Marcelino T Suzuki
email: suzuki@obs-banyuls.fr

Delphine Parrot

Gabriele Berg

Martin Grube

Sophie Tomasi

Lichens as natural sources of biotechnologically relevant bacteria

Keywords:

come

Introduction

Recently, the World Health Organization (WHO) produced a global map of antimicrobial resistance and issued a warning that a 'post-antibiotic' world could soon become a reality and has already started [START_REF] Woolhouse | Policy: An intergovernmental panel on antimicrobial resistance[END_REF]. Therefore, the quest for new drugs from microbial sources is currently at a crossroads, where on one hand it is widely thought that the discovery of novel compounds has passed its "golden era" with many pharmaceutical companies abandoning or downscaling their microbial natural products research, while on the other hand, full genomic sequencing and metagenomic research shows that a large number of "silent" or novel biosynthetic pathways exist (for reviews see [START_REF] Brady | Metagenomic approaches to natural products from free-living and symbiotic organisms[END_REF][START_REF] Ochi | New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters[END_REF], promising to lead to novel bioactive compounds produced via heterologous expression (e.g. [START_REF] Chang | Discovery of indolotryptoline antiproliferative agents by homology-guided metagenomic screening[END_REF]. The continued search for microorganisms from novel sources represents an effective strategy forward towards drug discovery. In this context microbial symbioses are of particular interest, since increasing evidence supports the hypothesis that microbial bioactive compounds, such as those used as antibiotics, might be involved in microbe-microbe and microbe-host communication [START_REF] Yim | Antibiotics as signalling molecules[END_REF][START_REF] Yoon | Activating secondary metabolism with stress and chemicals[END_REF]). An example supporting this hypothesis, is the fact that many interesting novel bioactive compounds of microbial origin have been discovered in marine sponges, known to harbor complex symbiotic microbial communities (see Fuerst 2014 for a review).

Lichens are a well-recognized, self-supporting, mutualistic symbiosis between a dominant fungal partner (mycobiont) that provides shelter for one or several photosynthetic green algae and/or cyanobacteria (photobionts) forming a unique symbiotic structure, the lichen thallus, (De [START_REF] Bary | Cyanobacterial toxins: biosynthetic routes and evolutionary roots[END_REF][START_REF] Frank | Ueber die biologischen Verhältnisse des Thallus einiger Krustenflechten[END_REF][START_REF] Schendener | Untersuchunger über des Flechtentallus[END_REF]reviewed in Honegger 2000).

Lichen-forming fungi have evolved for at least 400 million years [START_REF] Taylor | The oldest fossil lichen[END_REF]. The lichen thallus is well-adapted to a wide variety of specific ecological niches, often including extreme environmental conditions (e.g. extreme temperatures, periodic desiccation, high levels of UV radiation). At least some of the mechanisms to cope with these extreme conditions are of a chemical nature (e.g. UV screens, cryoprotectants, osmolites) providing leads and inspiration for biotechnological development. It also appears that allelopathic compounds by the holobiont could play a role in the persistence of lichens. Despite estimated individual ages of decades to centuries, there are no reports of destructive bacterial infections in lichens. The capacity of produce a broad range of interesting chemical compounds help explain the fact, that lichens have been historically also used as sources of pigments, perfumes, human medicines etc. [START_REF] Rikkinen | What's behind the pretty colours?: a study on the photobiology of lichens[END_REF].

A number of chemical structures have consistently been associated with lichens and due to their presence in axenic culture or other non-lichenizing fungi, it is generally accepted that they are produced by the mycobiont. Those include repeats of aromatic acids such as orsellinic acid (depsides, depsidones and usnic acids) that are believed to be synthesized by non-reducing fungal polyketide synthases [START_REF] Huneck | Identification of lichen substances[END_REF][START_REF] Robinson | The genetics of industrial microorganisms: the first half century[END_REF], that have been directly recovered from lichens (i.e. [START_REF] Grube | On the phylogeny of some polyketide synthase genes in the lichenized genus Lecanora[END_REF][START_REF] Muggia | Type III polyketide synthases in lichen mycobionts[END_REF][START_REF] Schmitt | 6-MSAS-like polyketide synthase genes occur in lichenized ascomycetes[END_REF]. The diversity of these major compounds have been subject of recent reviews and catalogues [START_REF] Boustie | Lichens, a promising source of bioactive secondary metabolites[END_REF][START_REF] Elix | A Catalogue of Standardized Chromatographic Data and Biosynthetic Relationships for Lichen Substances[END_REF][START_REF] Molnár | Current Results on Biological Activities of Lichen Secondary Metabolites: a Review[END_REF][START_REF] Shrestha | Lichens: a promising source of antibiotic and anticancer drugs[END_REF][START_REF] Shukla | Lichens as a potential natural source of bioactive compounds: a review[END_REF][START_REF] Stocker-Woergoetter | Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimate metabolite production, and PKS genes[END_REF]) and therefore we will focus here on those produced by other microorganisms, in particular Bacteria, although we cannot rule out that these bacteria can be also involved in the biosynthesis or conversion of major compounds found in lichens.

Culturable bacteria of biotechnological interest

While the presence of microorganisms other than the mycobiont and the photobiont in the lichen holobiont has long been known by isolation techniques [START_REF] Uphof | Purple bacteria as symbionts of a lichen[END_REF]reviewed in Cardinale et al. 2006), systematic attempts to study these organisms have only recently been carried out. One early study [START_REF] Gonzalez | Actinomycetes isolated from lichens: Evaluation of their diversity and detection of biosynthetic gene sequences[END_REF] published 10 years ago clearly demonstrated that lichens are sources of bioactive microorganisms. That study focused on Actinobacteria by employing a targeted isolation strategy using nalidixic acid (an inhibitor of Gram-negative bacteria) as well as heat-treatment prior to cultivation. A standard medium (Yeast Malt Extract) was used for isolation from 25 terrestrial lichen samples from Hawaii, Alaska and the Reunion Islands. The lichens species were not identified and the samples were just classified as saxicolous (i.e. rock associated; 6 from Alaska, 5 from Hawaii) or arboricolous (i.e tree-associated; 5 from Hawaii and 9 from the Reunion Islands). A large number (337) of strains were obtained, and identified based on DNA fingerprinting and fatty acid analysis. Among these strains, 110 belonged to the family Streptomycetaceae, wellknown to produce bioactive compounds. Other actinobacterial families also know for the production of bioactive compounds, some belonging to the "rare Actinomycetes" sensu [START_REF] Subramani | Culturable rare Actinomycetes: diversity, isolation and marine natural product discovery[END_REF], such as Micromonosporaceae (142 strains), Pseudonocardiaceae (30 strains) and Thermomonosporaceae (7 strains) were also isolated. These strains were screened for genetic potential for biosynthesis (PCR targeting genes involved in polyketide, small peptides, and isoprenoid biosynthesis), as well as antimicrobial activity (against Escherichia coli, Staphylococcus aureus and Candida albicans). A high percentage of strains (over 60%) potentially contained at least one biosynthetic cluster and about 30% of the strains had antimicrobial activity against at least one of the target microorganisms.

Since the pioneering study by Gonzalez and colleagues (2005), many other studies have focused on lichen-associated bacteria via cultivation. A number of these studies have targeted individual isolates or species, especially for taxonomic purposes or for full genomic sequencing (Table 1). A particular interest was given to biotechnological potential of strains, since in several cases isolation selected for Actinobacteria by nalidixic acid addition (9 out of 23 strains). Finally in some cases [Actinoplanes sp. ATCC55532 [START_REF] Singh | Inhibitor compounds of farnesyl-protein transferase and chemotherapeutic compositions containing the same, produced by strain ATCC 55532[END_REF], [START_REF] Davies | Uncialamycin, a new enediyne antibiotic[END_REF], Streptomyces sp. [START_REF] Motohashi | A new angucycline and a new butenolide isolated from lichen-derived Streptomyces spp[END_REF], S. cyaneofuscatus [START_REF] Brana | Two Streptomyces species producing antibiotic, antitumor, and anti-inflammatory compounds are widespread among intertidal macroalgae and deep-sea coral reef invertebrates from the central Cantabrian sea[END_REF] and Streptomyces sp. [START_REF] Lavallee | Antipain and its Analogues, Natural Product Inhibitors of Cathepsin K Isolated from Streptomyces[END_REF]] the focus was primarily on the discovery of novel bioactive molecules, with the first strain described in a patent [START_REF] Singh | Inhibitor compounds of farnesyl-protein transferase and chemotherapeutic compositions containing the same, produced by strain ATCC 55532[END_REF]. These strains will be discussed in a later section. It is remarkable that in several of these studies there is only a poor description or a complete lack of identification of the source lichens.

Streptomyces uncialis

A different class of culture-based studies performed a more comprehensive isolation of lichen associated bacteria, basing the isolation on criteria other than bioactivity and, except for our recent study [START_REF] Parrot | Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep in press Reichenbach H[END_REF], did not target Actinobacteria (Table 2). The results of these studies offer a less biased view of the cultivable bacterial diversity associated with lichens, and it is clear that certain bacterial phyla and classes, in particular Alpha-and Gammaproteobacteria, Firmicutes and Actinobacteria are frequently isolated from lichens [START_REF] Cardinale | Molecular analysis of lichen-associated bacterial communities[END_REF][START_REF] Grube | Species-specific structural and functional diversity of bacterial communities in lichen symbioses[END_REF][START_REF] Selbmann | Culturable bacteria associated with Antarctic lichens: affiliation and psychrotolerance[END_REF]. In addition Bacteroidetes seem to be isolated from marine or littoral lichens [START_REF] Parrot | Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep in press Reichenbach H[END_REF][START_REF] Sigurbjornsdottir | Novel bacteria associated with Arctic seashore lichens have potential roles in nutrient scavenging Can[END_REF]). Among all isolates, many belong to bacterial families known to contain strains know to produce bioactive compounds, enzymes of biotechnological interest such as lipases proteases and antagonistic molecules (Table 2). In a few cases this was experimentally tested [START_REF] Grube | Species-specific structural and functional diversity of bacterial communities in lichen symbioses[END_REF][START_REF] Lee | Biodiversity and physiological characteristics of Antarctic and Arctic lichens-associated bacteria[END_REF], while in others studies this potential was inferred by the presence of genes involved in the biosynthesis of polyketides or 16S rRNA similarity to known bioactive strains [START_REF] Parrot | Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep in press Reichenbach H[END_REF]. In the latter study the authors focused on the isolation of Actinobacteria from marine and littoral lichens, and showed that these lichens were sources of bacteria of biotechnological interest, and that the use of Marine Agar, a medium used to the isolation of a broad diversity of marine bacteria, yielded many strains that were not recovered with the commonly used Actinomycete Isolation Agar medium, and this did not appear to be an effect of salinity [START_REF] Parrot | Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep in press Reichenbach H[END_REF]. Among these stains some appeared to represent novel species or even genera. Finally that study compared the 16S rRNA genes to those in the NCBI Genbank from bacteria associated with lichens studied using culture independent methods and as expected none of the strains appeared to be prevalent in the studied lichens.

To obtain a larger representation of lichen associated strains, including strains for which only 16S rRNA sequences exist, we searched all 16S rRNA gene sequences present in the NCBI Genbank nt database with the query "lichen AND bacteria [orgn] NOT Cyanobacteria [orgn] NOT uncultured" and recovered the taxonomy information associated with these sequences (associated or not to publications), and obtained somewhat similar results, which again might be a reflection of the targeting of Actinobacteria in isolation efforts even though this analysis did not include the study of Gonzalez and colleagues and Parrot and colleagues that have focused on Actinobacteria [START_REF] Gonzalez | Actinomycetes isolated from lichens: Evaluation of their diversity and detection of biosynthetic gene sequences[END_REF][START_REF] Parrot | Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep in press Reichenbach H[END_REF].

Among 511 bacterial strains retrieved, nearly half of isolates ( 246 Finally, a number of studies have attempted to cultivate cyanobacteria (mostly the main photobionts) from lichens (Table 3). In addition to the search of bioactive compounds of pharmacological the main interest a few of these studies was the production of toxins or other metabolites by these cyanobacteria strains, and in particular hepatoxins relevant to public health (see Dittmann et al. 2013 for a review). Extensive genetic and chemical screening of lichens of the lichen order Peltigerales, known of for their association with the cyanobacterial genus Nostoc have clearly shown that a non-negligible (about 5 % of specimens) contain microcystins or nodularins [START_REF] Kaasalainen | Lichen species identity and diversity of cyanobacterial toxins in symbiosis[END_REF]) most likely produced by symbiotic cyanobacteria. In addition to the original interest for public health, the use of microcystin congeners has also recently been proposed for cancer therapy [START_REF] Niedermeyer | Selectivity and Potency of Microcystin Congeners against OATP1B1 and OATP1B3 Expressing[END_REF], demonstrating the interest in these lichen associated cyanobacteria.

Summarizing, existing information from culture-based studies clearly show that lichens can be a source of bacterial strains of biotechnological interest such as Bacillus, Paenibacillus, Pseudomonas, Burkholderia and Nostoc. Regarding Actinobacteria, interesting strains in this phylum were isolated even without selective approaches. Studies using selective approaches yielded a higher diversity of Actinobacteria and in particular of non-Streptomyces genera, including possible new species. It also appears that cyanobacterial symbionts can be a promising source of bioactive molecules of biotechnological interest.

Strains known to produce bioactive compounds

Despite the large number of bacterial strains of relevance to drug discovery isolated from lichens, the presence of structurally-identified bioactive molecules have only been reported for a few of these strains (Streptomyces uncialis and Actinoplanes sp. ATCC55532,

Streptomyces sp. RI104-LiC106, Streptomyces sp. RI104-LiB101, Streptomyces sp. L-91-3
Nostoc sp., Nostoc sp. ATCC 53789 and Nostoc sp. IO-102-I Tables 1 and3). In addition, identified molecular structures have also been reported for strain S. cyaneofuscatus T178 reported as being phenotypically and metabolically indistinguishable from S. cyaneofuscatus M-27. The relative small number of reported molecules is partly due to the fact that interest in lichen-associated bacteria is relatively recent but also likely due to the fact that publications tend to focus on novel chemical structures, and thus presence of known compounds, even in novel organisms, is seldom reported. In the following paragraphs we describe several lichenassociated bacteria for which bioactive compounds have been identified. The molecular structures of selected compounds are presented in Figure 1.

Actinoplanes sp. ATCC55532 was isolated from a non-identified arboricolous lichen from Spain has been reported to produce actinoplanic acids A and B [START_REF] Singh | Inhibitor compounds of farnesyl-protein transferase and chemotherapeutic compositions containing the same, produced by strain ATCC 55532[END_REF] . This patent also claims that actinoplanic acid B (1) inhibits farnesyl-protein transferase (FPTase) and the farnesylation of the oncogene protein Ras, in vitro at (IC 50 at 50 nM) and thus have a potential for the treatment of colorectal carcinoma, exocrine pancreatic carcinoma and myeloid leukemias.

Streptomyces uncialis, isolated by Davies and co-workers from a Cladonia uncialis, has been the been well characterized and reported to produce the enediyne antibiotic uncialamycin (2) highly active against Escherichia coli, Staphylococcus aureus as well as Burkholderia cepacia [START_REF] Davies | Uncialamycin, a new enediyne antibiotic[END_REF]. The same strain produces the alkaloids Cladoniamides A-G with the latter (3) showing significant in vitro toxicity against MCF-7 breast cancer cells at 10 µg/mL [START_REF] Williams | Cladoniamides A-G, tryptophan-derived alkaloids produced in culture by Streptomyces uncialis[END_REF]. A second lichen-derived Streptomyces strain isolated from lichens by the Davies' group (L-91-3) was reported to produce several inhibitors of Catepsin K (a protease identified as a drug target for osteoporosis therapy [START_REF] Yasuda | The role of cathepsins in osteoporosis and arthritis: Rationale for the design of new therapeutics[END_REF]. These molecules included the oligopeptide Antipain and two analogues described as vince-2 and lichostatinal [START_REF] Lavallee | Antipain and its Analogues, Natural Product Inhibitors of Cathepsin K Isolated from Streptomyces[END_REF].

Strains RI104-LiC106 and RI104-LiB101 reported as belonging to a new Streptomyces species have been shown to produce JBIR-88 (4), a 1,1-dichlorocyclopropane-containing angucycline and JBIR-89 (5), a butenolide. JBIR-88 was cytotoxic to HeLa and ACC-MESO-1 cells IC50 36 and 52 mM, respectively. It also showed antibacterial activity against Micrococcus luteus, but not against Candida albicans and E. coli [START_REF] Motohashi | A new angucycline and a new butenolide isolated from lichen-derived Streptomyces spp[END_REF].

S. cyaneofuscatus M-27 was isolated from the algae Fucus spiralis and reported to be metabolically indistinguishable from S. cyaneofuscatus T-178 isolated from an unidentified lichen. This strain was reported to produce a number of anthracycline family antitumor antibiotics daunorubicin, cosmomycin B (6) and galtamycin B and the antifungal macrolactam maltophilin (7). Daunorubicin is already used as a drug for cancer chemotherapy, cosmomycin B shows in vitro cytotoxicity against the KB human epidermoid carcinoma cell line [START_REF] Ando | The Structures of Anthracycline Antibiotics, Cosmomycins A and B[END_REF] and galtamycin B shows in vitro cytotoxicity against gastric adenocarcinoma (HMO2), breast carcinoma (MCF7), and hepatocellular carcinoma (HepG) cell lines at (10 µg/mL [START_REF] Antal | Retymicin, galtamycin B, saquayamycin Z and ribofuranosyllumichrome, novel secondary metabolites from Micromonospora sp Tu 6368 -I. Taxonomy, fermentation, isolation and biological activities[END_REF]. Maltophilin has been shown to inhibit the growth of a number of fungi [START_REF] Jakobi | Maltophilin: A new antifungal compound produced by Stenotrophomonas maltophilia R3089[END_REF]. It has to be noted however that all molecules reported to be produced by S. cyaneofuscatus M-27 were only identified based on their HPLC retention time and UV-vis spectra [START_REF] Brana | Two Streptomyces species producing antibiotic, antitumor, and anti-inflammatory compounds are widespread among intertidal macroalgae and deep-sea coral reef invertebrates from the central Cantabrian sea[END_REF].

Nostoc strain. ATCC 53789 was isolated from lichens and was shown to produce cryptophycin 1 (8), which was originally patented for its antifungal properties [START_REF] Moore | Method for producing Cryptocins by cultivating a Nostoc sp[END_REF]. Further analysis of other molecules in this class resulted in the discovery of it biosynthetic pathway, and of a strategy for chemoenzymatic synthesis of analogous molecules [START_REF] Magarvey | Biosynthetic characterization and chemoenzymatic assembly of the cryptophycins. Potent anticancer agents from Nostoc cyanobionts[END_REF], Furthermore, a synthetic analog (cryptophycin 52) achieved Phase II clinical trials for lung cancer therapy [START_REF] Edelman | Phase 2 study of cryptophycin 52 (LY355703) in patients previously treated with platinum based chemotherapy for advanced non-small cell lung cancer[END_REF]) at which stage development was stopped. More recently, the use of cryptophycin analogs in antibody-drug conjugates has also been proposed [START_REF] Verma | Trastuzumab Emtansine for HER2-Positive Advanced Breast[END_REF]).

An unnamed Nostoc sp. strain isolated from Peltigera canina was shown to produce the chlorine containing nostoclides I and II, which have moderate toxicity (10 µg/ml) against cell lines Neuro-Pa CCL 131 and KB CCL17. Nostoc sp. strain IO-102-I isolated from a Pannaria pezizoides from Finland, appears in turn to be the only lichen-derived isolate shown to produce a number of microcystins [START_REF] Oksanen | Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp strain IO-102-I[END_REF], despite the report that many lichen specimens contain this class of molecules.

Finally, a recent study has reported that a Nostoc sp. strain N6 isolated from Peltigera membranaceae produces nosperin (9) a pederin like-molecule, produced by a predominantly trans-AT polyketide synthase biosynthetic pathway [START_REF] Kampa | Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses[END_REF]. In this remarkable study Kampa and colleagues predicted -using shotgun metagenomic sequencing and sequence-based biosynthetic analysis -that a Nostoc strain was likely to produce a pederinlike molecule. Based in this information the authors were able to perform targeted isolation of the cyanobacterium that was followed by mass cultivation and isotopic incorporation to finally determine the structure of this molecule present in very low abundance, linking it to its most likely biosynthetic pathway [START_REF] Kampa | Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses[END_REF].

In summary, although interest in lichen-associated biotechnologically relevant bacteria is relatively recent, a number of bioactive and structurally identified molecules were produced by Actinomycetes and Cyanobacteria isolated from lichens, with some novel structures of high potential and some already in drug development.

The yet untapped potential seen through cultivation independent approaches

For the past 10 years, lichen-associated bacteria have been subject of an increasing number of cultivation-independent molecular surveys. These studies employed a number of techniques including fingerprinting of rRNA genes, fluorescence in situ hybridization and sequencing from mixed microbial populations (e.g., [START_REF] Cardinale | Molecular analysis of lichen-associated bacterial communities[END_REF][START_REF] Stocker-Woergoetter | Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimate metabolite production, and PKS genes[END_REF][START_REF] Grube | Species-specific structural and functional diversity of bacterial communities in lichen symbioses[END_REF]). The cultivation-independent approaches used DGGE, SSCP, 16S rRNA gene cloning and Sanger sequencing, or next generation sequencing (454 pyrosequencing, Illumina sequencing) either of PCR amplified 16S rRNA genes or of shotgun fragments (Table 4).

These studies clearly show a difference in abundance and diversity of culture-dependent versus -independent fractions of the lichen associated bacterial communities and demonstrated that the bacterial communities of most lichens studied so far are dominated by bacteria belonging to the Alphaproteobacteria class of the Proteobacteria, with other taxa such as the Beta-, Gamma-and Deltaproteobacteria and the phyla Bacteroidetes, Actinobacteria, Firmicutes and Verrucomicrobia frequently present [START_REF] Grube | Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics[END_REF]. The general relationships between microbiomes, lichen morphology, photobiont and substrates have recently been summarized by Aschenbrenner and colleagues, indicate that most lichens do have Alphaproteobacteria as predominant symbionts, although at a finer taxonomic resolution some patterns emerge such as the importance of Acidobacteria and Acetobacteraceae (Rhodospirillales) in acid rock and soil lichens, and higher actinobacterial percentages in intertidal lichens [START_REF] Aschenbrenner | Microbial cargo: do bacteria on symbiotic propagules reinforce the microbiome of lichens?[END_REF].

Even though lichens are hosts of phyla and classes known to contain species with potential of producing bioactive compounds, such as the Actinobacteria and Firmicutes, an analysis of diversity at a finer resolution indicates that although present, bacterial families of biotechnological interest (apart from the Cyanobacteria) are relatively minor members of the community, with the Burkholderiaceae and Paenibacillaceae representing at a maximum one percent of the community, while the other families are about 10 to 100 times less abundant (Table 4). That does not decrease the potential for biodiscovery based on cultivation approaches, as it has been clearly pointed out in the previous sections. However, lower relative abundances have to be taken in account when evaluating the role of these organisms and their bioactive molecules to the lichen symbiosis (i.e. their spatial localization and aggregation becomes important), and during the design of culture independent strategies to uncover the biotechnological potential of these microorganisms (i.e. via metagenomics).

One interesting point emerging from cultivation independent studies, is the presence Myxococcales in relative abundances of the same range or sometimes higher than those of Actinobacteria (Table 4). This group of microorganisms is particularly interesting with large genomes and potential to the production of bioactive compounds [START_REF] Dworkin | Myxobacteria[END_REF].

Myxobacteria have a particular life style [START_REF] Dworkin | Myxobacteria[END_REF] which is also conducive to the use of targeted isolation (e.g. [START_REF] Zhang | Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains[END_REF], but to date a single lichen-associated Myxobacteria [ATCC 25944 (M 155)] has been described [START_REF] Mccurdy | Studies on the Taxonomy of the Myxobacterales: IV. Melittangium[END_REF]. This is interesting, since in the original description of the Myxobacteriaceae Thaxter indicates that a number of species including Corallococcus (Myxococcus) coralloides and Melittangium (Chondromyces) lichenicolus were associated with lichens (Thaxter 1892), and myxobacteria have been reported as associated to decaying lichens (Reichenbach 1999) .

Regarding the application of metagenomic techniques in biotechnological development of lichen-associated bacteria, the nosperin example discussed above [START_REF] Kampa | Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses[END_REF]) clearly shows that the direct shotgun sequencing and assembly and analysis of cyanobacterial genomes can lead to the discovery of potentially new novel interesting molecules and biosynthetic pathways, as well as guide targeted cultivation of microorganisms of interest. Other recent studies have applied shotgun sequencing targeting lichen associated bacteria [START_REF] Grube | Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics[END_REF][START_REF] Sigurbjornsdottir | Analysis of the Peltigera membranacea metagenome indicates that lichen-associated bacteria are involved in phosphate solubilization[END_REF], but the complexity of community did not allow the assembly of contigs long enough to permit analyses of biosynthetic pathways. A focus on lichens with chlorophyte symbionts, with a possible physical separation of bacteria (or bacterial DNA) would increase the chance of assembling larger genomic fragments and allow a better access to biosynthetic pathways, in particular those possibly present in the genomes of dominant Alphaproteobacteria symbionts (with the caveat that these organisms are not the most prolific in terms of the biosynthesis of bioactive molecules).

Other techniques such as the construction and massive screening of fosmid libraries (e.g. [START_REF] Freeman | Metagenome Mining Reveals Polytheonamides as Posttranslationally Modified Ribosomal Peptides[END_REF] or single cell genomics (e.g. [START_REF] Wilson | An environmental bacterial taxon with a large and distinct metabolic repertoire[END_REF]) likely represent better alternatives to access the genomes of rarer bacteria from lichens. However, the fact that many of the bacteria with biosynthetic potential present in lichens belong to groups for which targeted cultivation methods do exist, seem to indicate that for the near future, cultivation based-methods might be in fact a more promising approach.

In a first metaproteomic study, proteomics using one-dimensional gel electrophoresis combined with LC-MS/MS and normalized spectral counting was applied to characterize the active proteins of the Lobaria pulmonaria symbiosis [START_REF] Schneider | Structure and function of the symbiosis partners of the lung lichen (Lobaria pulmonaria L. Hoffm.) analyzed by metaproteomics[END_REF]. Interestingly, not only the fungal partner was characterized by a rich secondary metabolism, also the bacterial partners showed a high activity supporting the hypothesis of biotechnologically relevant bacteria in symbioses.

Further biotechnological potential of lichen-associated bacteria

Irrespective of their capacity to produce small metabolites, lichen-associated bacteria have a wider biotechnological potential. According to a pioneering study by [START_REF] Gasser | Lichen-associated bacteria antagonistic to phytopathogens and their potential to accumulate polyhydroxyalkanoates[END_REF], one third of the detected lichen-associated bacteria have the potential to produce PHA biopolymers, yet, the strains isolated also showed a remarkable high antagonistic potential against plant pathogens. Up to 100% were antagonistic especially against leave pathogens, such as Alternaria alternata. Similarly, antagonistic properties were also detected by [START_REF] Kim | Antimicrobial properties of the bacterial associates of the Arctic lichen Stereocaulon sp[END_REF]Kim et al. ( , 2014)). Cernava et al. (2015) studied the antagonistic potential of a lichen-associated bacterial community of the lung lichen (Lobaria pulmonaria) with an integrative approach combining isolate screening, multi-omics techniques and high resolution mass spectrometry.

The highly diverse microbiome contained an abundant antagonistic community dominated by Stenotrophomonas, Pseudomonas and Burkholderia. In these examples, the precise principle of antagonisms, and whether this also comprises interesting novel compounds, still needs to be established. The examples for antagonisms will be continued with future studies, especially after [START_REF] Zachow | Catch the Best: Novel screening strategy to select stress protecting agents for crop plants[END_REF] proposed a promising screening strategy using plant rhizospheres as bait for lichen-associated bacteria. These studies further support lichens as important reservoirs of bacteria that are biotechnologically attractive.

Considering the high estimate of 18500 described lichens, much work apparently lays ahead of us to exploit their biotechnological potential. A more focused search for the most promising habitats and species may be guided by ecological knowledge. This possibly includes habitats where microbial regulation and competition becomes a major factor due to certain abiotic parameters, such as temperature and water availability. Thus expected hot spots for further research will include warmer zones such as tropical rain forests, or those, which are more frequently exposed to water inundation, including water streams or intertidal habitats.

  ) belong to Actinobacteria, 185 to Alphaproteobacteria, 85 to Firmicutes and 59 to the Gammaproteobacteria. Focusing on bacterial families of biotechnological interest, about 30% (150 in 511) belong to the families Bacillaceae (40), Paenibacillaceae (37), Burkholderiaceae (24), Pseudomonadaceae (19), Streptomycetaceae (18), Micromonosporaceae (5), Nocardioidaceae (5) and Pseudonocardiaceae (2) known for the production of bioactive molecules.

Figure 1 .

 1 Figure 1. Structures of some molecules produced by lichen associated bacteria (1) actinoplanic acid B, (2) uncialamycin, (3) cladoniamide G (4) JBIR-88 (5) JBIR-89 (6) cosmomycin B (7) maltophilin (8) cryptophycin 1 (9) nosperin

Table 1 .

 1 Bacterial strains isolated from lichens that 1) are officially described and/or 2) have a fully sequenced genome and/or 3) produced molecules that have been identified

	Strain	Phylum or Class -Famlly	Location, Lichen	Isolation	Nalidixic	Reference
				Medium	Acid
	Corallococcus sp.	Deltaproteobacteria -	n.r.	n.r.	no	McCurdy et al, 1971
	ATCC 25944	Myxococcaceae
	Actinoplanes sp.	Actinobacteria -	Spain, Madrid	ISP3	no	Singh et al 1997
	ATCC 55532	Actinoplanacee	Unidentified
	Streptomyces	Actinobacteria -	Canada, British Columbia	n.r.	no	Davies et al. 2005
	uncialis "ex Julian	Streptomycetaceae	Surface of Cladonia uncialis
	Davis et al."
	Herminiimonas	Betaproteobacteria -	Turkey, Mugla	Potassium	no	Lang et al. 2007
	saxobsidens NS11	Oxalobacteraceae	Lichen-Rock interface	Oxalate
	Nocardioides	Actinobacteria -	Japan, Izu Oshima Island	IAM-A1	no	Li et al 2007
	exalbidus RC825	Nocardioidaceae	unidentified
	Schumannella	Actinobacteria -	Japan, Tokyo	Modified	no	An et al 2008
	luteola KHIA	Microbacteriaceae	unidentified	Dettmer
	Leifsonia lichenia	Actinobacteria -	Japan, Tokyo	Modified	no	An et al 2009
	2Sb	Microbacteriacee	unidentified	Dettmer
	Mucilaginibacter	Bacteroidetes-	Finland, Lapland	Lichenin	no	Mänistö et al 2010
	lappiensis ANJLI2	Sphingobacteriaceae	Decaying Lichen
	Streptomyces sp.	Actinobacteria -	Japan, Rishiri Island	HV agar	yes	Motohashi et al 2010
	RI104-LiC106	Streptomycetaceae	unidentified

Table 2 .

 2 A list of studies that surveyed the diversity of microbial isolates associated with lichens

	Lichen species	Lichen origin	target of	Medium	Potentially interesting families,	Reference
			isolation		activities or others caracteristics of
					biotechnological interest
	Not identified	Sitka and Kodiak,	Biotechnology	soil	Streptomycetaceae,	Gonzalez et al.
		Alaska Hawaii and		extract	Micromonosporaceae,	2005
		Reunion Islands		agar,	Pseudonocardiaceae,
				humic	Thermomonosporaceae.
				acid agar	Antimicrobial activity, Genes
				and	involved in biosynthesis of
				glycerol	secondary metabolites.
				asparagi
				ne agar
	Cladonia digitata, C.	Styria, Austria	Untargeted,	TYE,	Bacillaceae, Paenibacillaceae,	Cardinale et al.
	rangiferina, C coniocracea,	and Normandy,	Diazotrophs	Sugar-	Burkholderiaceae,	2006
	C. pyxidata, C. coccifera,	France*		rich N	Micromonosporaceae,
	Pseudoevernia furfuracea,			free	Streptomycetaceae,
	Hypogminia physodes,				Streptosporangiaceae
	Rocella phycopsis*, R.
	fuciformis*
	Canoparmelia caroliniana,	Rain Forest, Brazil Diazotrophs	N-free	Pseudomonadaceae	Liba et al. 2006
	Canoparmelia crozalsiana,			NFb
	Canoparmelia texana,
	Parmotrema sancti-angeli
	Parmotrema tinctorum

Table 3 .

 3 Cyanobacteria strains isolated from lichens with characterized secondary metabolites

	Strain	Lichen species	Lichen origin	Metabolites Described	Reference
	Nostoc sp. ATCC 53789 Not described	Lichen, Arron Island,	Cryptophycin 1	Hisrsch et al 1990
			Scotland
	Nostoc sp.	Peltigera canina	Not reported	Nostoclide I and II	Yang et al 1993
	Nostoc sp. ATCC 53789 Not described	Lichen, Arron Island,	Nostocyclopeptide A1 and A2	Golakoti et al 2001
			Scotland
	Nostoc sp. IO-102-I	Pannaria pezizoides	Rock Southern	[ADMAdda5]microcystin-LR	Oksanen et al. 2004
			Finland	[DMAdda5]microcystin-LR
				[D-Asp3,ADMAdda5]microcystin-
				LR [ADMAdda5]-XR type variants
	Nostoc sp N6	Peltigera membranacea	Iceland	Nosperin	Kampa et al 2013

Table 4 .

 4 Cultivation independent studies broadly targeting the diversity of lichen associated bacteria Estimated from PCR amplification with primers targeting these clades; b.Estimated from OTU table graciously provided by S. Bates; c Estimated from figure 3B in the reference; d. Estimated from PCR amplification with primers targeting this clade; e.Estimated from Figure 2A in the reference; f Estimated from the OTU table associated with the publication; g. Estimated from OTU table associated to the publication; h. Used as reference genomes i. based on blastx analysis of contigs * Order in the case of Myxococcales †Cyanobacteria excluded

	Lichen species	Lichen origin	Techniques	Phyla/Class described, average and	Potentially interesting	Reference
			applied	range of percentage of Total ¶,	families*, activities or others
				Prokaryotes, Bacteria §	caracteristics of
					biotechnological interest
	Cladonia digitata, C.	Styria, Austria	ITS	Gammaproteobacteria	Bacillaceae	Cardinale et al.
	rangiferina, C.	and Normandy,	fingerprinti	Actinobacteria		2006
	coniocracea, C.	France*	ng,	Betaproteobacteria
	pyxidata, C. coccifera,		Sequencin
	Pseudevernia		g of bands
	furfuracea,
	Hypogymnia
	physodes, Roccella
	phycopsis*, R.
	fuciformis*
	Cladonia arbuscula	Styria, Austria	FISH,	Alphaproteobacteria 75%	Not described	Cardinale et al.
			SSCP	Actinobacteria 6%		2008
				Betaproteobacteria 4%
	Cladonia cristatella, C.	Virginia and N.	Direct	Alphaproteobacteria	None	Hodkinson and
	cryptochlorophaea, C.	Carolina, USA	Sanger	Acidobacteria		Lutzoni 2009
	cf. sobolescens, C.		sequencing	Gammaproteobacteria
	peziziformis, C.		of PCR
	subtenuis,		products
	Flavoparmelia		using
	caperata, Parmotrema		universal
	perforatum, Peltigera		primers
	phyllidiosa, Lasallia		(RHAPSA-
	pensylvanica,		D)
	Umbilicaria

Acknowledgments

This work was partly supported by EMR, a partnership between the UPMC, the Laboratoires Pierre Fabre and CNRS and by the project MALICA (10-INBS-02-01) of the Agence Nationale de la Recherche, France, as well as by the Fonds zur Förderung der wissenschaftlichen Forschung (FWF I882), and the Wissenschaftlich-Technische Zusammenarbeit, Austria. We thank Sanjay Antony-Babu, Nyree West, Laurent Intertaglia for fruitful discussions and Scott T. Bates for sending OTU tables from his previous publication.

Conflict of Interest:

The authors declare that they have no conflict of interest.


Sarah Bouchemousse
email: sbouchemousse@sb-roscoff.fr

Laurent Lévêque

Guillaume Dubois

Frédérique Viard
email: viard@sb-roscoff.fr

Umr7144

Co-occurrence and reproductive synchrony do not ensure hybridization between an alien tunicate and its interfertile native congener

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Biological introductions (i.e. transport of species away from their native range via human activities) are modifying species ranges at an unprecedented rate and on global scales (e.g. in the marine environment, [START_REF] Molnar | Assessing the global threat of invasive species to marine biodiversity[END_REF]). Among the various ecological and evolutionary consequences of species introduction, hybridization between native and nonnative species can dramatically alter the evolutionary trajectories of both species [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF]. Hybridization sensu lato commonly refers to interspecific gene flow, including first-generation hybrids and repeated backcrossing of their offspring with parental species [START_REF] Harrison | The language of speciation[END_REF]. Such interspecific gene flow between formerly allopatric taxa is not rare and can have diverse consequences [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF], including an increased risk of extinction of the native species [START_REF] Rhymer | Extinction by hybridization and introgression[END_REF][START_REF] Fitzpatrick | Rapid spread of invasive genes into a threatened native species[END_REF], increased invasiveness through, for instance, adaptive introgression mechanisms [START_REF] Currat | The hidden side of invasions: Massive introgression by local genes[END_REF][START_REF] Schierenbeck | Hybridization and the evolution of invasiveness in plants and other organisms[END_REF] and the emergence of new species [START_REF] Abbott | Plant invasion, interspecific hybridization and the evolution of new plant taxa[END_REF].

Hybridization facilitated by human activities has been studied mainly in plant species (e.g. [START_REF] Schierenbeck | Hybridization and the evolution of invasiveness in plants and other organisms[END_REF][START_REF] Guo | Plant hybridization: the role of human disturbance and biological invasion[END_REF]) and to a lesser extent in animals (e.g. [START_REF] Fitzpatrick | Rapid spread of invasive genes into a threatened native species[END_REF][START_REF] Steeves | Genetic analyses reveal hybridization but no hybrid swarm in one of the world's rarest birds[END_REF], with only a few cases reported in marine systems (but see, for instance, Abudefduf vaigiensis and A. abdominalis, [START_REF] Coleman | Large-scale introduction of the Indo-Pacific damselfish Abudefduf vaigiensis into Hawai'i promotes genetic swamping of the endemic congener A. abdominalis[END_REF]. However, marine species live in a dispersive environment and there is a large number of cryptic, co-existing native and non-native marine species (e.g. in tunicates; [START_REF] Bock | Multilocus genetic analyses differentiate between widespread and spatially restricted cryptic species in a model ascidian[END_REF][START_REF] Perez-Portela | Cryptic speciation or global spread? The case of a cosmopolitan marine invertebrate with limited dispersal capabilities[END_REF]. Furthermore, the increasing rate of marine biological invasions on the global scale facilitates numerous secondary contacts between allopatric taxa [START_REF] Geller | Genetic perspectives on marine biological invasions[END_REF].

The recently described Ciona intestinalis species complex presents an interesting case to study. The nominal species C. intestinalis (Linnaeus, 1767) covers a complex of four cryptic species [START_REF] Zhan | Invasion genetics of the Ciona intestinalis species complex: from regional endemism to global homogeneity[END_REF]. Two of them, C. intestinalis species A and C. intestinalis species B (hereafter referred to species A and species B, respectively) are now considered pseudo-cryptic species since the advent of morphological criteria to distinguish them [START_REF] Sato | Field identification of 'types' A and B of the ascidian Ciona intestinalis in a region of sympatry[END_REF]). In addition, very recently, [START_REF] Brunetti | Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis[END_REF] showed that species A displays morphological features specific to the formerly described Japanese species Ciona robusta Hoshino and Tokioka 1967, later synonymized under C. intestinalis, whereas species B fits with the C. intestinalis description by [START_REF] Millar | Ciona[END_REF]. These new alpha-taxonomy discoveries

have not yet been implemented in the World Register of Marine Species (e.g. C. robusta is still a non-accepted name) and we will use 'species A' and 'species B' in the following text.

Despite a substantial time elapsed since their divergence (estimated at around 4 Mya; [START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF][START_REF] Roux | Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species[END_REF], the two species are not reproductively isolated: viable and fertile F1 hybrids are easily produced under laboratory conditions [START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF]. As in many other tunicates [START_REF] Shenkar | Global diversity of Ascidiacea[END_REF], the distribution range of these two species has expanded in the last two centuries due to their accidental introduction via human activities [START_REF] Zhan | Invasion genetics of the Ciona intestinalis species complex: from regional endemism to global homogeneity[END_REF]. In particular, species A, has been recently introduced (ca. 15-20 years ago; [START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF]J.D.D. Bishop, personal communication) in the native European range of species B. Although the two species are widely distributed around the world, the western English Channel and the South of Brittany in the Northeast Atlantic (hereafter referred to as WEC) constitute the only confirmed region in which both species have been reported in sympatry.

Like many other ascidians [START_REF] Airoldi | Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale[END_REF], the nominal species C. intestinalis often forms well-established populations in artificial habitats (e.g. marinas with floating docks, commercial harbors and aquaculture installations). In their area of sympatrythe WECspecies A and species B can also live in syntopy (i.e. in the same locality, Fig. 1).

However, because of the cryptic nature of these two species, most published ecological studies do not distinguish between them [START_REF] Procaccini | A new animal model for merging Ecology and Evolution[END_REF]. Furthermore, C. intestinalis spp. is a model organism for evolutionary developmental biology research as well as phylogenetic studies. Nonetheless, ecological data are still lacking with regard to the recent discovery of the cryptic species. Due to its recent introduction in the WEC, species A experiences numerous, unprecedented biotic interactions and environmental changes that may affect its invasion dynamics [START_REF] Blackburn | A unified classification of alien species based on the magnitude of their environmental impacts[END_REF]. Consequently, there are many issues regarding the history, dynamics and fate of the recent introduction of species A in the native range of species B, including the stability of co-occurrence in species that share the same habitat and localities, the intensity of interspecific competitive interactions, and the occurrence of introgression or a hybrid swarm in sympatry.

To address these issues, we surveyed 11 marinas located along the coasts of Brittany for three years, and examine six distinct generations (i.e. the spring and autumn generations of the same year). We carried out genetic analyses to investigate 1) regional and local variation in the relative abundance of species A and species B; 2) their potential to mate; and 3) the rate and direction of interspecific crosses in the wild.

Materials and Methods

Field survey: sampling, morphological species identification and determination of reproductive status

We selected 11 marinas (out of 27) with floating pontoons located along the 1600 km long coastline of Brittany, France. These marinas were representative of a range of characteristics (i.e. open to the sea or closed during low tide (by means of tidal or sill gates)), fully marine or under freshwater (estuarine) influence; see Table S1 for details of each locality). In spring 2012, Ciona spp. populations were sampled in 10 marinas over two weeks (Fig. 2a). The same sampling was repeated five times, with an additional site (no. 11 in Fig. 2) in autumn 2012, spring and autumn 2013, and without sites nos. 10 and 11 (see Fig. 2) in spring and autumn 2014. The six sampling dates correspond to at least six different adult generations in European waters (e.g. [START_REF] Dybern | The life cycle of Ciona intestinalis (L.) f. typica in relation to the environmental temperature[END_REF][START_REF] Caputi | Long-term demographic and reproductive trends in Ciona intestinalis sp[END_REF]. In each locality, ca. 200-300 adult individuals (Table 1 for details) were randomly collected along two pontoons during SCUBA diving operations over ca. 50 m. Additionally, in most of the studied marinas (all except nos. 1, 2, 10 and 11, Fig. 2), seawater temperature was recorded using data loggers (TidbiT ® v2) located at 1.5 m depth.

Following their collection, individuals were sedated with phenoxy-2-propanol (1:1000) for 20-30 min to facilitate species identification and excision of tissue samples. Species were identified using morphological criteria according to [START_REF] Sato | Field identification of 'types' A and B of the ascidian Ciona intestinalis in a region of sympatry[END_REF]. For each site, a random subset of 87 specimens (SD ± 12.6; sample size detailed in Table S2

Molecular identification of hybrids

F1 hybrids and introgressed individuals (the two categories being referred to "admixed individuals" in the following text) cannot be discriminated morphologically [START_REF] Sato | Field identification of 'types' A and B of the ascidian Ciona intestinalis in a region of sympatry[END_REF]).

To identify them, species-diagnostic molecular markers were used on a random subset of individuals (average of 60 individuals ± 6.9 (SD) per location and sampling date; Table 2) collected in 2012 and 2013. DNA extraction was performed with Nucleospin® 96 Tissue Kit according to the manufacturer's protocol (Macherey-Nagel, Germany).

Four species-diagnostic markers were used: one mitochondrial marker (mtCOI, cytochrome oxidase I) to identify the maternal lineage, and three nuclear loci, namely vAChTP (vesicular acetylcholine transporter), CesA (cellulose synthase) and Hox5 (intronic nuclear regions of Hox5 gene) to identify the nuclear background. The Hox5 locus was amplified using the primers and PCR protocol described in [START_REF] Caputi | Cryptic speciation in a model invertebrate chordate[END_REF]. For this locus, species-diagnostic alleles differ in their length and allele size was scored on simple agarose gels (2%). For mtCOI, vAChTP and CesA, amplifications (protocols detailed in Nydam andHarrison, 2007, 2010) were followed by enzymatic digestions as explained in [START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF].

For each site and sampling date, the proportion of admixed individuals (i.e. displaying at least one alternative species-diagnostic allele) was computed across the four loci. To test for stability of admixture across samples, a Pearson's chi-squared test was used, and P-values were calculated using a Monte Carlo simulation with 5000 replicates. To further examine admixture patterns, a hybrid index per maternal lineage was computed for each sampling date using the above-described random subset and additional selected individuals from the field samples to reach a minimum number of 20 individuals per species when possible. The hybrid index was calculated by adding, over the three nuclear loci, the number of alleles specific to species B. It varies from zero to six: zero if each of the three nuclear loci are homozygous for the species A allele and six if the three nuclear loci display only species B alleles.

Laboratory crosses

Although the two species have been previously shown to be interfertile (e.g. [START_REF] Suzuki | Genomic approaches reveal unexpected genetic divergence within Ciona intestinalis[END_REF][START_REF] Caputi | Cryptic speciation in a model invertebrate chordate[END_REF][START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF], we carried out additional fertilization tests to ascertain that F1 hybrids could be easily produced with individuals from our study area. A series of bi-parental crosses (47 conspecific, 38 heterospecific and 23 self-crosses) were carried out using individuals sampled in autumn 2013 in the marinas of Aber Wrac'h (species B) and Moulin Blanc (species A and species B) (site nos. 6 and 7 in Fig. 2, respectively) following protocols available in [START_REF] Cirino | Laboratory culture of the ascidian Ciona intestinalis (L.): a model system for molecular developmental biology research[END_REF] and [START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF]. Fertilization success was estimated by computing the proportion of eggs with cellular division after 1 h (i.e. fertilization rate). Following [START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF], a linear mixed-effects model and pairwise comparisons were used to investigate differences in fertilization rate according to the type of cross (i.e. six categories: two heterospecific (with reverse parental roles for each species), two conspecific and two self-crosses) as a fixed factor. Because the same individuals were used in different crosses, a random effect of male genotype nested by female genotype was added.

Results

Significant variation in the relative abundance of the two species across generations

In the first study generation (i.e. spring 2012), species A and species B co-occurred in 8 localities out of the 10 surveyed, although species A was never abundant (Fig. 2). Detailed values per site and sampling date are given in Table 1. Although abundance varied across localities, regionally 6.0% of the 2,316 individuals were morphologically identified as species A in spring 2012. This spatial pattern changed dramatically in autumn 2012 with 32.2% of the individuals identified as species A out of the 3,678 individuals collected in 11 marinas (Fig.

2

). Similar patterns were observed in 2013 and 2014 with an increase in the relative abundance of species A in autumn (Table 1, Fig. 2).

At the locality level, 7 of the 11 studied marinas showed temporal variation similar to the regional pattern, with some showing particularly strong contrasts between spring and autumn generations (e.g. Perros Guirec; no. 3 in Fig. 2). The remaining four localities showed distinct features. Moulin Blanc (no. 7, Fig. 2) showed a different pattern in 2013, with spring abundance of species A (35.8%) being much higher than in autumn (2.2%). In Roscoff (no. 5, Fig. 2), a regular decrease in species A was observed over the course of the survey, starting in spring 2012 with 8.8% of species A dropping to 0% during the last four sampling dates.

Conversely, the presence of species A was first recorded in St Malo (no. 1, Fig. 2) during the last sampling date (autumn 2014; 1.1% of species A). Finally, species A was absent in one site (Aber Wrac'h, no. 6 in Fig. 2) on all sampling dates.

Altogether, a significant interaction was found between season and year (Table 3a) which reflects 1) an increase in the relative abundance of species A in autumn compared with spring of the same year and 2) a higher relative abundance of species A in 2012 compared with the following two years (Fig. 2).

Synchronous gamete production and successful fertilization between the two species

The reproductive status of individuals at each sampling date for both study species is illustrated with ternary diagrams (Fig. 3): the distribution of the three categories varied among species and localities. When comparing species for the proportion of individuals with sperm S2.

There were significant differences in fertilization rates when comparing cross type (Fig. 4, Table 3b). In particular, selfing success was very poor. Fertilization rates of heterospecific crosses with oocytes from species B did not differ from those of the two conspecific crosses (ca. 80%, Fig. 4). Fertilization rates dropped to very low values (<6%)

when species A was the female parent (Fig. 4).

mtDNA-based species identification

Altogether, 3,048 individuals collected during the four surveys carried out in 2012 and 2013 were genotyped over four loci including one mitochondrial marker for identifying the maternal lineage. Only 33 of them (1.08%) showed discordance between morphological and mitochondrial identification, indicating a close association between maternal type and species assignment based on morphology. During the 2012 and 2013 field surveys, 90 individuals out of 15,463 (0.59%) were morphologically recorded as "unidentified" (Table 1); most (82%) of these showed a species B maternal lineage.

A unique putative F1 hybrid and little genetic admixture in the wild

Only 131 of the 3,048 individuals (4.3%) showed an admixed genome and all of them displayed very low hybrid index values (Fig. 5). In addition, only one (0.03%) individual was possibly a first-generation hybrid (i.e. heterozygote across the three nuclear loci). This putative F1 hybrid displayed a species B maternal lineage and was found in Perros Guirec (no. 3, Fig. 2) in autumn 2013.

Regardless of maternal type, at the regional level, the proportion of admixture varied from 0.8% (spring 2013) to 6.3% (autumn 2012). A slightly higher rate of admixture was found in the autumn generations compared with the spring generations (Table 1). The admixture rate varied slightly across localities (Table 1). In particular, in localities where the two species co-occurred, admixed individuals were frequently found. For instance during the first sampling date 1.6% in St Quay, Trébeurden and Moulin Blanc (nos. 2, 4 and 7, Fig. 2) and 9.7% in Perros Guirec and Roscoff (nos. 3 and 5, Fig. 2). In contrast, no admixed individuals were observed in the locality where species A was absent throughout the survey (Aber Wrac'h, no. 6 in Fig. 2). These variations are, however, explained by differences in admixture patterns between individuals with different maternal backgrounds ( 1) is due to variation in the relative abundance of species A, which showed the highest admixture rate.

Discussion

The Despite the sustainable regional establishment of species A, there was local population instability: for instance, the Roscoff marina showed a steady decline leading to complete disappearance. Local population dynamics are likely to be unstable in this short-lived and recently introduced NNS, as observed in other introduced regions (e.g. Saldanha Bay in South Africa, [START_REF] Rius | Long-term coexistence of non-indigenous species in aquaculture facilities[END_REF]. Such major changes in site occupancy and local abundance in just a few years have already been documented in other NNS discovered in the English Channel (e.g. [START_REF] Bishop | Repeated rapid assessment surveys reveal contrasting trends in occupancy of marinas by non-indigenous species on opposite sides of the western English Channel[END_REF]. Many environmental and biotic mechanisms influence the successful and durable establishment of NNS [START_REF] Blackburn | A unified classification of alien species based on the magnitude of their environmental impacts[END_REF], including competitive interactions which may exacerbate invasion dynamics [START_REF] Simberloff | How risky is biological control?[END_REF][START_REF] Alpert | The advantages and disadvantages of being introduced[END_REF]. This mechanism may be particularly important here because the non-native species A co-occurs with its congener (Fig. 1). showed that spring and autumn generations are two separate generations, for the two study species. This is in agreement with the few data documenting the number of generations per year in the study area [START_REF] Dybern | The life cycle of Ciona intestinalis (L.) f. typica in relation to the environmental temperature[END_REF] in the North Atlantic for species B). By examining these two distinct generations of the same year, during three years, we documented a significant increase in the relative abundance of species A in autumn. Adults sampled in autumn likely correspond to juveniles that had settled from spring to early summer, when sea water temperatures increase from ca. 12°C in April to ca. 18°C in August-September in the study area (Figure S1). Conversely, adults sampled in spring likely correspond to juveniles that have settled in late summer / early fall, and which survive across the winter season (S.

Seasonal variation in species abundance

Bouchemousse, L. Lévêque, F. Viard, unpublished data). Seawater temperature greatly influences the development, growth and survival of Ciona intestinalis spp. [START_REF] Dybern | The life cycle of Ciona intestinalis (L.) f. typica in relation to the environmental temperature[END_REF][START_REF] Marin | Thermo-haline tolerance of Ciona intestinalis (L., 1767) at different developmental stages[END_REF], with species A and species B reported as warm-water and cold-water species, respectively [START_REF] Procaccini | A new animal model for merging Ecology and Evolution[END_REF][START_REF] Caputi | Long-term demographic and reproductive trends in Ciona intestinalis sp[END_REF]. We observed higher growth rates at 17°C, for species A than species B during laboratory experiments (data not shown), confirming previous findings [START_REF] Petersen | Growth and energetics in the ascidian Ciona intestinalis[END_REF] based on the growth rate of the nominal species C. intestinalis in localities where only species A or species B are now reported (e.g. at 15°C, growth rate varies between 2 and 3% in length per day for Japanese populations (species A) and between 0.7 and 1.1% per day for individuals from Scotland (species B)). In the study area, temperatures above 15°C are only observed from June to October (Figure S1).

It is also noteworthy that in the localities with the lowest maximum values of sea water temperatures (i.e. Aber Wrac'h and Roscoff, with a maximum always below 17°C) C.

intestinalis species A has never been observed or declined up to disappearance. The observed seasonal variations may thus partly result from spatial competition among species A and B.

The autumn generation of species A may have a competitive advantage, due to its faster growth rate during the warmer season.

Although temperature is likely the main environmental driver of the seasonal dynamics of the two congeners at a regional scale, other factors can drive local population dynamics. In particular, episodes of low salinity, due to rainfall and river inputs, can decrease survival of C. intestinalis [START_REF] Lambert | Non-indigenous ascidians in southern California harbors and marinas[END_REF]. Such perturbation events due to abrupt variations in environmental parameters have been recorded in our study area, as illustrated in Figure S2 for the marina of Moulin Blanc (no. 7 in Fig. 2), where additional monitoring (independent of our study; L. Lévêque, unpublished data) for C. intestinalis spp.

abundance and salinity was carried out in 2013-2014. Salinity declined sharply and subsequently Ciona spp. died off massively during winter months from January to March 2014. Synergistic effects between temperature and salinity may explain the very high percentage of species A observed in some localities such as Perros-Guirec (up to 90%; Fig.

2): this marina is closed off from the sea at low tide and remains several days without seawater renewal during neap tides such that 1) salinity can rapidly decrease in winter (due to rainfall) and 2) temperature can increase on the surface in summer (Figure S1). After winter die-offs of both species, due both to low temperatures and low salinity episodes, increasing seawater temperature may favor a more rapid colonization of species A over species B, taking into account its faster growth rate, on pontoons from adjacent sources (e.g. pillars, sea-wall),

giving locally and at some particular season, a competitive advantage to species A over species B. Conversely, Roscoff is an open, fully marine marina in the coldest part of the Brittany coastline (i.e. 13°C annual average, [START_REF] Gallon | Twenty years of observed and predicted changes in subtidal red seaweed assemblages along a biogeographical transition zone: inferring potential causes from environmental data[END_REF]. No massive mortality of Ciona intestinalis spp. has ever been observed and low temperatures may favor the development of species B, leading to competitive exclusion of species A.

Altogether, our study suggests that, with a few exceptions, environmental conditions in the study sites meet the niche requirements of the NNS species, though local variations in environmental parameters may favor one or the other of the two species, and thus may influence the outcome of competitive interactions among them. In particular, species A, thanks to its faster growth rate, may have a better adaptive potential than species B for (re)colonizing substrates during warmer seasons after disturbances due to variations in environmental factors, like a sharp decline in salinity after rains. Over larger temporal scales, the predicted increase of sea surface temperature (i.e. 0.35°C per decade; [START_REF] Gallon | Twenty years of observed and predicted changes in subtidal red seaweed assemblages along a biogeographical transition zone: inferring potential causes from environmental data[END_REF] and winter rainfalls [START_REF] Ouzeau | Scénarios régionalisés -édition 2014 -pour la métropole et les régions d'outre-mer[END_REF] in Brittany, due to climate change, could promote a substantial increase of the relative abundance of the NNS compared with its native congener in its introduced range.

Efficient barriers prevent hybridization in the wild

Sexually mature, gamete-producing individuals occurred simultaneously in the two study species which are interfertile and live in syntopy in most of the study localities. Despite these features that are expected to favor hybridization, the admixture rate was very low (4.3%) and only one putative F1 hybrid was observed in this study on more than 3,000 individuals, confirming previous studies (4.2% in localities of the WEC, [START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF]6.3% in one British locality, Sato et al. 2014). Ecological and/or genetic barriers thus seem to be at play and limit hybridization between the two species in the wild in Brittany. The success of interspecific fertilization in laboratory conditions (Fig. 4) seemingly suggests that post-zygotic isolation mechanisms act in the wild through a reduction in hybrid fitness [START_REF] Abbott | Hybridization and speciation[END_REF]). However, Sato et al. (2014) showed that F1 hybrids are viable and produce functional gametes, suggesting that hybrid depression, if any, is expressed at an earlier stage (e.g. larvae or juveniles). However, preliminary findings from a molecular study of juveniles of species A and species B in four marinas of the WEC (nos. 4, 5, 7 and 8; Fig. 2)

showed rates of admixture similar to those observed in adults (S. Bouchemousse, L. Lévêque, F. Viard, unpublished data). Pre-zygotic isolation mechanisms can enhance assortative mating; however, given the close contact (see Fig. 1), habitat segregation and behavior cannot be efficient reproductive isolation mechanisms. Differences in the timing of reproductive development, as suggested by [START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF], are also unlikely because we observed a high proportion of individuals producing gametes in both species and each sampling date. Gamete release occurs over several days or even weeks [START_REF] Carver | Strategies to mitigate the impact of Ciona intestinalis (L.) biofouling on shellfish production[END_REF], invalidating any consideration of slight shifts in the timing of gamete release in light of the short survival time of the gametes in seawater (i.e. 16 h after release for sperm and 30 h for oocytes; [START_REF] Svane | Spawning and dispersal in Ciona intestinalis (L.)[END_REF]. Assortative fertilization, facilitated by species-specific chemical attraction, can be a key mechanism for reproduction in broadcast-spawning marine invertebrates in marine systems [START_REF] Palumbi | Genetic divergence, Reproductive isolation and marine speciation[END_REF]. This mechanism has been described already within species complexes, for instance in two tropical sea urchins of the Echinometra species complex [START_REF] Geyer | Conspecific sperm precedence in two species of tropical sea urchins[END_REF] and in two mussels of the Mytilus species complex [START_REF] Bierne | Assortative fertilization and selection at larval stage in the mussels Mytilus edulis and M. galloprovincialis[END_REF]. Laboratory gamete choice experiments are needed to investigate this mechanism in the C. intestinalis species complex.

The source of admixture: historical or contemporary processes?

In the laboratory, F1 hybrids are easily obtained in one direction only, i.e. with species B oocytes (Fig. 4). Asymmetrical interspecific fertilization success is common, including in free-spawning marine invertebrates (e.g. [START_REF] Rawson | Patterns of gamete incompatibility between the blue mussels Mytilus edulis and M. trossulus[END_REF][START_REF] Geyer | Conspecific sperm precedence in two species of tropical sea urchins[END_REF]. The direction of this asymmetry was however unexpected based on the observed admixture rates that we computed according to the maternal lineage (82.4% of the 131 admixed individuals showed a species A maternal lineage). There are two possible explanations of this discrepancy between laboratory and field data. First, the few offspring produced by the least productive heterospecific crosses (i.e. a species A female crossed with a species B male) may be particularly fertile and vigorous. Similar situations with successful hybrids produced by parental combinations with low fertilization success have already been described, even in marine systems (e.g. [START_REF] Blum | Reproductive isolation and the expansion of an invasive hybrid swarm[END_REF]. This process may foster adaptive introgression of the NNS (species A) and/or drive hybrids to replace parental species (e.g. [START_REF] Rosenfield | The role of hybrid vigor in the replacement of Pecos pupfish by its hybrids with sheepshead minnow[END_REF][START_REF] Schierenbeck | Hybridization and the evolution of invasiveness in plants and other organisms[END_REF]. Second, conditions that promote successful crosses in the laboratory may not be the conditions found in the wild [START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF]. Monitoring survival of hybrid offspring obtained from experimental crosses is one way to test these two hypotheses. It is also interesting to note that the direction of the asymmetry is variable across studies: the direction observed here is in agreement with what has been reported by [START_REF] Caputi | Cryptic speciation in a model invertebrate chordate[END_REF] but not with [START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF], although in both studies some of the crosses involved individuals from the North Atlantic. Factors promoting (preventing) the hybridization success are potentially numerous, and among them the origin and the genetic background, in particular the introgression profiles (see below), may be important factors to consider in future studies.

Alternatively, from a conceptually different viewpoint, observed admixture may reflect the footprint of a previous secondary contact between species A and species B [START_REF] Roux | Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species[END_REF]. [START_REF] Roux | Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species[END_REF] compared various speciation models (e.g. divergence models with or without interspecific gene flow) using Approximate Bayesian Computations based on full transcriptomes of species A and species B (10 individuals for each). The best model to explain the data was a model of divergence with gene flow resulting from a secondary contact between the two species after their primary divergence (4 Mya ago). During this secondary contact, ca. 20% of loci presumably crossed the species barrier in both directions [START_REF] Roux | Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species[END_REF]. The authors then estimated the age of this secondary contact between 4,300 and 56,800 years ago. Many secondary contacts have been documented in marine systems during this period which corresponds to the end of the Pleistocene [START_REF] Maggs | Evaluating signatures of glacial refugia for North Atlantic benthic marine taxa[END_REF][START_REF] Geller | Genetic perspectives on marine biological invasions[END_REF].

The number of studies showing interspecific gene flow following these secondary contacts also recently increased [START_REF] Abbott | Hybridization and speciation[END_REF], see Becquet et al 2012 for an example). In this context, past hybridization is very plausible explanation of our results because it can account for 1) the discrepancy observed between laboratory and field experiments; 2) the temporal and spatial stability of the proportion of admixed individuals for species A; and 3) the complex evolutionary history of the two study cryptic species [START_REF] Roux | Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species[END_REF]. Although historical processes may explain most of the admixture observed, contemporary interspecific gene flow remains possible based on our results in two localities: in St Malo, no admixed individuals were observed until autumn 2013 following the colonization of this locality by species A and in Roscoff, admixed individuals were observed in 2012, but none afterwards when species A disappeared. Finally, at Aber Wrac'h, the only locality where no species A specimens were ever observed, did not show any admixed specimens. Furthermoreand interestinglyin St Malo and Aber Wrac'h, all admixed individuals displayed a species B maternal lineage, as did the only putative F1 hybrid found in our study (in Perros Guirec).

Altogether, our results suggest that past secondary contacts influenced the introgression rates measured here and rare contemporary hybridizations in the wild account mainly for the admixed individuals with a species B maternal background, in agreement with laboratory assays. The status of the presumed species diagnostic markers used so far [START_REF] Caputi | Cryptic speciation in a model invertebrate chordate[END_REF][START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF][START_REF] Sato | Symmetrical reproductive compatibility of the two species in the Ciona intestinalis (Acidiacea) species complex, a model for marine genomics and developmental biology[END_REF] this study) need to be re-evaluated because the three loci behave differently: CesA and vAChTP highlight admixture in species B maternal backgrounds whereas Hox5 revealed admixture with species A maternal backgrounds (see Table S3). Although not discernable in our laboratory crosses (or in the literature, [START_REF] Caputi | Cryptic speciation in a model invertebrate chordate[END_REF], we suggest that Hox5 crossed the species barrier a long time ago, in contrast to the two other loci (similar to the Glu locus which was mistakenly considered as a diagnostic marker of Mytilus species, [START_REF] Borsa | Genetics and taxonomy of Chilean smooth-shelled mussels, Mytilus spp. (Bivalvia: Mytilidae)[END_REF]. High-throughput genotyping based on SNPs are currently in progress to better investigate the fate of the introgression hot-spots defined by [START_REF] Roux | Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species[END_REF] and the true extent of contemporary hybridization in wild Ciona populations.

In conclusion, our study highlights the importance of temporal monitoring adapted to the life cycle and the generation time of short-lived NNS. This type of monitoring is critical for early-warning and accurate assessments of the durable establishment of introduced species. Our results also illustrate how difficult it is to predict the outcome of humanmediated introductions, even in supposedly well-known model species: we expected to find a large proportion of hybrids and introgression events between species A and species B, but only rare events (if any) were uncovered despite extensive sampling. Our results pave the way for future research on the reproductive isolation mechanisms acting in the wild between these model organisms, as well as on the processes that can sustain long-term co-occurrence of two functionally similar and congeneric species living in syntopy.

  non-native tunicate Ciona intestinalis species A is well establishedBased on the morphological examination of 23,000 individuals covering six different generations, we documented the durable establishment of the non-native species (NNS) C. intestinalis species A along the coasts of Brittany, ca. 15-20 years after the first reports of its introduction in the WEC. The first detailed study on the co-occurrence of species A and species B in the English Channel[START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF] revealed no samples of species A specimens during a follow-up survey (in 2009, two years after their first survey), interpreted as a possible decline of the NNS in the localities surveyed. This decline was not confirmed in our study. Our data, combined with those of[START_REF] Nydam | Introgression despite substantial divergence in a broadcast spawning marine invertebrate[END_REF], illustrate the importance of temporal monitoring over a substantial time window, particularly during the establishment stage, as well as during different seasons for short-lived species.

  : the outcome of competition in a changing environment Regular field work, sampling and monitoring as well as panels experiments examining settlement dynamics of Ciona sp. (S. Bouchemousse, L. Lévêque, F. Viard, unpublished data)

Figure 1 .

 1 Figure 1. Artificial plastic substrates are heavily colonized in the study area, including by

Figure 2 .

 2 Figure 2. Relative abundance of Ciona intestinalis species A (in black) and species B (in

Figure 3 .

 3 Figure 3. Ternary diagrams representing the proportion of the three recorded reproductive

Figure 4 .

 4 Figure 4. Fertilization rate for conspecific crosses (i.e. using female (Fe) and male (Ma)

Figure 5 .

 5 Figure 5. Hybrid index computed for each sampling date for individuals with a Ciona

  Figure 1.

  Figure 2.

  only, there were significant differences in spring and autumn 2013 (P<0.001 and P<0.001, respectively) and in autumn 2014 (P=0.032). The proportion of individuals with both types of gametes showed a similar pattern with no significant difference except for two dates (in

	autumn 2012 and autumn 2014; P<0.001). Altogether, regardless of the species, a high
	proportion of individuals produced gametes on all sampling dates. For example, in
	Trébeurden (no. 4, Fig. 2) in spring 2012, 100% of species A individuals and 84% of species
	B individuals showed both sperm and oocytes. Detailed values per site and sampling date are
	provided in Table

Table 2

 2

	). Large

2013: P=0.129). Altogether, these results show that most of the variation in admixture among localities or sampling dates (Table

Table 1 .

 1 Sample size (N tot ), number of individuals unidentified based on morphological criteria (N uni ), and percentage of Ciona intestinalis species A specimens (based on morphology; % spA ), for each study site and sampling date. The overall introgression rate (i.e. proportion of individuals showing an admixed multilocus genotype whatever their maternal lineage; % mix ) is indicated for the four surveys for which there were molecular data (i.e. all except in 2014).

		Spring 2012	Autumn 2012	Spring 2013	Autumn 2013	Spring 2014	Autumn 2014
		26 April -10 May	26 Oct. -6 Nov.	15 -25 April	14 -24 Oct.	15 -22 May	24 -31 Oct.
	Site	N tot N

uni % spA % mix N tot N uni % spA % mix N tot N uni % spA % mix N tot N uni % spA % mix N tot % spA

																				N tot	% spA
	1-St Malo	311	1	0.0	0.0	375	1	0.0	0.0	736	1	0.0	0.0	490	1	0.0	1.6	399	0.0	823	1.10
	2-St Quay	178 14	5.6	1.6	335	0 33.1	3.3	269	2	3.0	0.0	341	1 12.6	0.0	157	0.6	763	14.2
	3-Perros Guirec	297	6 13.5	9.7	305	0 90.2 10.9	223	3	8.5	0.0	531	8 67.0	3.2	217 14.3	495	96.0
	4-Trébeurden	228	4	6.6	1.6	262	0 61.1 12.7	436	0	6.7	0.0	474	4 43.2	8.1	281	6.4	809	16.4
	5-Roscoff	160	8	8.8	9.7	203	0	2.5	0.0	329	5	0.0	0.0	626	0	0.0	0.0	311	0.0	508	0.0
	6-Aber Wrac'h	171	3	0.0	0.0	236	2	0.0	0.0	290	1	0.0	0.0	523	0	0.0	0.0	36	0.0	315	0.0
	7-Moulin Blanc	168	1 18.5	1.6	234	1 42.7 13.8	120	1 35.8	7.8	543	1	2.2	1.7	99	0.3	424	3.3
	8-Château	205	0	3.4	0.0	721	6 19.1	8.3	300	0	0.0	2.2	778	0	6.0	7.9	295	0.0	906	8.0
	9-Camaret-sur-Mer	260	4	6.5	0.0	306	1 30.4 10.0	214	2	5.1	0.0	543	4 20.3	9.4	236	4.7	463	38.2
	10-Concarneau	338	1	1.5	0.0	474	1 60.1	9.7	264	0	0.0	0.0	712	0 27.2	3.2	-	-	-	-
	11-Quiberon	-	-	-	-	227	1	8.4	0.0	367	1	0.5	0.0	360	0	3.6	3.2	-	-	-	-
	Mean	232 4.2	6.4	2.4	334 1.2 33.9	6.2	323 1.5	5.9	0.9	538 1.7 17.9	3.5	226	2.9	612	19.7
	Standard deviation	66 4.3	5.9	3.9	150 1.7 29.6	5.6	160 1.5 10.5	2.4	131 2.6 21.7	3.5	113	4.9	213	31.1
	Total	2316 42	6.0	2.0	3678 13 32.2	6.3	3548 16	3.1	0.8	5921 19 16.6	3.5	2031	3.1	5506	22.0

Table 2 .

 2 Proportion of admixed individuals, i.e. individuals showing a mixture of alleles diagnostic for Ciona intestinalis species A and C. intestinalis species B, for each maternal lineage separately (% mix spA and % mix spB for individuals diagnosed with species A mitochondria and a species B mitochondria, respectively), for each site and sampling date studied in 2012 and 2013. N spA and N spB are the number of specimens for which admixture was examined.

		Spring 2012	Autumn 2012	Spring 2013	Autumn 2013
	Site	N spA %

mix spA N spB % mix spB N spA % mix spA N spB % mix spB N spA % mix spA N spB % mix spB N spA % mix spA N spB % mix spB

	1-St Malo	0	-56	0.0	0	-62	0.0	0	-60	0.0	0	-64	1.6
	2-St Quay	5	20.0 58	0.0	28	7.1 33	0.0	2	0.0 55	0.0	19	0.0 44	0.0
	3-Perros Guirec	18	33.3 44	0.0	56	12.5	8	0.0	4	0.0 44	0.0	50	4.0 12	0.0
	4-Trébeurden	4	25.0 59	0.0	40	15.0 23	8.7	5	0.0 54	0.0	29	10.3 33	6.1
	5-Roscoff	5	40.0 26	3.8	1	0.0 57	0.0	0	-77	0.0	0	-60	0.0
	6-Aber Wrac'h	0	-62	0.0	0	-58	0.0	0	-63	0.0	0	-62	0.0
	7-Moulin Blanc	11	9.1 51	0.0	33	18.2 25	8.0	19	21.1 32	0.0	3	33.3 56	0.0
	8-Château	0	-64	0.0	23	21.7 37	0.0	0	-46	2.2	6	16.7 57	7.0
	9-Camaret-sur-Mer	8	0.0 56	0.0	28	17.9 32	3.1	3	0.0 46	0.0	17	29.4 47	2.1
	10-Concarneau	1	0.0 63	0.0	48	12.5 14	0.0	0	-60	0.0	20	5.0 42	2.4
	11-Quiberon	-	-	-	-	5	0.0 59	0.0	0	-55	0.0	5	40.0 56	0.0
	Mean	5.2	18.2 53.9	0.4	23.8	11.7 37.1	1.8	3.0	4.2 53.8	0.2	13.6	17.3 48.5	1.7
	Standard deviation	5.8	15.8 11.5	1.2	20.0	7.8 19.2	3.4	5.6	9.4 11.8	0.7	15.6	15.1 15.4	2.6

Table 3 .

 3 Results of linear mixed models testing a) the effects of season and sampling year on the relative abundance of Ciona intestinalis species A and C. intestinalis species B in syntopic localities and b) the effect of cross type on the fertilization rate. Degrees of freedom (d.f.), residual d.f., residual deviation and P-value (from Chi-squared test) are given. Significant Pvalues are shown in italics.

	Variable	Model	d.f. Residual d.f. Residual deviation P-value
	a) Relative species abundance Null model		46	2968.1
		Year	2	44	2804.9	<0.01
		Season	1	45	1667.9 <0.001
		Year x Season	5	41	1319.2 < 0.001
	b) Fertilization rate	Null model		105	720.85
		Cross type	5	100	203.44 <0.001

Acknowledgments

We are very grateful to the divers of the Marine Operations department (Service Mer & Observation) at the Roscoff Biological Station for help in the field and to M. Danielo for assistance in data acquisition. FV thanks M. Nydam for her recommendations regarding molecular protocols. We are thankful for the numerous marina operators who provided access to pontoons and permission to carry out this study. We acknowledge S. Le Cam and T.

Broquet for advices regarding statistical analyses, N. Bierne for stimulating discussions about hybridization and introgression processes, and C. Lejeusne and T. Comtet for comments on earlier versions of this manuscript. This work was supported by the Interreg IVa Marinexus program and the ANR project HYSEA (no. ANR-12-BSV7-0011).

Site

Coordinates Open/ Closed Estuarine/Marine 1-St Malo 48.638617; -2.026411 Open Marine 2-St Quay 48.646136; -2.820925 Open Marine 3-Perros Guirec 48.804877; -3.441954 Closed Marine 4-Trébeurden 48.769510; -3.585811 Closed Marine 5-Roscoff 48.717500; -3.965401 Open Marine 6-Aber Wrac'h 48.598626; -4.562438 Open Estuarine 7-Moulin Blanc 48.390365; -4.432140 Open Estuarine 8-Château 48.379489; -4.489469 Open Estuarine 9-Camaret-sur-Mer 48.279674; -4.596113 Open Marine 10-Concarneau 47.870150; -3.914604 Open Estuarine 11-Quiberon 47.488223; -3.103269

Supplementary Information for online appendix

Table S1. Geographical coordinates and characteristics of each site, i.e. open or closed marinas (by means of tidal or sill gates) at low tide, freshwater (estuarine) or marine influence. Open Marine Table S2. Detailed sampling size used for analyzing the reproductive status and proportions of the observed reproductive stages for C. intestinalis species A (N spA ) and species B (N spB ) at each study site and sampling date. Three categories were recorded: 1) immature individuals (imm), 2) individuals with sperm only (sp) and 3) individuals with both sperm and oocytes (sp+ooc).