2 s -1 was sufficient for photosynthesis to exceed respiration in C1 specimens growing at 45 m depth. Similarly, the saturating irradiance Ek was only 25 µmol photons m -2 s -1 in deeper specimens, being 3.5-fold lower than in shallower specimens. The initial slope α was 2-fold higher in specimens from 45 m depth than in those from 28 m depth. Here, we demonstrated changes in Ek, Ec and α as an acclimation to ambient light within one cryptic species (C1). Photo-acclimation within species has already been observed in soft red macroalgae (Gomez et al., 1997;[START_REF] Marquardt | Light acclimation strategies of three commercially important red algal species[END_REF] and CCA [START_REF] Payri | Photoacclimation in the tropical coralline alga Hydrolithon onkodes (Rhodophyta, Corallinacea) from a French Polynesian reef[END_REF] from different light environments. This ability for photo-acclimation allows the L.

stictiforme cryptic species C1 to grow in a wide range of light environments, as confirmed by its broad vertical distribution, at least from 20 to 46 m depth, and its ubiquitous distribution in the region of Marseille, where it is dominant in all sites sampled (De Jode et al., 2019). Its ability to acclimate to various light niches may thus contribute to its large spatial distribution in this region.

Plasticity could not be studied in the cryptic species C4, which is very rare in the deepest sites along the French Mediterranean coasts (De Jode et al., 2019). The C4 species appeared less adapted to low light environments than C1, as indicated by its high light requirements to saturate photosynthesis (Ek: 105 µmol photons m -2 s -1 ). In shallower waters of the Bay of Marseille (LC), where irradiance can reach up to 135 photons m -2 s -1 at noon in July, the cryptic species C4 appeared particularly efficient, as shown by the 30% higher rate of maximum gross photosynthesis (Pg max) and the two-fold higher ratio of Pg max/Rd relative to C1. Such increased photosynthetic performance under higher irradiance levels could give the C4 cryptic species a competitive advantage in situations with higher incident irradiance, such as in summer, when algae are exposed to irradiance levels of more than 100 photons m -2 s -1 for several hours during daytime at 28 m depth in the Bay of Marseille. Accordingly, on a 24 h basis, we estimated diel net primary production to be approximately 30% higher in C4 than in C1 in shallow waters (LC). The C4 cryptic species is the second most abundant species after C1 in shallower waters in the northern area of the Bay of Marseille (De Jode et al., 2019). Unsurprisingly, this species is relatively abundant at depths of less than 30 m (> 90% of all C4 individuals sampled along the French Mediterranean coastline), but rare at depths below 37 m, where ~20% of all C1 individuals were sampled (Table S1). In contrast, C1 is nearly absent (~1%) in the relatively shallow waters, from 15 to 20 m depth, where 30% of C4 were sampled along the French Mediterranean coastline.

We also observed differences among the three algal groups in their ability to calcify in response to light. The C4 cryptic species appeared well adapted to calcifying under in situ irradiance levels of 50-100 µmol photons m -2 s -1 , with the highest rates of calcification found in the L. stictiforme species complex, 20-30% higher than C1 collected in a similar light environment, and 2-to 3-fold higher than C1 sampled at 45 m depth. Conversely, C4 was the least efficient species for dark calcification, with negative rates of net calcification (dissolution) in the dark, where the C1 cryptic species from both depths exhibited positive rates of net dark calcification. Differences in calcification rates can be explained by species-specific differences in the ability of coralline algae to physiologically control the calcification process [START_REF] Cornwall | Coralline algae elevate pH at the site of calcification under ocean acidification[END_REF]. In particular, recent observations support a species-specific control of calcification traits in Mediterranean Lithophyllum cryptic species. Differences in cell-wall skeletal ultrastructure have recently been demonstrated in two morphologically similar, but genetically distinct sympatric species, L. racemus and L. pseudoracemus, even though they grow in similar conditions and depths [START_REF] Basso | Calcification traits for cryptic species identification: Insights into coralline biomineralization[END_REF]. In the light, strong correlations between calcification and photosynthetic processes have been reported in coralline algae [START_REF] Pentecost | Calcification and photosynthesis in Corallina officinalis L. using 14 CO2 method[END_REF][START_REF] Borowitzka | Calcification in algae: Mechanisms and the role of metabolism[END_REF][START_REF] Chisholm | Calcification by crustose coralline algae on the northern Great Barrier[END_REF][START_REF] Martin | Primary production, respiration and calcification of the temperate free-living coralline alga Lithothamnion corallioides[END_REF][START_REF] Martin | Community metabolism in temperate maerl beds. I. Carbon and carbonate fluxes[END_REF]Martin et al. , 2013a,b;,b;[START_REF] Williamson | The regulation of coralline algal physiology, an in situ study of Corallina officinalis (Corallinales, Rhodophyta)[END_REF], and in particular in species of the L. stictiforme complex (Martin et al., 2013a,b).

Photosynthesis can effectively influence calcification by providing energy and substrates [START_REF] Chisholm | Primary productivity of reef-building crustose coralline algae[END_REF] or favourable conditions for CaCO3 precipitation by locally raising the pH at the calcification sites and in the boundary layer [START_REF] Koch | Climate change and ocean acidification effects on seagrasses and marine macroalgae[END_REF][START_REF] Hofmann | Biotic control of surface pH and evidence of light-induced H + pumping and Ca 2+ -H + exchange in a tropical crustose coralline alga[END_REF][START_REF] Cornwall | Coralline algae elevate pH at the site of calcification under ocean acidification[END_REF]. Indeed, light calcification responses among our three L. stictiforme algal groups exhibited patterns similar to those of photosynthesis (Pg max), with maximal rates observed in C4, minimal rates in C1 from 45 m depth and intermediate rates in C1 from 28 m depth. The highest calcification rates in C4 appear to confirm the competitive advantage of this cryptic species in high light conditions. Accordingly, C4 is dominant over C1 in the relatively shallow waters, from 15 to 20 m depth, along the French Mediterranean coastline (Table S1).

At 28 m depth, C4 is less abundant than C1 in spite of notable light availability and could thus be restricted by other factors.

We demonstrated here that L. stictiforme calcification rates increase with increasing light intensity and with a concomitant increase in photosynthetic or light-triggered processes.

However, the calcification process is not totally light-dependent because calcification can also occur in darkness. The continuation of calcification in the dark apparently relies on accumulated energy stored during periods of irradiance [START_REF] Mccoy | Coralline algae (Rhodophyta) in a changing world: integrating ecological, physiological, and geochemical responses to global change[END_REF]. We have previously shown that calcification continues to occur in the dark in species of the L. stictiforme complex (as L. cabiochae) both during day and night (Martin et al., 2013b). Here, net calcification rates in the dark were reduced, but remained positive in C1, while they became net negative (net CaCO3 dissolution) in C4. The deeper specimens of C1 carried out calcification the most efficiently in the dark, with the highest rates of dark calcification. They were able to maintain 20% of their calcification rates in the dark, whereas specimens from 28 m depth had calcification rates that were more than 10-fold lower in the dark, compared with in the light.

The better performance of the deeper specimens (45 m depth) for dark calcification confirms that they have very low light requirements for growth and are better acclimated to grow in deeper waters. Conversely, the shallower-depth C4 cryptic species was not able to maintain positive net calcification in the dark in comparison to C1. However, on a 24 h basis, diel net carbonate production did not significantly differ between C1 and C4 from shallow waters, being 3-to 4-fold higher than in specimens from 45 m depth. The high rate of diel net calcification estimated for C4 is explained by its ability to compensate for night-time dissolution by high calcification rates during daylight. This can occur in summer under favourable conditions of light intensity and photoperiod length. However, C4 may be disadvantaged at the other seasons, when exposed to lower light levels and shorter photoperiod.

Despite physiological acclimation to low light conditions, specimens from deeper waters showed reduced metabolic activities and significantly lower diel (24 h) net carbonate production than those found in shallower waters. L. stictiforme thus appears to grow faster at 28 m depth (LC) than at 45 m depth (RM). Associated differences in morphological traits observed between C1 from different depths likely result from acclimation to depth, with reduced thallus thickness (higher surface-to-volume or -weight ratio) in the deeper specimens.

Thallus thickness in coralline algae can reflect their growth strategies, i.e. vertical versus horizontal growth. The capacity of fast horizontal (marginal) growth of thin thalli, such as those found at 45 m depth, may thus be an advantage in deep water where light is reduced (Hanelt & Figueroa, 2012).

In conclusion, our results revealed physiological plasticity within the L. stictiforme cryptic species C1, which may contribute to its large spatial distribution from shallow to deep habitats. Physiological differences between cryptic species are compatible with the observed depth preferences of each species. C4 indeed displays a preference for relatively shallow waters, where it may hold an advantage over C1 through its better photosynthetic performance under high irradiance conditions (higher rate of maximum gross photosynthesis). Conversely, C4 is rare in deeper environments where it may be disadvantaged due to lower calcification rates where light is less available (lower ability to compensate for night-time dissolution).

To increase our knowledge on acclimation and differential adaptations in cryptic L.

stictiforme species, future research should focus on physiological performances on an annual basis, considering seasonal changes in daylength, temperature and irradiance levels. The physiological activities under optimal summer conditions of temperature and irradiance need to be high enough to promote growth, reproduction and energy storage to cope with the reduced temperature and light availability during less favourable seasons (Hanelt & Figueroa, 2012).

Cryptic L. stictiformee species may also differ in terms of their competitive ability or resistance to environmental stresses, which can also affect their distribution. Identifying cryptic speciesspecific physiological and ecological traits can help determine species' susceptibility to local and global environmental stresses [START_REF] Kato | Negative effects of ocean acidification on two crustose coralline species using genetically homogeneous samples[END_REF]. Studies on the specific cryptic species responses to environmental changes of L. stictiforme are thus needed for a better prediction of future changes in coralligenous habitats and Mediterranean coastal ecosystems.

Table 1

Comparison of morphological and physiological characteristics between the three algal groups (LC-C4, LC-C1 and RM-C1) of the species complex Lithophyllum stictiforme. Results are expressed as mean ± SE (n = 12 for morphological characteristics, P n -E curves parameters and diel P n , and n =6 for net calcification rates and diel G). G d are rates of net calcification (in µmol CaCO 3 cm -2 thallus h -1 ) measured in the dark (E = 0 µmol photons m -2 s -1 ) and G are rates of net calcification measured in the light under 50 or 100 µmol photons m -2 s -1 . P g max (µmol O 2 cm -2 thallus h -1 ) is the maximum rate of gross photosynthesis, E k (µmol photons m -2 s -1 ) is the saturating irradiance, E c (µmol photons m -2 s -1 ) is the compensation irradiance and α is the initial slope of the P n -E curve. Diel P n and diel G are 24-h net photosynthesis and calcification rates (in µmol C or CaCO 3 cm -2 thallus d -1 ).

The p-values from one-way analyses of variance (ANOVAs) (df = 2,33 for morphological characteristics, P n -E curves parameters and diel P n and df = 2,15 for net calcification rates and diel G) are shown at right. Bold type indicates significance (p < 0.05). Different subscripts (a and b) indicate significant difference between algal groups (p < 0.05, Tukey HSD post hoc tests); ns, non-significant difference (p > 0.05).

Transformed data are indicated: ¥ log (x). Since E k data did not meet the assumptions of equality of variance (Levene test), differences between groups were assessed by Kruskal-Wallis ANOVA H-tests followed by Mann-Whitney U-tests to separate sets of homogeneous data.

Fig. 1 .

 1 Fig. 1. Map of the study area, showing the locations of the two sampling sites in the Bay of Marseille: La Couronne (LC) at 28 m depth and Riou-Moyade (RM) at 45 m depth. SOMLIT: Frioul station (c. 65 m depth) of the French national network of coastal observation CNRS-INSU SOMLIT (Service d'Observation en Milieu Littoral).

Fig. 2 .

 2 Fig. 2. Daily mean changes in incident photosynthetically available radiation (PAR) at the surface (left axis) and at 28 and 45 m depth (right axis) in July in the Bay of Marseille.

Fig. 3 .

 3 Fig. 3. Morphology of the Lithophyllum stictiforme cryptic species C1 and C4 from "La Couronne" (LC) at 28 m depth and "Riou-Moyade" (RM) at 45 m depth. Scale = 5 cm. (Photo © S. Martin).

  and frozen at -20°C until DNA extraction after physiological experiments were achieved. DNA extraction, PCR (of partial 28S ribosomal DNA) and Sanger sequencing were performed according to De Jode et al. (2019).

(

  photons m -2 s -1 with an Apogee SQ-120 quantum sensor under a controlled photoperiod of 12 h. The light was provided by a combination of Blue (450 nm) and Cool White (6500 K) LEDs (Alpheus Radiometrix EVO) above the aquaria.

Fig. 4 .

 4 Fig. 4. Net photosynthesis versus irradiance curves in the three algal groups of the Lithophyllum stictiforme species complex: clade C4 from LC (LC-C4), clade C1 from LC (LC-C1) and clade C1 from RM (RM-C1). LC = 28 m depth and RM = 45 m depth.

Figs

  Figs 5-7. Net calcification in the dark (Gd) and light (G) under irradiances (E) of 50 and 100 µmol photons m -2 s -1 in the three algal groups (LC-C4, LC-C1 and RM-C1) of the Lithophyllum stictiforme species complex.Fig. 5. Gd.Fig. 6. G under 50 µmol photons m -2 s -1 . Fig. 7. G under 100 µmol photons m -2 s -1 . Each box plot has mean ("+" red cross in the boxplot), median (solid bar in the box plot), 25 th -75 th percentile (rectangular box) and 1.5*interquartile range (non-outlier range of the box whiskers).

Fig. S2 .

 S2 Fig. S2. Photosynthetic parameters of the P n -E curves in the three algal groups (LC-C4, LC-C1, and RM-C1) of the species complex Lithophyllum stictiforme: (a) respiration rate (Rd), (b) maximal rate of gross photosynthesis (Pg max), (c) saturating irradiance (Ek) and (d) initial slope of the photosynthesis-irradiance curve (α). LC = 28-m depth and RM = 45-m depth.

s -1 ). The initial slope of the Pn-E curve (α) was 2-fold higher in specimens from RM than those from LC (Table1).

ACKNOWLEDGEMENTS

We thank Dorian Guillemain from the diving service of the Observatory of Sciences of the Universe (OSU) Pytheas Institute and Bruno Belloni, Pascal Mirleau, and Sandrine Ruitton for their help in sample collection by scuba diving. We also thank Sacha Molinari for technical help during lyophilization and Térence Legrand for providing a map image file he has previously built. Data used in this work were partly produced through the technical facilities of the Molecular laboratory facility (SCBM) and the Experimental Aquaria facility (SCDE) of the IMBE (Marseille). This work was funded by the CNRS-INEE initiative "Projets Exploratoires Premier Soutien (PEPS Adaptation 2017)".

No potential conflict of interest was reported by the authors.

AUTHOR CONTRIBUTIONS

S. Martin: original concept, designed and performed the experiment, physiological measurements, data analyses, drafting and editing manuscript; V. Calvert: set up and performed the experiment; A. Chenuil: original concept, molecular analyses, editing manuscript.

De Jode, A. (2018). A study of coralligenous habitats biodiversity and of the influence of environmental factors using genetic tools: from engineer species populations to communities.

Supplementary Material Table S1

Abundances of the eight cryptic species (or clades) of the Lithophyllum stictiforme complex found (a) along the French Mediterranean coastline and (b) along the Marseille coastline according to depth (data from Fig. 2 and Fig. 3 of De Jode et al., 2019). The cryptic species C1 and C4 are presented in bold. We emphasize that sampling effort was much higher for sites between 28 and 31 m depth so the relevant information in this table is the relative abundances among depth categories. This does not represent absolute depth distributions.