Insights into species diversity of associated crustose coralline algae ( Corallinophycidae , Rhodophyta ) with Atlantic European maerl beds using DNA barcoding

Atlantic European rhodolith beds, also known as maerl beds, are marine coastal habitats of high diversity composed of unattached non-geniculate coralline red algae —maerl/rhodolith— mixed with gravel, shells, and pebbles overgrown by crustose coralline algae (CCA; v.gr., Cabioch 1969, 1970; Bosence 1976; Peña & Bárbara 2009; Hall-Spencer & al. 2010; Adey & al. 2015). The life-history of coralline red algae involves an alternation among three phases: a haploid gametophyte, a diploid carposporophyte —carried by the haploid female gametophyte after fertilization of carpogonium—, and a diploid tetrasporophyte (Irvine & Chamberlain 1994). In two major Atlantic European maerl species, Phymatolithon calcareum (Pallas) W.H.Adey & D.L.McKibbin and Lithothamnion corallioides (P.L.Crouan & H.M.Crouan) P.L.Crouan & H.M.Crouan, sporangial conceptacles have been occasionally reported —usually in free-living Abstract. DNA barcoding in combination with morpho-anatomical analysis was applied to study the diversity of crustose coralline algae associated to two maerl beds from two protected Atlantic European areas from Brittany and Galicia —France and Spain, respectively—. Given the records of gametophytes of the maerl species Phymatolithon calcareum under crustose growth-forms, and that associated crustose coralline algae appear to be involved in the recruitment of new maerl plants, we compared the species composition between the associated crustose coralline algae to Breton and Galician maerl beds with the maerl species identified in these beds in previous DNA barcoding surveys. Our molecular results revealed higher species diversity in associated crustose coralline algae than in maerl-forming species. Nine taxa of crustose coralline algae were found in both study areas: four in Brittany and five in Galicia. Three species from Brittany were identified as Phymatolithon calcareum, Phymatolithon lamii, and Lithophyllum hibernicum. The remaining six ones were assigned to the genera Phymatolithon and Mesophyllum, along with Lithothamnion and Lithophyllum. Morpho-anatomical examination of diagnostic characters corroborated our molecular identification. Our results showed that the most representative genus of crustose coralline algae in Brittany was Phymatolithon, while in Galicia was Mesophyllum. In Brittany, Phymatolithon calcareum was found under both growth-forms, maerl and crustose coralline algae, the latter assigned to the gametophyte stage by the presence of uniporate conceptacles. The recruitment of new maerl plants involving associated crustose coralline algae with maerl beds may occur, but only we can affirm it for Phymatolithon calcareum in Brittany. By contrast, the different species composition between both growth-forms in the Galician maerl beds would indicate that the fragmentation of own free-living maerl species appears to be the most common propagation mechanism.

Several authors (Lemoine 1910;Cabioch 1969Cabioch , 1970;;Freiwald 1995;Mendoza & Cabioch 1998) have pointed out the contribution of the CCA to the development of further unattached maerl plants: after germination of the carpospores produced in CCA, juvenile tetrasporophytic plants grow also as crusts, and afterwards erect branches formed by these crusts break off, and give rise to unattached maerl plants.In Breton maerl beds, this type of recruitment was reported as the dominant (Cabioch 1969(Cabioch , 1970;;Mendoza & Cabioch 1998).Nonetheless, others authors (Bosence 1976;Woelkerling 1988;Peña & al. 2014) have reported that fragmentation of free-living maerl thalli is the main method of propagation in the maerl beds.
In recent years, several molecular studies have been focused on the diversity and systematics of maerl-forming species in Atlantic Europe (Carro & al. 2014;Hernández-Kantún & al. 2014, 2015a, 2015b;Pardo & al. 2014;Peña & al. 2015b).However, the diversity and specific composition of their associated CCA are little-known.In Brittany, two DNA barcoding works found crustose plants of Phymatolithon calcareum and Phymatolithon lamii (Me.Lemoine) Y.M.Chamb., which showed that species composition of associated CCA could share some similarities with unattached maerl plants (Peña & al. 2014(Peña & al. , 2015b)).Based on previous results obtained for maerlforming species of two study areas from Brittany and Galicia (Pardo & al. 2014), we identified their associated CCA using a combination of DNA barcoding -COI-5Pand morpho-anatomical features.Apart from the species diversity of associated CCA, we compared the composition of both growth-forms (maerl and CCA) in order to know if the associated CCA could be involved in the recruitment of new unattached maerl plants.

Molecular studies
For the DNA extraction, a subsample was obtained by grounding of a small portion of each plant selected, choosing free epiphytes areas under stereomicroscope.DNA was extracted and purified using the DNeasy® Blood & Tissue Kit -Qiagen, Valencia, CA-following manufacture's recommendations.A fragment of 664 bp of the 5' end of the mitochondrial gene cytochrome oxidase I -COI-5P-was amplified using primers GazF1 and GazR1 (Saunders 2005).Amplifications PCR were performed in a Biometra TProfesional Basic thermocycler following Saunders & McDevit (2012).Amplification success was evaluated by running the reactions in agarose gels.After excess of primers and nucleotides were removed with shrimp alkaline phosphatase and exonuclease I enzymes.PCR products were sequenced using the Sanger method at Macrogen facilities -http://www.macrogen.com-.The sequences obtained were checked, edited and aligned with the program Geneious v. 5.6.6 -Biomatters, New Zealand-, and lodged in BOLD and GenBank (table 1).Haplotypes obtained were searched for matches in BOLD and GenBank databases to their molecular taxonomic identification (table 2).For the molecular analyses, publicly available COI-5P sequences for both maerl-forming and CCA taxa, particularly from Atlantic Europe, were included (Carro & al. 2014;Pardo & al. 2014;Peña & al. 2014Peña & al. , 2015aPeña & al. , 2015b;;Hernández-Kantún & al. 2015a), as well as a sequence of Corallina officinalis L. as outgroup (Pardo & al. 2015;table 3).In total, 30 sequences were used to generate a Maximum Likelihood -ML-tree.A bootstrap with 1,000 replicates was applied in Mega v. 6.0 with defaults settings (Tamura & al. 2013), using the General Time-Reversible model, Gamma distributed with Invariant sites -GTR+G+I-, identified previously in ModelTest v. 2.1.3(Darriba & al. 2012) as the best-fitting substitution model using an Akaike Information Criterion -AIC-and Bayesian Information Criterion -BIC.

Morphological studies
CCA selected for molecular studies were examined under stereomicroscope, and representative fragments were examined by Scanning Electron Microscope -SEM, model JEOL JSM 6400, Univ.da Coruña-.Vegetative and reproductive features considered diagnostic and anatomical terminology followed Irvine & Chamberlain (1994).

DISCUSSION
In our study, nine CCA species were recorded associated with two Atlantic European maerl beds: Molène -Brittany-and Ons -Galicia-Archipelagos.Despite that these CCA collections consisted on epilithic crustose corallines growing partial or entirely over pebbles, we are aware that could be interpreted as "rhodolith" given that this term is extensively applied to unattached nodules with non-algal core (Irvine & Chamberlain 1994;v.gr.see fig. 6 in Basso & al. 2009).While the four CCA species associated with the Breton maerl bed matched with available sequences from northern Atlantic coast -Phymatolithon calcareum, Phymatolithon lamii, Lithophyllum hibernicum, and Phymatolithon sp.3ATcrust-, the five CCA taxa from the Galician maerl bed did not obtain any record.However, according to the molecular and morphological data obtained, CCA collected in Galicia were identified up to genus level as Phymatolithon sp. 5, Lithothamnion sp. 2, Mesophyllum sp. 3, Mesophyllum sp. 4, and Lithophyllum sp. 3. The absence of any match with public databases containing more than 4,000 COI-5P sequences of coralline red algae pointed out the cryptic diversity still uncovered in coralline algae.
In Molène Archipelago the dominant genus in the CCA studied was Phymatolithon, while in Galicia was Mesophyllum, this latter having a southern distribution along the Atlantic European coasts (Guiry & Guiry 2016).This gradual replacement of both species with latitude has been also observed in the composition of major maerl-Species diversity of crustose coralline algae in Atlantic European maerl beds 7 Anales del Jardín Botánico de Madrid 74 (2): e059.http://dx.doi.org/10.3989/ajbm.2459a, b, c, g, h, j, 1cm; i, o, q, 2 cm.] forming species in the OSPAR maritime area, including Brittany and Galicia (Carro & al. 2014;Pardo & al. 2014).
According to the literature, Phymatolithon calcareum is considered as a major maerl-forming species in Atlantic Europe (Irvine & Chamberlain 1994;Carro & al. 2014;Pardo & al. 2014).Our record of CCA with uniporate sexual conceptacles in the Breton maerl bed agrees with previous reports of gametophytes -including the carposporophyte-under crustose forms, all of them from this region (Mendoza & Cabioch 1998;Peña & al. 2014).This observation would corroborate that, at least in Brittany, the gametangial stage of Phymatolithon calcareum occurs as associated crusts -i.e.attached-, while unattached maerl plants of this species are sporophytes (Cabioch 1969(Cabioch , 1970;;Mendoza & Cabioch 1998).However, in adjacent areas such as the British Isles or Atlantic Iberian Peninsula where maerl beds have been intensively studied, fertile gametophytes -crusts or unattached-have been never recorded yet (Irvine & Chamberlain 1994;Peña & al. 2014Peña & al. , 2015b)).Given that all Atlantic European records of gametangial stages of Phymatolithon calcareum are still restricted to Brittany (Mendoza & Cabioch 1998;Peña & al. 2014), we presume that this Atlantic region plays an important role in the life-history of this major maerlforming species.
Phymatolithon lamii is a crustose species occurring from the intertidal to subtidal rocky shores of the Atlantic coasts from Arctic Norway to South Atlantic Iberian Peninsula (Chamberlain 1991;Irvine & Chamberlain 1994;Peña & al. 2015b).Our collection in the Breton maerl bed as associated CCA corroborated recent records of this species from the same maerl bed -Molène archipelago (Peña & al. 2015b).

CONCLUSION
The present study carried out in two protected Atlantic European maerl beds pointed out that maerl beds harbours a high species diversity of associated CCA, which may not share the same species composition that maerl-forming algae.Also a high cryptic diversity was found in the associated CCA, as occurs in the maerl-forming species (Carro & al. 2014;Pardo & al. 2014) and in geniculate coralline algae (Walker & al. 2009;Pardo & al. 2015).Further extensive studies in other European maerl beds are necessary to understand this unexpected diversity of associated CCA.Nonetheless, according to our results, the crustose growth-forms should be taken into account in management actions of Atlantic European maerl beds.

Table 1 .
Information and collection data of the epilithic CCA collected in Brittany and Galicia.

Table 2 .
Matches with our sequences in public databases (BOLD and GenBank).Only hits with an identity beyond a minimum threshold are reported in this table (98%).