Emmanuelle Sachon
email: emmanuelle.sachon@upmc.fr

Photocross-linked peptide-protein complexes analysis: A comparative study of CID and ETD fragmentation modes

Protein-protein interactions are among the keys to organizing cellular processes in space and time.

One of the only direct ways to identify such interactions in their cellular environment is to covalently bond the interacting partners to fix the interaction. Photocross-linking in living cells is thus a very promising technique. The feasibility of in cellulo photocross-linking reactions has been shown and mass spectrometry is a tool of choice to analyze photocross-linked proteins. However, the interpretation of the MS and MS/MS spectra of photocross-linked peptides remains one of the most important bottlenecks of the method and still limits its potential for large-scale applications (interactomics). Fundamental studies are still necessary to understand and characterize the fragmentation behavior of photocross-linked peptides. Here, we report the successful identification of the interaction sites in a well-characterized model of in vitro interaction between a protein and a peptide. We describe in details the fragmentation pattern of these photocross-linked species in order to identify trends that could be generalized. In particular, we compare CID and ETD fragmentation modes (and HCD in a lesser extent), demonstrating the complementarity of both methods and the advantage of ETD for the analysis of photocross-linked species. The information should help further developing dedicated softwares to properly score MS/MS spectra of photocross-linked species.

Introduction

The relative maturation of the proteomics technologies for protein identification led to the emergence of ever more complex biological questions in order to understand the dynamics of living organisms. Among these, one crucial point is the elucidation of how these proteins interact in vivo with each other (interactome). Classical bottom-up proteomics is not well suited to determine these interactions which are, by nature, fragile and transient. The developments of the so-called native mass spectrometry could be a breakthrough, but the technologies are for now not ready for largescale applications. Cross-linking has been developed as a way to use bottom-up proteomics for interaction studies. The elegant principle is based on the use of a cross-linker that can covalently fix the interaction between proteins in order to preserve them all along the sample preparation and up to the MS and MS/MS analysis. In chemical cross-linking, the cross-linker is a small molecule whose extremities are chemically reactive and tailored to target chosen amino acids, like lysine or cysteine residues [START_REF] Singh | Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique[END_REF]. Chemical cross-linking coupled to mass spectrometry has already been used as lowresolution protein structure determination technique [START_REF] Singh | Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique[END_REF]. Solvent accessibility of residues can be directly checked, similarly to the H/D exchange. However, chemical cross-linking has several limitations such as i) the cross-linker selectivity towards specific residues which limits the interactions that can be preserved; ii) the low yield of cross-linking reaction; iii) the rapid hydrolysis of cross-linker reactive groups in solution (in vitro or in cellulo); iv) the uncontrolled reactivity of the cross-linker in time and space; (v) the limited ability of some cross-linker to pass the cell membrane for in cellulo applications. Therefore, it is often used in vitro but rarely found in the literature for in cellulo or in vivo experiments except for a few examples using formaldehyde, a non selective and also toxic reagent, resulting in extensive and complex cross-linking [START_REF] Vasilescu | Identification of protein-protein interactions using in vivo crosslinking and mass spectrometry[END_REF]. To overcome these issues, photoinduced cross-linking has been developed in chemical biology by using photo-reactive groups which can now be routinely incorporated into the proteins either chemically or metabolically [START_REF] Davis | Designer proteins: applications of genetic code expansion in cell biology[END_REF][START_REF] Suchanek | Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells[END_REF] and then used in the native environment [START_REF] Tanaka | Photocrosslinkers illuminate interactions in living cells[END_REF]. Scheme A1 (electronic Supplemental Material) shows three commonly used photoprobes. However, unlike chemical cross-linking which targets specific residues, photocross-linking can involve any C-H bond of any amino acids which drastically increases the number of possible photocross-linked products in a reaction mixture and gives the opportunity to get information about the covalent link at the atomic level. Therefore, the data interpretation still remains very challenging in a classical database search strategy, because it requires considering the possibility of any link between all the potential peptides, leading to an explosion in search space and in false positive identifications.

The average molecular weight of tryptic photocross-linked peptides corresponds to the sum of the two digested peptides linked and is thus typically > 2000 u. This high mass and thus the large number of degrees of freedom is not in favor of an effective fragmentation by classical CID through low energy collision. In addition, fragmentation spectra obtained for a precursor ion where two peptides are covalently linked are much more complex as these spectra contain fragmentation patterns of both peptides, as well as possible internal fragmentations if the covalent bond formed remains intact. This is common to all cross-linking strategies and requires dedicated softwares taking into account special features of cross-linked peptides such as their fragmentation pathways. StavroX is, to our knowledge, the first software able to deal with MS/MS spectra of photocross-linked species [START_REF] Gotze | StavroX--a software for analyzing crosslinked products in protein interaction studies[END_REF].

CID, the most commonly used fragmentation method for tandem mass spectrometry (MS/MS) analyses of peptides and proteins, relies on activation by energetic collisions of the protonated analytes with a neutral gas. In high-energy CID (keV), the collision gas pressure is low enough (≤ 10 -7 Torr) that only a few collisions can occur, while in low-energy CID (eV) a collision gas pressure of 10 -3 Torr allows multiple collisions with the ions to be fragmented, which increases internal energy and yields extensive dissociation. CID typically induces fragmentation of the amide bonds CO-NH of the peptides generating the b-and y-fragment ions series [START_REF] Biemann | Contributions of mass spectrometry to peptide and protein structure[END_REF][START_REF] Roepstorff | Proposal for a common nomenclature for sequence ions in mass spectra of peptides[END_REF].

In general, CID is more effective for small, low-charged peptides [START_REF] Wells | Collision-induced dissociation (CID) of peptides and proteins[END_REF]. Therefore, one can suppose that with CID fragmentation, the amount of energy required to fragment large cross-linked peptide ions would be high due to the isomerization of the internal energy over a larger number of degrees of freedom.

Electron-transfer dissociation (ETD), developed by Hunt et al. [START_REF] Syka | Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry[END_REF], has also shown much promise for the sequencing of peptides and proteins. Compared to CID, ETD is described as more favorable to analyze large peptides, proteins, and labile post-translational modifications (PTMs) [START_REF] Good | Performance characteristics of electron transfer dissociation mass spectrometry[END_REF][START_REF] Mikesh | The utility of ETD mass spectrometry in proteomic analysis[END_REF][START_REF] Molina | Comprehensive comparison of collision induced dissociation and electron transfer dissociation[END_REF][START_REF] Swaney | Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors[END_REF]. This gasphase radical process leads to a rapid homolytic fragmentation mainly at the site where the originally formed radical was trapped [START_REF] Li | The effect of fixed charge modifications on electron capture dissociation[END_REF] thereby promoting N-C fragmentation that yield c-and z • -type fragment ions, leaving side-chains as well as PTMs intact.

In this study, we performed an advanced evaluation of photocross-linked peptides CID and ETD fragmentation spectra obtained for a model interacting system composed of BSA protein and (R/W) 9 cell penetrating peptide bearing here a benzophenone (Bp) photoactivable group [START_REF] Clavier | An integrated cross-linking-MS approach to investigate cell penetrating peptides interacting partners[END_REF][START_REF] Delaroche | Cell-penetrating peptides with intracellular actinremodeling activity in malignant fibroblasts[END_REF]. We used two different mass spectrometer instruments and four activation modes for the study of photocross-linked peptides fragmentation. We applied either high-energy CID-based fragmentation (keV) using a MALDI-TOF/TOF instrument or low energy CID (eV), HCD (Higher-energy Collisional Dissociation) (eV range) and ETD fragmentation using a nanoLC ESI-LTQ/Orbitrap system enabling rapid cycling between alternate CID and ETD or CID and HCD fragmentations on the same set of precursor ions in a data-dependent manner. This study allowed us to assess the suitability of the different fragmentation modes for the analysis of photocross-linked peptides. ETD revealed to be particularly adapted for the determination of the residues involved in the photocross-linking reaction. We hope that such a study will help future developments to implement new scoring methods and improve the identification and characterization of photocross-linked peptides by mass spectrometry in order to investigate later biologically relevant interacting systems in cellulo (interactomic).

Experimental

Peptide synthesis: The CPP biotin(O 2 )-G 5 -K(Bp)-RRWWRRWRR (photo(R/W) 9 ) was synthesized in both deuterated and non deuterated versions, incorporating or not 2 deuterium atoms to each of the 5 glycines in the spacer arm by R. Marquant from our laboratory. The peptide synthesis was realized as previously published in ref [START_REF] Sachon | Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway[END_REF] with some modifications. Briefly, the biologically active (R/W) 9 peptide sequence NH 2 -RRWWRRWRR was first synthesized with a peptide synthesizer (Applied Biosystems, Darmstadt, Germany, model 433A) using a solid phase butyloxicarbonyl (Boc) chemistry in the 0.1 mmol scale, starting from a p-methylbenzhydrylamine resin (MBHA resin). Standard protocols were used, with DCC/HOBt activation (10 eq. excess for standard Boc amino acids). After the removal of the last N-Boc protecting group, the synthesis was manually completed by adding the protected Lysine residue (Boc-Lys(Fmoc)-OH). The resin was dried in vacuo and separated into two batches for the manual addition of the 5 Boc-Gly-OH either bideuterated or not followed by the addition of the biotinyl sulfone. The Lysine(Boc-Arg(Tosyl)-OH) and tryptophane (Boc-Trp(Formyl)-OH) residues were then deprotected using 20 % piperidin. Then, the carboxybenzophenone was coupled to the lateral chain of the deprotected lysine residue. Finally, both peptidyl-MBHA-resins were treated with liquid HF at 0°C (2h30) under stirring, in the presence of anisole and dimethylsulfide. After evaporation of HF and of the solvents in vacuo, the resins were washed three times with Et 2 O and then subsequently extracted three times with 10 % AcOH. Lyophilization of the extracts gave crude peptides, which were purified by HPLC and purity was checked by UV (> 95 %) and by MALDI-TOF MS.

Photocross-linking reaction: 100 µL of a solution of 10 µM BSA (Sigma, Saint Louis, Missouri, USA) and 10 µM of both the deuterated and the non-deuterated form of photo(R/W) 9 in NH 4 HCO 3 50 mM and NaCl 150 mM to mimic physiological conditions was prepared and placed into two Eppendorf tubes. One of the tube was wrapped into aluminum foil (negative control) while the other was kept opened. These tubes were placed in a water/ice bath to avoid heating since the light emitted by the UV lamp is absorbed by the sample and introduced in the photoirradiation chamber at a distance of about 5 cm of the lamp (HPR 125W-mercury lamp, Philips) and irradiated for 30 min.

Enzymatic trypsin digestion: After irradiation, the photocross-linking reaction mixtures were submitted to in-solution trypsin digestion. Disulfide bridges were reduced (5 mM dithiothreitol, 15 min, 50 °C) and cysteines were alkylated (20 mM iodoacetamide, 30 min, room temperature in the dark). Trypsin (Promega, gold, mass spectrometry grade) digestion was conducted at 37 °C overnight (1:30 (w:w) protease-to-protein ratio).

Biotinylated photocross-linked peptides affinity purification:

A volume of tryptic digest corresponding to 80 pmol of photo(R/W) 9 CPP was incubated with 200 µg of streptavidin magnetic beads (Dynabeads M280 streptavidin coated magnetic beads from Invitrogen, Carlsbad, New Mexico, USA) for 60 min at RT under gentle stirring. Beads were conditioned before use and washed after incubation [START_REF] Sachon | Cgamma H2 of Met174 side chain is the site of covalent attachment of a substance P analog photoactivable in position 5[END_REF]. Peptides were eluted from the beads with 5 µL of 0.1 M hydrochloric acid (HCl) under gentle stirring (30 min).

MS and MS/MS analysis of photocross-linked peptides:

MALDI-TOF/TOF: Spectra were obtained with an AB4700 Proteomics Analyzer mass spectrometer (AB Sciex) in positive ions reflector mode and delayed extraction. CHCA (Sigma) was used as the matrix and solubilized at 5 mg/mL in 1/1 ACN/0.1 % trifluoroacetic acid (TFA). 0.5 µL of the biotinylated photocross-linked peptides solution eluted from the beads was mixed with 0.5 µL of matrix and deposited on the MALDI plate. For MALDI-TOF spectra a total of 10,000 laser shots were acquired in the m/z range 500-5000. MALDI-TOF/TOF spectra were acquired simultaneously for the two peaks of doublets by centering the selection window on the average m/z value with a low resolution R = 30.

The metastable suppressor ion mode was first turned off for a few shots to check if both ions were correctly selected and then turned on for the rest of the MS/MS acquisition. On average, 30,000 laser shots were accumulated for each MS/MS spectrum.

NanoLC-ESI-Orbitrap:

The analysis of the photocross-linked peptides was performed using an Ultimate 3000 Nano-HPLC system (Dionex) coupled with a LTQ-Orbitrap-XL mass spectrometer (Thermo Scientific). The biotinylated photocross-linked peptides solution eluted from the beads was diluted by two in deionized H 2 O and transferred into a HPLC vial. 5 µL were injected and concentrated on a trapping column (C 18 , 300 µm × 5 mm, 5 µm, 100 Å, Dionex) with 99% solvent A (2 % acetonitrile (ACN),0.1 % formic acid) at a flow rate of 20 μL/min for 10 min. The peptides were separated on a column (C 18 , 75 µm × 150 mm, 3 µm, 100 Å, Dionex) equilibrated with 98 % solvent A. Peptides were separated at a flow rate of 300 nL/min using the following gradient: 0-90 min 2-40 % solvent B (98 % ACN, 0.1 % formic acid), 90-93 min 40-60 % solvent B, 93-110 min 60 % solvent B.

The LTQ-Orbitrap mass spectrometer was outfitted with a nano ESI interface. Electrospray emitters were 360/20 µm o.d. × 10 µm i.d. fused-silica tips (PicoTip Emitter, standard Coated SilicaTip, New Objective). The heated capillary temperature and spray voltage were 200 °C and 1.5 kV, respectively.

The LTQ Orbitrap XL was operated in positive ion and data dependent acquisition mode. Orbitrap full scan spectra (automated gain control (AGC) 2.10 5 ) were collected in the range m/z 300-2000 at a resolution of 30,000 in the profile mode followed by sequential ion trap CID and ETD MS/MS spectra of the three most intense ions (AGC target : 2500, isolation width CID 4.0 m/z, ETD 2.0 m/z, ETD reagent ion AGC target 3.10 5 , ETD reaction time 100 ms) using the Masstag option corresponding to the mass difference of 10.06 u. and with various charge states (∆ m/z 10.06; 5.03; 3.35; 2.52; 2.01; 1.68; partner intensity tolerance 70-100 % and choice of both partners fragmentation). For ETD fragmentation a supplemental activation (SA) of 20 % was used.

Results and Discussion

We previously characterized the interaction between the BSA protein and a modified version of the cell-penetrating peptide (CPP), namely (R/W) 9 containing a Lys residue at the N-terminus to allow chemical cross-linking [START_REF] Clavier | An integrated cross-linking-MS approach to investigate cell penetrating peptides interacting partners[END_REF]. In the present study we developed a general workflow using photocrosslinking to characterize interacting systems. We validated this technique using the interacting model system (R/W) 9 /BSA by designing a photoactivable CPP analog of (R/W) 9 , namely photo(R/W) 9 , which has the following sequence: biotin(O) 2 -G 5 -K(Bp)RRWWRRWRR. The NH 2 of the lateral chain of the Lys residue is substituted with a benzophenone (Bp) moiety. The biotin added at the N-terminus allows purification of the photocross-linked species. This biotin is oxidized to biotin sulfone (biot(O 2 ), to avoid further oxidation during the sample preparation which would dilute the MS signal of the photocross-linked species (more species with lower intensity each). The photo(R/W) 9 peptide was synthesized in heavy and light forms thanks to the presence of the 5 Gly residues that can be deuterated. Performing the photocross-linking reaction with a 1:1 mixture of the heavy and light photo(R/W) 9 should allow an easier MS1 determination of the ions corresponding to photocrosslinked species which should appear as 1:1 doublets separated by a fixed 10.06/z difference.

The Bp moiety, present on the photo(R/W) 9 , once activated via UV-irradiation at 365 nm (creation of a biradical), can lead to the formation of a covalent bond at any of the C-H or heteroatom-H bonds of the interacting partner, as long as these atoms are sufficiently close to the photoprobe, by a well known and controlled reaction as shown in Figure 1. However, the knowledge of the cross-linking site and residue remains difficult to extract from the list of branched peptide fragments even with known interacting partners in vitro. After UV-irradiation of the photo(R/W) 9 / BSA partners in the conditions described above, complexes obtained were trypsin digested and the biotinylated species -corresponding to the unreacted photo(R/W) 9 or cross-linked photo(R/W) 9 with BSA peptides -were purified using streptavidin coated magnetic beads. As shown on the MALDI-TOF spectrum presented in Figure 2a, this purification step was very efficient and led to the specific observation of only 1:1 doublets separated by 10.06/z mass units. Fig. 2 The presence of these doublets shows that the processes (photocross-linking reaction, digestion, purification, analysis) are those expected and without artifacts for both forms of photo(R/W) 9 . The base peak at m/z 1054-1064 (doublet) corresponds to the photo(R/W) 9 entirely digested by trypsin (namely photo(R/W) 9 pept(1-8): biotin(O) 2 -GGGGG-K(Bp)-R). The negative control (Figure 2b), corresponding to the same sample without photoirradiation, only contained this doublet, indicating that all the ions with higher m/z in Figure 1 are specific of the photocross-linking reaction. The prevalence of unreacted photo(R/W) 9 after photocross-linking revealed the incomplete reaction and the efficient trypsin digestion of photo(R/W) 9 since no partial digestion products were observed. However, we can assume that the photocross-linking reaction occurred with a relatively high yield, taking into account the high number of different photocross-linked peptide products with a contribution of more than 10 different species that dilute the signal in the MS spectrum (Figure 2). This result is in good agreement with the high yield of photocross-linking described in the literature using the Bp photoprobe [START_REF] Mourey | Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands[END_REF].

The m/z of the potential photocross-linked peptides resulting from the interaction between photo(R/W) 9 and BSA followed by trypsin digestion can be easily computed. For this simple model system, it was possible to identify most of the photocross-linking product ions of the MALDI-TOF MS spectrum (Figure 2) by manually comparing theoretical and measured m/z of the doublets (Table 1).

Table 1

However, the localization of the cross-linked residues requires complete series of relevant MS2 fragments and time consuming manual fragments interpretation. In a broader context, fragmentation will obviously be necessary for identification of unknown partners in more complex interacting systems (in cellulo). All the enriched (biotin/streptavidin affinity purification) photocrosslinked peptide sequences identified in this study (Table 1) were submitted to MS2. We applied either high-energy CID-based fragmentation (keV) using a MALDI-TOF/TOF instrument or low-energy CID (eV) and ETD fragmentation using an ESI-LTQ Orbitrap XL system. HCD (eV range) was also performed and evaluated for different photocross-linked peptides. This fragmentation type did not generate reproducibly informative spectra for the various photocross-linked peptides probably due to the low amount of material and mainly to the energy function of the HCD cell on the LTQ-Orbitrap -XL instrument which is not optimized. Spectra appeared extremely noisy with no valuable sequence information. However, when sensitivity was not a limiting factor, HCD could lead to informative MS2 spectra.

The study was conducted on all photocross-linked peptides and for the sake of simplicity, the comparison of the different fragmentation types (low-and high-energy CID, ETD and in a lesser extent HCD) will be illustrated on doublet precursor ion identified as the tryptic BSA peptide containing 3 carbamidomethylcystein residues : 267 ECCHGDLLECADDR 280 , covalently linked to the photo(R/W) 9 pept(1-8) (Figure 2c). This doublet ion has one of the highest m/z in the MALDI-TOF spectrum (Figure 2a) and is relatively abundant, existing as both singly and doubly protonated ions The nomenclature that will be used to describe the CID and ETD fragment ions of both peptides covalently linked in the photocross-linked species, namely sequence  and  for the photo(R/W) 9 pept(1-8) and the BSA trypsic peptide respectively, is presented in Figure 3. Unlinked y-and b-or cand z-fragment ions correspond to linear peptides arising from the photo(R/W) 9 pept(1-8) (sequence , in blue) or from the BSA peptide (sequence , in red). Interlinked y-and b-or c-and z-fragment ions contain residues from both sequences  and  covalently linked by the Bp moiety with a fragmentation that occurred along the  or  sequence (in purple). Fragmentation occurring at a C-C bond was also observed and named C-C or C-C depending on the fragment ion that is observed.

Fig. 3

High-energy CID using a MALDI-TOF/TOF instrument: Keeping in mind that the upper limit for CID at high energy is approximately m/z 3000 [START_REF] Wells | Collision-induced dissociation (CID) of peptides and proteins[END_REF], fragmentation of a precursor ion at m/z 2803-2813 ([M+H] + ) requires manual optimization as well as relatively high amount of material allowing averaging of a high number of spectra (typically more than 30,000 laser shots). In addition, the selection window in the MALDI-TOF/TOF is relatively broad and does not allow selecting only one ion of the doublet without an important loss of precursor ions (already enriched by streptavidin affinity purification and therefore present in limiting amount). Therefore, we manually centered the selection window on the m/z ratio 2809 with a low resolution R = 30, allowing the entire doublet to be selected and fragmented, minimizing the loss of material and combining heavy and light ions structural information in the same spectrum presented in Figure 4a.

Fig. 4

However, in this MALDI-TOF/TOF spectrum, the most intense ions observed did not correspond to bor y-fragment ions but to the unexpected fragmentation of C-C bonds which theoretically require high energy to be broken. For instance, the ion at m/z 1054-1064 (photo(R/W) 9 pept(1-8)) and its complementary ion at m/z 1749.69 (BSA peptide) correspond to the fragmentation of the created covalent bond between the carbon of the Bp and the carbon of the photocross-linked Leu residue (C-C or C-C) (Figure 4b). The steric hindrance due to the presence of the two aromatic rings of the benzophenone (tertiary alcohol function with bulky substituents) probably weaken the C-C bond. This C-C fragmentation corresponding to the release of the intact photo(R/W) 9 pept(1-8) (m/z 1054-1064) was reproducibly the predominant fragment observed in the MALDI-TOF/TOF spectra of every photocross-linked peptides fragmented in this study. In this MALDI-TOF/TOF experiment, another possible explanation could be the absorption of the Bp moiety at the wavelength of the MALDI UV laser ( = 355 nm). The resulting excitation of the Bp could contribute to a local increase of internal energy, leading to the fragmentation of the C-C bond. Another C-C bond was fragmented in this example (fragment C-C at m/z 1693.65), corresponding to the fragmentation in the lateral chain of the cross-linked Leu residue, between the C and the C (Figure 4b) was generated, likely for the same reasons. This indicates that the site of photocross-linking is either on the C,  or  of the Leu side chain but not on the C. Both ions at m/z 1749.69 and 1693.65 also appeared with their neutral losses (water and/or water and ammonia).

The MALDI-TOF/TOF spectrum of the m/z 2803-2813 ions was therefore very informative and led to the identification of the covalent bond on the C,  or  of the lateral chain of Leu273 or 274 of the BSA sequence 267 ECCHGDLLECADDR 280 (in bold). With another photocross-linked peptide, observed at m/z 2453-2463 in Figure 2 and corresponding to the BSA peptide 569 TVMENFVAFVDK 580 photocrosslinked to the photo(R/W) 9 pept(1-8), we determined precisely that the covalent bond occurred on the C of the Met571 (Figure A1, electronic Supplemental Material) thanks to the C-C fragments information. The fragment at m/z 1115-1125 was attributed to the C-C fragmentation between the photocross-linked carbon of the Bp and the C of the lateral chain of the Met571. However, the ability to get information on the carbon involved in the photocross-linking reaction cannot be generalized to every photocross-linked peptides manually inspected in this study as fragmentation in the lateral chain of the cross-linked residue is not always observed. Another point that is worth mentioning here is that Met was not always identified as the covalently linked residue in this interacting system showing that Bp reactivity is not always biased towards Met residues [START_REF] Rihakova | Methionine proximity assay, a novel method for exploring peptide ligandreceptor interaction[END_REF].

Low-energy CID and ETD using a nanoLC-ESI-LTQ/Orbitrap XL instrument: Low energy CID-and ETDbased fragmentation spectra were acquired using a nanoLC ESI-LTQ Orbitrap XL system. CID and ETD fragmentation were systematically alternated on the same precursor ions (multiprotonated ions produced by the ESI ion source) in an automatic data-dependent manner. Fragments were analyzed using the LTQ in both modes of fragmentation. To optimize fragmentation time and allow fragmenting only informative ions, the Masstag option of the Xcalibur software was used to select only doublets with the same intensity and a defined m/z (10.06/z) difference. We also chose to fragment successively both peaks of the doublets, corresponding to the light and heavy forms of the photocross-linked peptides. It is worth noting here that the Masstag option is based on mass difference (10.06/z) and on the relative abundance (%) of doublet intensity that should be between 70 and 100 % to take into account experimental error in 1:1 mixture of both the light and the heavy forms of the peptides as well as difference of retention times due to deuteration. As usually observed with ETD, general efficiency of fragmentation is low since the most abundant ions are the precursor ion **, the charge-reduced precursor * (see Table A2, electronic Supplemental Material), irrespective of the use of supplemental activation (SA = 20 %) [START_REF] Swaney | Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors[END_REF]. In addition, peaks corresponding to entire or partial side chain losses issued from charge-reduced precursor ions or z +• fragment ions were observed as summarized in Table A2, electronic Supplemental Material. For example, a loss of C 2 H 4 NOS • (90 mass units) from z +• or z 2+• fragment ions containing a carbamidomethylated Cys in the N-terminal region of the fragment was observed as described by Sun R-X. et al. [START_REF] Sun | Improved peptide identification for proteomic analysis based on comprehensive characterization of electron transfer dissociation spectra[END_REF]. The same loss of C 2 H 4 NOS • (90 mass units) was observed from the chargereduced ions whereas the loss of C 4 H 8 (56 mass units) from a Leu residue side-chain was only observed from the charge-reduced 2+ and 3+ precursor ions. Possible mechanisms leading to these neutral losses are proposed in Scheme I.

Scheme I

Besides these intense ions, an almost complete c and z • ions series, corresponding to unlinked and interlinked fragments were observed. Unlinked ions only appeared as monoprotonated c-or zfragment ions whereas interlinked ions appeared as doubly charged c-or z-ions. No higher charge states were observed, as expected from the ETD mechanism where one charge is lost due to electron-transfer to the 4+ peptide ion, leaving three charges which are distributed on the resulting fragments. As expected, the interlinked fragments which usually have the higher m/z ratio, have the higher charge state. This was observed with other photocross-linked peptides examples such as in the ETD spectrum of the 4+ precursor ion corresponding to the covalent bond between the photo(R/W) 9 pept(1-8) and Ser89 of the BSA sequence 89 SLHTLFGDELCK 100 (in bold), doublet observed at m/z 2473.13-2483.17 ([M+H] + ) in Figure 2. In this example, the c-interlinked fragment ions appear as 2+ and/or 1+ ions whereas the unlinked z-fragment ions were always observed as 1+ ions (Fig. A2, electronic Supplemental Material).

Besides the conventional c-and z-fragment ions observed with the chosen example from Figure 5, and as already mentioned with high-energy (keV) CID, a fragment which could correspond to the fragmentation of a C-C bond, was observed in the ETD spectrum from both the light and heavy forms at m/z 847.37. Based on the m/z ratio, this fragment could be the result of the loss of the photo(R/W) 9 pept(1-8) moiety (as a radical cation) from the z  7 2+• due to the fragmentation between the C  and the C  of the Leu274 side chain as represented in Figure 5d.

Crossing information given by these different fragments allowed confident assignment of the site of photocross-linking on Leu274 of the BSA peptide 267 ECCHGDLLECADDR 280 (in bold). The presence of interlinked fragments ions was observed for every ETD spectra manually checked in this study and in most examples the series of c-and/or z-ions were almost complete. However, the possible C-C bond fragmentation was only seen in the ETD spectrum of the chosen example shown in Figure 5.

Low-energy CID:

From the same LC-MS/MS run we obtained an independent CID dataset (Figure A3, electronic Supplemental Material). In the case of the chosen example (peptide [M+H] + at m/z 2803-2813), it is important to note that CID-based fragmentation of the 3+ and 2+ precursor ions was not efficient enough and that only the MS2 spectrum of the 4+ precursor ion (Figure A3, Table A3, electronic Supplemental Material) revealed informative (higher internal energy due to a higher Coulomb repulsion). However, the signal-to-noise ratio of the CID spectra for both heavy and light versions of the photocross-linked peptide appeared much lower compared to ETD, and the number and intensity of unassigned species ("noise peaks", i.e. ions which are not part of the main b/y fragment ion series) was much higher.

Whatever the sequence of the photocross-linked peptide, low-energy CID spectra mostly presented singly charged unlinked fragment ions (only one example was found containing a doubly charged interlinked y ion, see Figure A4, electronic Supplemental Material). In similar experimental conditions, we could observe multiprotonated ions either charge-reduced ions or fragment ions in ETD. Therefore, the absence of multi charged fragment ions was quite unexpected for multiprotonated precursor ions, such as a 4+ ion. This could be the result of further consecutive fragmentation of the higher charge state fragment ions.

An extensive fragmentation of the biotin(O) 2 -G 5 ( 1 H/ 2 H)-K(Bp)-R tag ( sequence) led to b-diagnostic ions series at m/z 259.08 (Biot), 316.10 (Biot-G), 373.12 (Biot-GG), 430.14 (Biot-GGG), 487.16 (Biot-GGGG) and 544.18 (Biot-GGGGG) (values are given here for the light version) which are the signature of the presence of the photo(R/W) 9 pept [START_REF] Singh | Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique[END_REF][START_REF] Vasilescu | Identification of protein-protein interactions using in vivo crosslinking and mass spectrometry[END_REF][START_REF] Davis | Designer proteins: applications of genetic code expansion in cell biology[END_REF][START_REF] Suchanek | Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells[END_REF][START_REF] Tanaka | Photocrosslinkers illuminate interactions in living cells[END_REF][START_REF] Gotze | StavroX--a software for analyzing crosslinked products in protein interaction studies[END_REF][START_REF] Biemann | Contributions of mass spectrometry to peptide and protein structure[END_REF][START_REF] Roepstorff | Proposal for a common nomenclature for sequence ions in mass spectra of peptides[END_REF] in the sequence of the precursor ion. Generally no yions were observed in the  sequence for the m/z range (200-1500) of the MS2 spectra. In the case of the chosen example (peptide [M+H] + at m/z 2803-2813), some of the expected y-and b-unlinked fragment ions of the  sequence were present. No interlinked ions were observed in the low-energy CID MS2 spectrum. These ions should however exist as multiprotonated ions and were possibly further fragmented leading to low m/z fragments which could not be easily attributed in the MS2 spectrum and contributed to the noise observed in the spectrum. Crossing information given by the unlinked y-and b-fragment ions was not sufficient to precisely determine the photocross-linked residue. However, we could restrict the interaction zone to the residues L273 or L274 from the  sequence. In all other manually inspected MS/MS spectra mostly unlinked fragments were observed and most of the time these unlinked fragments were not sufficient to unambiguously determine the cross-linking position (additional examples are shown in Figures A2a andA4a, electronic Supplemental Material).

Compared to low-energy CID, ETD fragmentation led to the production of numerous and relatively abundant interlinked ions which constitute the unambiguous signature of the existence of a covalent bond between the two (photocross-linked) peptides. The presence of these interlinked ions in ETD spectra was reproducible whatever the sequence of the photocross-linked peptide species and revealed particularly useful to narrow down the number of residues possibly involved in the crosslinking reaction (Figures A2,A3,A4, electronic Supplemental Material). The signal-to-noise ratio of the ETD spectra appeared much improved compared to CID and the number and intensity of unassigned species was much lower. Taken together, these features of ETD may contribute to an increase in confidence of assignment of fragment ions. Based on our results obtained with different photocross-linked peptide sequences, we can conclude that ETD is more suitable for the identification and characterization of covalently linked peptides. However, when fragmentation is not complete, both CID and ETD fragmentation can be complementary in obtaining information about the characterization of the interaction zones. It is worth noting here that even if HCD could not lead to informative spectra for most of the enriched photocross-linked peptides, HCD MS2 spectra of the quadruply charged ion at m/z 701.5-704.5 ([M+H] + at m/z 2803-2813 in Figure 2) allowed the characterization of this photocross-linked peptide and the localization of the covalent bond on Leu274 of the BSA sequence 267 ECCHGDLLECADDR 280 (in bold) (Table A4, electronic Supplemental Material), only based on the information given by the 1+ unlinked b-and y-fragment ions since no interlinked ions were present in the spectrum.

Finally, low-energy CID did not allow fragmentation of the C-C bond of the cross-linker in our study. This fragmentation was always easily observed using keV collision in MALDI-TOF/TOF and can be observed in some ETD spectra (only one example in this study, corresponding to a consecutive fragmentation). Therefore, even if MALDI-TOF/TOF cannot be easily automated in an on-line workflow (nanoLC coupling and automatic acquisition) and thus would not be suitable for high throughput proteomic/interactomic analysis, it can provide occasional and very valuable information in the case of a cross-linking-based structural studies. However, low-energy CID fragmentation of the C-C bond has been recently reported in a similar study concerning the interaction between the photoreactive peptide M13 and its partner protein CaM using the para-benzoyl phenylalanine as the photoprobe [START_REF] Pettelkau | Reliable identification of cross-linked products in protein interaction studies by 13C-labeled p-benzoylphenylalanine[END_REF]. In this case the fragmentation of the newly formed C-C bond is believed to be specific of cross-linked products with N-terminal and oxidized methionines and results from consecutive fragmentation.

Conclusions

In this study, we optimized a very efficient affinity purification step that can be realized with photocross-linking reaction mixtures obtained in vitro or in cellulo. Moreover, we show that the number of MS2 data acquired can be considerably restricted using a targeted approach involving isotopic labeling coupled to the Masstag option of Xcalibur which allows fragmentation of only relevant isotopically labelled ions. These two strategies, both aiming at a targeting of the generated data, can reveal extremely useful in the case of complex photocross-linking mixtures involving unknown partners. Actually this approach aiming at the identification of in vitro or in cellulo interaction partners of a molecule/peptide of interest can be applied to numerous others peptides providing that the biotin and the photoprobe are not dissociated during the enzymatic digestion. The photoactivable peptide under study must thus be carefully designed avoiding for example the presence of lysine or arginine residue between the biotin and the photoprobe in the case of a tryptic digestion. In addition, it will be necessary to ensure that these biotin and photoprobe are not modifying the peptide original biological properties. In order to limit the effect of these features on the peptide activity we suggest to position the biotin and the photoactivable probe either N-term or C-term of the peptide of interest not to affect the core structure of the latter.

Besides these considerations, it is important to note that, even if not trivial, manual identification and characterization of the photocross-linked species was feasible in the present study since we worked in vitro, with a simple context of two known partner sequences, one of them being a short biotinylated and isotopically labelled photoreactive peptide. This manual interpretation of the MS2 spectra revealed the high potential of ETD for the identification and characterization of photocrosslinked peptides due to the production of interlinked ions that are usually not observed in CID spectra. However, in the case of a low yield of ETD or CID fragmentation, we showed that combining information given by both fragmentation techniques helped for the identification of the cross-linked residues. Consequently, we describe a general procedure to facilitate the identification of interacting partners using photocross-linking that can be extended to more complex in vitro or in cellulo interacting systems. The whole strategy is summarized in Figure 6. To conclude, automation of the data treatment is absolutely required to extend this strategy to unknown interacting partners. Most of the widely applied searching programs, such as Mascot are not developed and thus are not suitable, for analyzing fragmentation spectra originating from photocross-linked peptides even when, as in our case, at least one of the peptide partners is known and can thus be defined as a peptide modification (an example is shown in Figure A5, electronic Supplemental Material) where the photo(R/W) 9 pept(1-8) was defined as a modification occurring on selective residues for the Mascot search. We consider that StavroX, an open source software developed by M. Gotze et al. [START_REF] Gotze | StavroX--a software for analyzing crosslinked products in protein interaction studies[END_REF] is very promising for the search of photocross-linking products. It actually allowed us to confirm most of the manually identified photocross-linked peptides. We performed the search with CID data only, ETD data only or with both CID and ETD data. Searching CID and ETD data at the same time led to significant improvements of the cross-link candidates score.

However we noticed that the localization of residue involved in the cross-linking reaction was difficult solely based on the cross-link candidate score. Indeed scores are usually proportional to the number of identified fragments for a peptide. However some fragments such as short interlinked fragments may be more important than others to precisely determine a cross-linking position.

Consequently combining CID and ETD data in a specific search algorithm using information given by the shorter interlinked and the longer unlinked ions observed in the MS2 spectra (as proposed in summarized strategy from Fig. 6) should allow to really reduce the number of possible cross-linked residues and thus to gain in structural resolution. A recent study from Liu, F. et al. [START_REF] Liu | Facilitating Protein Disulfide Mapping by a Combination of Pepsin Digestion, Electron Transfer Higher Energy Dissociation (EThcD), and a Dedicated Search Algorithm SlinkS[END_REF] describes EThcD fragmentation for other type of covalently bond peptides: disulfide bridged peptides. In this study, ETD preferentially leads to the cleavage of the S-S bonds, generating two disulfide cleaved peptides and the subsequent HCD fragmentation primarily targets unreacted and charge reduced precursor ions, inducing peptide fragmentation. Therefore, according to the results obtained in our study, this type of analysis could be the next step for the efficient identification/characterization of enriched photocross-linked peptides with ETD generating interlinked c-and z-fragment ions and HCD targeting the unreacted species to produce unlinked b-and y ions.

Tables Table 1: Attribution of the doublet ions observed in the MALDI-TOF spectrum from Figure 1. The Bp is located on the Lys side chain of photo(R/W) 9 . Under UV irradiation at 365 nm, the carbonyl group of the Bp is excited to give a biradical C=O --> C • -O • extremely reactive towards C-H or heteroatom-H bonds (triplet state) of the protein/peptide partner. After hydrogen abstraction and radicals recombination, a covalent bond is formed between photo(R/W) 9 and its protein partner (BSA). The covalent complex is labelled in purple.

Fig. 1

 1 Fig. 1

(

  doublets at m/z 2803-2813 and 1402-1407, respectively). Other examples are presented in the Supplementary Information.

  ETD fragmentation: The ETD spectrum of the quadruply charged precursor ion [M+4H] 4+ at m/z 701.51-704.03 corresponding to our previous example ([M+H] + ion at m/z 2803.07-2813.13) is shown in Figure 5.

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

  Fig. A1 High-energy CID (MALDI-TOF/TOF) spectrum of the singly charged parent ion at m/z 2453-

Fig. 2 aFig. 3

 23 Fig. 2 a) MALDI-TOF spectrum (m/z range 1000-3000) in positive ions reflector mode of the photocross-linking mixture obtained with BSA and photo(R/W) 9 ( 1 H/ 2 H) in 1:1 ratio, after trypsin

Fig. 6

 6 Fig. 6 Proposed strategy to identify photocross-linked peptides and restrict the number of residues

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

Acknowledgments

We acknowledge Dr. L. Matheron for proof reading and scientific discussions.

Both unlinked and interlinked y-and b-fragment ions (Figure 3) were identified (Table A1, electronic Supplemental Material), with possible neutral losses such as ammonia and/or water. They are localized with vertical bars in the photocross-linked peptide 2803-2813 presented in Figure 4b. They appeared either as singlet (s) or doublet (d) ions helping the reading of the peptide sequence, since the doublet revealed the presence of at least the photo(R/W) 9 pept(1-8) in the fragment ion (presence of the isotopically labelled Gly). No fragmentation was observed along the sequence of the photo(R/W) 9 pept [START_REF] Singh | Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique[END_REF][START_REF] Vasilescu | Identification of protein-protein interactions using in vivo crosslinking and mass spectrometry[END_REF][START_REF] Davis | Designer proteins: applications of genetic code expansion in cell biology[END_REF][START_REF] Suchanek | Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells[END_REF][START_REF] Tanaka | Photocrosslinkers illuminate interactions in living cells[END_REF][START_REF] Gotze | StavroX--a software for analyzing crosslinked products in protein interaction studies[END_REF][START_REF] Biemann | Contributions of mass spectrometry to peptide and protein structure[END_REF][START_REF] Roepstorff | Proposal for a common nomenclature for sequence ions in mass spectra of peptides[END_REF]. The ion at m/z 175, corresponding to the y1 ion (Arg) could arise from both peptide sequences ( or ). As expected for high-energy CID, immonium ions corresponding to Arg and His at m/z 129 and 110 respectively were also observed.

Combining the information given by the y-and b-unlinked and interlinked fragment ions, we concluded that the created covalent bond occurred between photo(R/W) 9 pept(1-8) and Leu273 or Leu274 residues of the BSA peptide 267 ECCHGDLLECADDR 280 (in bold).


Alexandre Geoffroy

Stéphane Mauger

Line Aurélien De Jode

Le Gall

Line Le Gall

Christophe Destombe
email: christophe.destombe@sb-roscoff.fr

O De Clerck

Molecular evidence for the coexistence of two sibling species in Pylaiella littoralis

Keywords: gene flow, microsatellite, multilocus barcode, Pylaiella littoralis, sibling species PCA, principal component analysis FCA, factorial correspondence analysis cox1, cytochrome

INTRODUCTION

Most species descriptions in brown algal groups follow the morphological species concept and the delimitation of species is therefore essentially based on discontinuities in morphological characters [START_REF] Mayden | A hierarchy of species concepts : the denouement in the saga of the species problem[END_REF]. However, due to phenotypic plasticity and the lack of diagnostic characters in some groups, species delimitation using conventional

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methods is often problematic and has led to erroneous taxonomic assignments [START_REF] Tellier | Phylogeographic analyses of the 30°S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: vicariance or parapatry?[END_REF], Tronholm et al. 2010). Analyses of molecular data have become the closest proxy of species delimitation (for review, [START_REF] Leliaert | DNA-based species delimitation in algae[END_REF]. The combination of phylogenetic and population genetics approaches can help determine algal diversity and study boundaries between closely related species. As a phylogenetic approach, DNA barcoding -based on discontinuity in sequence variation as diagnostic character -has proven to be an effective tool for delineating species [START_REF] Sites | Delimiting species: a Renaissance issue in systematic biology[END_REF] and has been successfully applied in algae to aggregate individuals into different closely related species [START_REF] Leliaert | DNA-based species delimitation in algae[END_REF]). The population genetic approach has been used in brown algae to detect gene flow discontinuity among taxa [START_REF] Tellier | Possible role of a mitochondrial genome rearrangement in maintaining the spatial segregation of two cryptic species of the Lessonia nigrescens species complex[END_REF]) and to identify hybridization or introgression events [START_REF] Engel | Genetic entities and mating system in hermaphroditic Fucus spiralis and its close dioecious relative F. vesiculosus (Fucaceae, Phaeophyceae)[END_REF]. Population genetic data acquired from microsatellite loci are extremely useful to estimate the extent of gene flow between closely related species.

The brown algal genus Pylaiella includes species characterized by a filamentous structure with various branching patterns. As in most algal groups, systematics in Pylaiella has been centered on morphological species concept. However, Pylaiella species are known for their disconcerting phenotypic plasticity and their lack of diagnostic characters, making species delimitation very problematic (Fritch 1952, Bolton 1979a). More than 90 specific and infraspecific names have been reported for this genus, of which only seven are currently accepted taxonomically [START_REF] Guiry | [END_REF]. Pylaiella littoralis (Linnaeus) Kjellman is the most common accepted species and has been reported worldwide in the littoral zones of cold and cold temperate coasts of both hemispheres (Müller andStache 1989, Silva et al. 1999). More than 50 forms or varieties have been described on the basis of morphology, habitat and seasonal distribution characteristics [START_REF] Guiry | [END_REF]. A plethora of authors have suggested that these various forms and varieties can possibly be attributed to

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phenotypic plasticity (i.e., [START_REF] Knight | Studies in the Ectocarpaceae I. The life-history and cytology of Pylaiella littoralis (L.) Kjellm[END_REF][START_REF] Newton | A handbook of the British seaweeds[END_REF][START_REF] Russell | A study in populations of Pylaiella littoralis[END_REF][START_REF] Russell | The ecology of some free-living Ectocarpaceae[END_REF], Bolton 1979b, Müller and Stache 1989). Therefore, no clear consensus has ever been adopted on species delimitation within Pylaiella [START_REF] Siemer | The taxonomic status of Pilayella littoralis, P. varia and P. macrocarpa (Pilayellaceae, Fucophyceae)[END_REF]. Molecular tools appear as a useful alternative to morphological observation for clarifying the systematics of the genus

Pylaiella.

Although various species, such as Pylaiella anglica Ruprecht and Pylaiella aquitanica Ruprecht, have been reported in England and in south-western France, respectively [START_REF] Ruprecht | Algae Ochotenses: Die ersten sicheren Nachrichten über die Tange des Ochotskischen Meeres[END_REF][START_REF] Feldmann | Inventaire de la flore marine de Roscoff. Algues, champignons, lichens et spermatophytes[END_REF], and most authors after him, recognized only P. littoralis at the species level in Brittany, France. In this region, the species is found both on rocky substrates and as an epiphyte of perennial seaweed species [START_REF] Feldmann | Inventaire de la flore marine de Roscoff. Algues, champignons, lichens et spermatophytes[END_REF]. In the northern Baltic Sea, two different seasonal cohorts have been observed, one characterized by rapid growth in early spring with a maximum cover at the beginning of May-June followed by degradation and detachment of filaments and one with a growth period in September-October [START_REF] Kiirikki | Life strategies of filamentous algae in the northern Baltic proper[END_REF]. [START_REF] Kornmann | Meeresalgen von Helgoland[END_REF] reported two different species in Helgoland-Northern Sea (P. littoralis and Pylaiella sp.). These authors observed that P. littoralis is found throughout the year whereas the second species (later identified as Pylaiella macrocarpa, Kornmann 1990), is observed only in spring.

The aims of the present study were to assess the diversity and its spatio-temporal pattern among populations of P. littoralis in Brittany. Toward these objectives, we first used a combination of five molecular markers characterized by different mutation rates and different effective sizes: the mitochondrial genes cox1, nad1 and atp9, and the two chloroplastic genes rbcL and atpB [START_REF] Silberfeld | A multi-locus time-calibrated phylogeny of the brown algae (Heterokonta, Ochrophyta, Phaeophyceae): investigating the evolutionary nature of the "brown algal crown radiation[END_REF]. In addition to the multilocus approach, we used a population genetics approach based on 10 microsatellite markers to assess gene flow between populations. Mitochondrial sequences combined with the

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population genetics approach revealed the occurrence of two sibling species in Brittany under the name of P. littoralis.

MATERIALS AND METHODS

Sampling

In total, 140 specimens of Pylaiella littoralis were sampled at four different localities (16-62 specimens per site) along the coast of Brittany, France (Fig. 1), from Dinard to Concarneau (about 380 km coastline). Sampling was conducted from April to October (Table 1) to compare spring and autumn samples. In the laboratory, a fragment of tissue from each individual was preserved in silica gel for molecular analysis. In addition, at least one specimen from each site was pressed and mounted on a herbarium sheet to preserve anatomical features (vouchers were deposited in the herbarium of the Museum National d'Histoire Naturelle, Paris, France, nos. PC0533607-PC0533613).

To complete the sampling, two strains of reference (1330/1 and 1330/2) of P. littoralis from the Culture Collection of Algae and Protozoa (CCAP) isolated respectively in Roscoff in 1970 and 2002, were included in the analysis.

DNA extraction, amplification and sequencing

DNA was extracted from 5-10 mg of dry algal tissue using the Nucleospin® Multi-96 plant kit (Macherey-Nagel GmbH and Co. KG) according to the manufacturer's protocol except for the cell lysis buffer in which samples were left at room temperature for one hour rather than heating to 65°C for 30 min. Sequences of three mitochondrial genes cox1 (658 nt), nad1 (800 nt) and atp9 (200 nt), and of the two plastid genes rbcL (1300 nt) and atpB (630 nt) were amplified and sequenced. Primers used and PCR reactions performed are described in [START_REF] Silberfeld | A multi-locus time-calibrated phylogeny of the brown algae (Heterokonta, Ochrophyta, Phaeophyceae): investigating the evolutionary nature of the "brown algal crown radiation[END_REF]. cox1 and rbcL genes were sequenced for all specimens (i.e., 140

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individuals of P. littoralis and two CCAP strains). For the other mitochondrial and chloroplast genes (nad1, atp9 and atpB) a subsample of 45 individuals of P. littoralis was used to confirm results obtained with the previous genes. PCR products were purified and sequenced by LGC genomics (Berlin, Germany). The obtained sequences were edited and aligned using Codoncode Aligner v. 3.7 (www.codoncode.com, Centerville, Massachusetts, USA).

Phylogenetic approach

Phylogenetic relationships among mitochondrial DNA (mtDNA) and chloroplastic DNA (cpDNA) haplotypes were reconstructed using median-joining networks with Network version 4.2.0.1 [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF]. Mismatch distribution were analyzed for cox1 and rbcL genes. Pairwise comparisons were calculated for each gene to determine the threshold between intraspecific and interspecific divergences [START_REF] Puillandre | ABGD, Automatic Barcode Gap Discovery for primary species delimitation[END_REF].

Population genetic approach

Microsatellite markers developed for Ectocarpus siliculosus (Dillwyn) Lyngbye [START_REF] Cock | The Ectocarpus genome and the independent evolution of multicellularity in brown algae[END_REF] were transferred to P. littoralis. Of the 60 microsatellites analyzed, 30 showed repeatable positive amplification, but only 10 showed a legible signal after sequencing (Table 2). These 10 markers were used for the population analysis on the 140 specimens of P. littoralis according to the following method. PCR was carried out in a 10 µL volume of the following components: 2 µL of DNA template diluted 1:25, 150 µM of dNTPs (Thermo Fisher Scientific Inc., Waltham, MA, USA), 30 pmol forward fluorescent-labelled primer FAM, Yakima Yellow (VIC), ATTO550 (NED) or ATTO565(PET) (Eurofins MWG Operon, Ebersberg, Germany), 30 pmol reverse primer (Eurofins MWG Operon, Ebersberg, Germany), 2.5 mM of MgCl 2 (Promega Corp.), 1 X GoTaq® Flexi Buffer (Promega Corp.), Accepted Article and 0.35 U GoTaq® DNA polymerase (Promega Corp.). PCR cycling included an initial denaturing step at 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 50 or 54°C for 30 s and, 72°C for 30 s with a final elongation step of 72°C for 10 min. PCR products were pooled and diluted (1:10) and 2 μL were added to a mixture containing 9.5 μL of Hi-Di formamide (Life Technologies Corporation) and 0.5 μL of SM594 size standard [START_REF] Mauger | A simple and cost-effective method to synthesize an internal size standard amenable to use with a 5-dye system[END_REF]). This loading mix was denatured (95ºC for 5 min) and run in a 3130XL Genetic Analyzer (Life Technologies Corporation). Alleles were scored using GeneMapper ® v. 4.0 (Applied Biosystems TM ).

Standard measures of genetic diversity were estimated using GenAlEx version 6.5 [START_REF] Peakall | GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research--an update[END_REF]: mean number of alleles per locus (N a ) was calculated in 10 populations corresponding to cluster of individuals assigned to different cytotypes for each sample (Table 1). Expected heterozygosity (H e ) was estimated when the number of individuals per cluster was higher than 10. Moreover, all pairs of loci across sites were tested for linkage disequilibrium using a Markov chain approximation of an exact test as implemented in GENEPOP web version 4.0.10 [START_REF] Raymond | GENEPOP (Version 1.2): Population Genetics Software for Exact Tests and Ecumenicism[END_REF] and the genetic structure between sampled populations was computed by calculating an estimate of F ST [START_REF] Weir | Estimating F-Statistics for the analysis of population structure[END_REF]. For each population, allele frequencies were calculated at all 10 loci. These data were summarized and statistically tested with a factorial correspondence analysis (FCA) performed using GENETIX software [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations[END_REF]) and a principal component analysis (PCA) performed using PCA-GEN software (version 1.2; Goudet 1999). The statistical significance associated with each axis was evaluated after 10,000 randomizations.

In a separate analysis, the most likely number of genetic clusters was inferred using STRUCTURE version 2.2 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] on the seven sampled sites. This program was run under the admixture and independent allele frequency model using a Markov chain

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Monte Carlo length of 250000 steps after a burn-in of 50,000. The number of clusters (K) was determined by estimating the probability of the data [log Pr (X|K)] for runs with K= 2-10, averaged over 20 independent runs. The value of K that most likely captured the major structure patterns in the data was determined as the smallest K at which average log Pr (X|K) estimates leveled out. For the selected K, results from replicate runs were averaged with CLUMPP version 1.1.2 [START_REF] Jakobsson | CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure[END_REF]) and displayed graphically using DISTRUCT version 1.1 [START_REF] Rosenberg | DISTRUCT: a program for the graphical display of population structure[END_REF]).

RESULTS

Haplotypic diversity

Sequence comparisons of the 5' end of the cox1 gene (658 nt) obtained for 142 specimens revealed a total of 30 polymorphic sites (up to 3.2% divergence) corresponding to 17 mitotypes (Fig. 2). These mitotypes were clearly separated into two distinct clusters, named MtA and MtB, with respectively 12 and 5 mitotypes (GenBank accession number: KM027317 to KM027333). Analysis of mismatch distributions of the cox1 sequence, considering all the analyzed individuals is shown in Figure 2 (inset). The bimodal distribution (first peak centered on 2 differences and the second on 18 differences) confirmed the existence of a barcode gap (i.e., intergroup differences greater than intragroup differences) between cytoplasmic clusters MtA and MtB. A minimum divergence of 16 nucleotides (2.4%) was observed between mitotypes of these two groups. Group MtB was mainly represented by three mitotypes (M1b, M2b and M3b) and one mitotype was unique, found in only one individual (M5b). In MtB, the intragroup haplotypic divergence ranged from 0 to 0.45%. Group MtA was mostly represented by four mitotypes (M1a, M2a, M3a and M4a), with two mitotypes found in only one individual (M8a and M9a) and the

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intragroup haplotypic divergence ranged from 0 to 0.9%. Similar results were obtained for other sequenced mitochondrial genes nad1 (1.9-2.1% divergence between mitotypes; GenBank accession number: KP058512, KP058513 and KM057703 to KM057706) and atp9 (1.5-2.0%; GenBank accession number: KM057708 to KM057710) with respectively GenBank accession number: KM057683 to KM057702) with 12 polymorphic sites (up to 0.7% divergence); nevertheless these chlorotypes did not cluster in conspicuous groups (Fig. 3). The two cytoplasmic groups MtA and MtB, resulting from cox1 clustering, shared 47% of the chlorotypes (7 of the 15 haplotypes found in at least two individuals), whereas six were unique to the MtA group (CL8, CL9, CL13, CL14, CL16 and CL17) and seven were unique to the MtB group (CL3, CL4, CL10, CL11, CL15, CL18 and CL20). Group MtB was mostly represented by two chlorotypes (CL3 and CL4), whereas Group MtA was represented mostly by a single haplotype (CL1). Results for the atpB gene (GenBank accession number: KM057707) were congruent with those of rbcL, showing one haplotype shared by MtA and MtB.

Temporal distribution of the two mitochondrial clusters

Although both mitotype clusters MtA or MtB were found together in the field (except in October), a seasonal shift was observed in their frequencies. The maximum frequency of

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individuals with MtA cytoplasm (83.6%) was found in spring (April), whereas the maximum frequency of individuals with MtB cytoplasm (63.3%) was observed in autumn (October; Table 1).

Structure of populations

All the microsatellite genotypes observed in this study were heterozygous for at least one microsatellite locus. Based on the mitochondrial clusters, individuals were assigned to the two groups, MtA and MtB (Table 1). The total number of alleles observed over all samples ranged from 18 to 52 from the least to the most polymorphic locus (M028-1 and M194, respectively, Table 2). For localities with more than 10 samples, the mean number of alleles per locus (N a ) ranged from 6.7 to 14 (Table 1). Over a total of 353 alleles, 153 (43%) were found only in the MtB cluster and 94 (26%) were found only in the MtA cluster (i.e. private alleles) with frequencies oscillating between 0.014 and 0.208. Gene diversity (H e ) ranged from 0.608 to 0.741. No repeat multilocus genotypes and no multilocus monomorphic genotypes were detected. There was no evidence of linkage disequilibrium between pairs of loci (data not shown) except for one pair of loci (M105-1/M194), which remained significant after sequential Bonferroni correction.

The results of the PCA (Fig. 4a) showed a significant overall F ST value of 0.22 (P = 0.0001).

The first two PCA axes were significant and explained 64% of the variance: axis 1 accounted for 50.8% of the variance and axis 2 accounted for 13.6% of the variance. Axis 1 separated MtA samples from MtB samples. The factorial correspondence analysis (FCA) confirmed the partitioning into two main groups of individuals along axis 1 corresponding to mitotypes MtA and MtB (Fig. 4b). Within the MtB mitotype group, individuals were partitioned according to sampling date, whatever their origin. Individuals sampled in autumn in St. Malo and in Roscoff clustered together, whereas individuals sampled in spring in these Accepted Article sites were intermediate between the MtB individuals and the MtA mitotype group (Fig. 4b).

Again, the two FCA axes were significant and explained 45% of the variance: axis 1 accounted for 28% of the variance (F ST = 0.11, P = 0.001) and axis 2 accounted for 17% of the variance (F ST = 0.03, P = 0.815). Genetic differentiation between pairs of samples (F ST ) (Table 3) ranged from 0.07 (samples 4 vs. 1) to 0.24 (samples 4 vs. 7). The lowest F ST values were observed between samples assigned to the same mitotype, whereas the highest values were observed for samples assigned to mitotypes sampled in spring and in autumn.

Regarding the analysis of structure, the ΔK criterion separated the seven spatiotemporal samples into two "clusters" (K=2). The proportion of genotypes assigned to each of the two clusters is given in Figure 5. The first cluster included virtually all the MtA samples and the second cluster includes MtB samples. This result confirms the PCA (and FCA) results, which grouped these samples along axis 1 (Fig. 4). However, when individuals were sampled together in the same location (2, 3 and 6), admixture was observed. Five individuals from Dinard (sample 3) with MtB mitotypes were assigned to the first (MtA-dominant) cluster (Fig. 5).

DISCUSSION

Our findings revealed that Pylaiella littoralis comprises two cryptic species that have thus far been overlooked in Brittany, since only one had been recognized after the work of [START_REF] Feldmann | Inventaire de la flore marine de Roscoff. Algues, champignons, lichens et spermatophytes[END_REF]. Both phylogenetic inferences and population genetic studies highlighted the existence of two genetic entities under the name of P. littoralis. The level of cox1 divergence observed between the two mitochondrial clusters (MtA and MtB) of P. littoralis (2.4%-3.2%), was substantially greater than values detected within a cluster (0.0%-0.9%).

Thus, our results are in agreement with the recent proposals on Desmaretiales [START_REF] Yang | Ligulate Desmarestia (Desmarestiales, Phaeophyceae) revisited: D. japonica sp. nov. and D. dudresnayi differ from D. ligulata[END_REF] and Ectocarpales (Peters et al. 2015 in press) that estimated species-level cut-off at Accepted Article divergence of about 1.2% and 1.8% respectively. Furthermore, the common chlorotypes observed in the two P. littoralis species suggest that differentiation is recent. Therefore, these two closely related species of the P. littoralis complex should be regarded as sibling species [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF].

Our results confirm that chloroplastic markers have lower mutation rates than mitochondrial markers (e.g., for brown algae: [START_REF] Presting | Identification of conserved regions in the plastid genome : implications for DNA barcoding and biological function[END_REF][START_REF] Engel | Conservation and polymorphism of mitochondrial intergenic sequences in brown algae (Phaeophyceae)[END_REF][START_REF] Tellier | Phylogeographic analyses of the 30°S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: vicariance or parapatry?[END_REF].

Difference in resolution found between mitochondrial and plastid markers can be explained by the difference in effective population size [START_REF] Palumbi | Predicting nuclear gene coalescence from mitochondrial data: the three-times rule[END_REF]. In Phaeophyceae, mitochondria are maternally inherited (but see [START_REF] Hoarau | Paternal leakage of mitochondrial DNA in a Fucus (Phaeophyceae) hybrid zone[END_REF] in Fucus species), whereas plastid may be partly biparentally inherited [START_REF] Peters | Inheritance of organelles in artificial hybrids of the isogamous multicellular chromist alga Ectocarpus siliculosus (Phaeophyceae)[END_REF]). [START_REF] Peters | Inheritance of organelles in artificial hybrids of the isogamous multicellular chromist alga Ectocarpus siliculosus (Phaeophyceae)[END_REF] suggested that the biparental chloroplast inheritance observed in Ectocarpus siliculosus may be widespread in isogamous brown algae. For example, biparental chloroplast inheritance is observed in Scytosiphon lomentaria (Lyngbye) Link [START_REF] Kato | Inheritance of mitochondrial and chloroplast genomes in the isogamous brown alga Scytosiphon lomentaria (Phaeophyceae)[END_REF]). However, although the genus of Pylaiella is phylogenetically related to Ectocarpus and Scytosiphon, no information is available on chloroplast inheritance.

Nuclear microsatellite markers consistently recovered the two well-supported cox1 mitochondrial clusters (i.e., MtA and MtB), suggesting that gene flow is limited between taxa. Likewise, we observed high values of genetic differentiation (F ST ) between pairs of allopatric populations (1, 4, 5, 7) that were assigned to different clusters. However, when their spatio-temporal distribution intersects (i.e., sympatric populations in which individuals MtA and individuals MtB were sampled simultaneously in spring and at the same site), admixtures was found (Fig. 5). The FCA showed that individuals with MtB mitotypes sampled in the spring were genotypically intermediate to the MtA group and the other MtB mitotypes (Fig. 4). These results suggest that there is some contemporary gene flow

Accepted Article

reflecting the fact that reproductive isolation is not complete. However, we argue that effective gene flow is too low to compromise the integrity of these two genetic entities.

Interspecific gene flow is well documented in some other brown algae, such as in the genus Fucus resulting from hybridization and introgression [START_REF] Wallace | Determining the affinities of salt marsh Fucoids using microsatellite markers: evidence of hybridization and introgression between two species of Fucus (Phaeophyta) in a Maine estuary[END_REF][START_REF] Billard | Genetic isolation between three closely related taxa: Fucus vesiculosus, F. spiralis, and F. ceranoïdes (Phaophyceae)[END_REF], 2010, Engel et al. 2005[START_REF] Coyer | Hybridization and introgression in a mixed population of the intertidal seaweeds Fucus evanescens and F. serratus[END_REF], 2011[START_REF] Zardi | Adaptive traits are maintained on steep selective gradients despite gene flow and hybridization in the intertidal zone[END_REF]. For example, [START_REF] Engel | Genetic entities and mating system in hermaphroditic Fucus spiralis and its close dioecious relative F. vesiculosus (Fucaceae, Phaeophyceae)[END_REF], using microsatellite markers, identified about 13% of hybrids in sympatric natural populations of Fucus spiralis Linnaeus and Fucus vesiculosus Linnaeus. [START_REF] Coyer | Evolution and diversification within the intertidal brown macroalgae Fucus spiralis/F. vesiculosus species complex in the North Atlantic[END_REF] suggest that despite introgression, the maintenance of species integrity of F. spiralis, F. vesiculosus and Fucus guiryi G.I. Zardi, K.R. Nicastro, E.S. Serrão & G.A.

Pearson is due to a combination of limited gamete dispersal capacities, reproductive system as well as ecological factors reinforcing the divergence between species. In externally fertilizing species, asynchronous gamete release between related taxa is considered the major prezygotic barrier to hybridization [START_REF] Levitan | Mechanisms of reproductive isolation among sympatric broadcast-spawning corals of the Montastraea annularis species complex[END_REF][START_REF] Monteiro | Prezygotic barriers to hybridization in marine broadcast spawners: reproductive timing and mating system variation[END_REF]).

In our study, although the two sibling species are broadly sympatric, a temporal niche segregation may promote reproductive isolation. Our observations showed that the species characterized by the MtA mitotype was present in spring and absent in autumn whereas, the other species characterized by the MtB mitotype, was mainly observed in autumn. Although more accurate information on reproduction is not available, these apparent phenological differences may act as a prezygotic barrier favoring reproductive isolation. Moreover, this difference in phenology implies that both species probably face different biotic (e.g., grazers, competition, host) and abiotic parameters (e.g. temperature, day length, irradiance) during their peak of abundance. Such differences probably have important evolutionary consequences for reproductive isolation and sympatric speciation. Furthermore, hybridization in natural populations depends on both intrinsic (i.e., environment independent) and extrinsic reproductive barriers. Extrinsic barriers can greatly

Accepted Article

affect the success of interspecific crosses, either by favoring prezygotic barriers (phenology, synchrony of spawning) or by limiting the success of hybrids via habitat-dependent or negative frequency-dependent selection [START_REF] Tellier | Possible role of a mitochondrial genome rearrangement in maintaining the spatial segregation of two cryptic species of the Lessonia nigrescens species complex[END_REF].

The life history of the genus Pylaiella remains poorly known and controversial, as several patterns have been observed (Müller and Stache 1989). Kylin (1933) noticed the occurrence of sporophytes and gametophytes in natural populations in Sweden and [START_REF] Knight | Studies in the Ectocarpaceae I. The life-history and cytology of Pylaiella littoralis (L.) Kjellm[END_REF] demonstrated the alternation sporophytes and gametophytes in culture, in material from Isle of Man (Irish Sea). However, in different strains from Helgoland (North Sea) and Chile (Pacific), this species reproduces only asexually (Müller and Stache 1989). The microsatellite results obtained in our study suggest that the two sibling species of Pylaiella littoralis reproduce mainly sexually. Moreover, it is noteworthy in our study that the two cryptic species showed high values of gene diversity (H e ), suggesting that these species are characterized by large populations. The range of genetic diversity within populations was similar to that of microsatellite data in kelps [START_REF] Valero | Using genetic tools for sustainable management of kelps: a literature review and the example of Laminaria digitata[END_REF].

In conclusion, our study illustrates that the combination of phylogenetic and population genetics is a powerful approach for understanding and delimiting species status in seaweed. Two sibling species were detected in P. littoralis as distinct genetic entities with limited gene flow. We nevertheless did not assign names to the two species uncovered in this study because the genus Pylaiella encompasses roughly 90 specific and intraspecific taxa [START_REF] Guiry | [END_REF]), all of which would have to be regarded as possible names of the two species in application of Article 11 of the International Code of Nomenclature for algae, fungi, and plants [START_REF] Mcneill | International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) adopted by the eighteenth international botanical congress[END_REF]. Unfortunately, we are presently unable to identify putatively diagnostic characters for either species uncovered in this study and or in the type materials, which prevents bridging the gap between the classical taxonomical approach and Accepted Article modern species delimitation methods. Our study highlights how complementary barcoding and population genetics analyses can help solve taxonomic questions at short phylogenetic distances. However, further understanding of speciation and of the maintenance of integrity of these different species requires phenological, biological, ecological and genetic experiments to document sexual reproduction and to reveal the differences in their ecological tolerance. 1).

Accepted Article

The values of F ST were calculated only for populations with more than 10 individuals (N>10). All F ST values were significant (P < 0.001). In grey, the highest F ST values between pairs of samples assigned to different mitotypes. indicate the s e name (see Table 1). axis is given in parentheses. For a key to sample names, see Table 1. (MtA, black; MtB, white). The seven samples are separated by vertical gray lines (see Table 1).

  20 and 4 polymorphic sites (Figs. S1 and S2 in the Supporting Information). The sequence of one CCAP strain of Pylaiella littoralis (1330/1) corresponded to haplotype M1b of the MtB cluster, whereas the sequence of the 1330/2 strain corresponded to the haplotype M5a of the MtA cluster. Individuals with the cytoplasm MtA and MtB were found in all locations except in Concarneau (site at which sampling was done only once, in spring) where all individuals analyzed only had MtA mitotypes. Analysis of rbcL sequences (1321 nt) showed high chlorotype diversity (20 chlorotypes;

Figure 1 .

 1 Figure 1. Map of the four sampled locations along the Brittany coast (France), Saint Malo

Figure 2 .

 2 Figure 2. Median-joining network of the relationships among haplotypes of the cox1 coding

Figure 3 .

 3 Figure 3. Median-joining network of the relationships among haplotypes of the rbcL coding

Figure 4 .

 4 Figure 4. (a) Principal component analysis (PCA) on population allele frequencies and (b)

Figure 5 .

 5 Figure 5. Bayesian clustering of microsatellite data obtained for all samples (considering all

Table 1 .

 1 Characteristics of Pylaiella littoralis samples; N T: total number of sampled individuals, N: number of individuals assigned to mitotype MtA or MtB according to cox1 sequences, N cox1: number of mitotypes cox1, N rbcL: number of chlorotypes rbcL, N a :

	mean number of alleles per microsatellite locus; H e : microsatellite multilocus non-biased
	expected heterozygosity; -: no data.
	Location	N T	Sample name	Date	Mitotype N	N cox1	N rbcL	N a H e
	Saint Malo (ST)	41
			1	15 October 2008	MtB	32 4	6 14 0.672
			2	15 June 2009 15 June 2009	MtB MtA	5 4	2 4	2 4.8 -4 3.9 -
	Dinard (DI)	16
			3	21 April 2010 21 April 2010	MtB MtA	12 2 4 2	6 6.7 0.694 3 3.6 -
	Roscoff (RO)	62
			4	26 October 2008	MtB	18 2	1 9.6 0.658
			5	29 April 2009	MtA	26 6	9 9.6 0.608
			6	14 May 2010 14 May 2010	MtB MtA	12 3 6 3	4 8.5 0.741 1 4.4 -
	Concarneau (CO) 21
			7	19 April 2011	MtA	21 2	1 9.6 0.617

Table 2 .

 2 List and characteristics of microsatellite makers used for population analysis. N a: number of alleles detected at each locus.

	Marker name Gene Location M-168 No Gene M-239-3 No Gene M-383-1 Intron M-392 Intron M-199-3 No Gene M-231 Exon M-028-1 Intron M-105-1 Intron M-138 No Gene M-194 Intron ccepted Article	Length Left primer (5'-3') 76 AAACTACACCAACGAAATCAA AACCAACCTCCTCCAATC Right primer (5'-3') 66 ATAAGACTCAGCAAGAGGCAG CATTGAACGACAAATCCC 76 GGGTGAGACAGTAGTAGCAGG CTCGGAACAGGAAGGTTT 137 CTTGTGTGCGTGCTGTTG GACTTAGATTTCCGTGTCCTC 54 Tm (°C) 50 50 50 42 GCAGTAGTAGCAGTAGTCGG TTCATTGTGTTCTTCTTCGTC 50 91 CGGTGACAGATAGACAGACAG CCCTCAGTTGTTCCTCAAGT 50 126 ATTACAGCAAGGACAAATGG TAAGAAGGACTGGTGGAGG 54 106 CTACTCTCCGCAATAAAGACA AGGTATGAACACGACGACAG 50 69 TGTCTGAATGTTGATGTGTGT AGGGTAGGCAAAGAATGG 50 91 GTCGGATGACGAGGACTT AATGTTTATTTAGGCTTTGCT 50	Product size (bp) 279 241 240 304 227 193 262 298 271 296	N a 37 42 50 21 18 38 18 45 33 51

Table 3 .

 3 Pairwise population genetic differentiation (pairwise F ST estimates). All F ST values were significant (P < 0.001). The numbers (1 to 7) refer to the sample name (see Table

Acknowledgments

We thank M. Valero, A. Peter, S. de Goer, B. de Reviers and C. Engel for helpful comments. We are also most grateful to the Biogenouest Genomics core facility for its technical support. This study was partially supported by grants from the French Ministry for Economy, Industry and Employment and the Brittany Regional Council through the project AQUACTIFS "Convention de soutien de l'état à des actions de recherche et d'innovation par voie de subvention -Fonds de compétitivité des entreprises", the project IDEALG (ANR-10-BTBR-04) 'Investissements d'avenir, Biotechnologies-Bioressources' and ASSEMBLE grant no. 227799. Special thanks to CCAP for providing strains of Pylaiella littoralis.

Accepted Article

Supplementary material