Andrés Meynard

Javier Zapata

Nicolás Salas

Claudia Betancourtt

Gabriel Pérez-Lara

María Francisco Castañeda

Eliana Ramírez

Cristian Bulboa Contador

Marie-Laure Guillemin

Loretto Contreras-Porcia

Andres ; Meynard

Andres Bello Universidad

Javier ; Bello Zapata

Nicolas ; Salas

Claudia ; Bello Betancourtt

Gabriel ; Bello Pérez-Lara

Francisco ; Bello Castañeda

Maria ; Bello Ramírez

Bulboa Bello

Universidad Andres

Bello Guillemin

Andrés Meynard

Nicolás Salas

María Eliana Ramírez

Meynard, Ramírez, Contreras-Porcia, sp. nov Pyropia Variabilis Zapata

Ramírez, Contreras-Porcia sp. nov Porphyra Luchensis Meynard

Ramírez, Contreras-Porcia, sp. nov. Porphyra Longissima Meynard

Porphyra Sp Fih

Pyropia Orbicularis

Pyropia Sp Chj

Kp781590 Kp781687

Porphyra Sp Chf

Genetic and morphological differentiation of Porphyra and Pyropia species (Bangiales, Rhodophyta) coexisting in a rocky intertidal in Central Chile

Keywords: taxonomy, Macroalgae, Rhodophyta Bangiales, taxonomy, COI, rbcL, Chile, Intertidal distribution COI, cytochrome oxidase I, ML, Maximum Likelihood, PCA, Principal Component Analysis, PCR-RFLP, polymerase chain reaction restriction fragment length polymorphism analyses, rbcL, large subunit ribulose Pyropia orbicularis Pyropia orbicularis Pyropia orbicularis Pyropia orbicularis Pyropia orbicularis Pyropia

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

Alternate Keywords: Intertidal distribution, Bangiales, COI, rbcL, Chile Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online.

INTRODUCTION

Prior to the last decade, all foliose Bangiales have been classified under the genus Porphyra sensu lato [START_REF] Blouin | Porphyra: a marine crop shaped by stress[END_REF][START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF]). Nevertheless, classical taxonomic methods, based mainly on morphological and life history traits, were shown to be highly misleading in this order [START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF]). Indeed, with the advent of molecular phylogenies, several cryptic genera of foliose Bangiales were discovered and various classical genera were redefined [START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF][START_REF] Sánchez | Diversity of bladed Bangiales (Rhodophyta) in western Mediterranean: recognition of the genus Themis and descriptions of T. iberica sp. nov., and Pyropia parva sp[END_REF], 2015[START_REF] Yang | A molecular phylogeny of the bladed Bangiales (Rhodophyta) in China provides insights into biodiversity and biogeography of the genus Pyropia[END_REF]. The existence of nine genera of bladed Bangiales was then proposed based on these molecular studies: Boreophyllum S.C Lindstrom N. Kikuchi, M. Miyata et Neefus, Clymene WA Nelson, Fuscifolium S.C Lindstrom, Miuraea N. Kikuchi, S. Arai, G. Yoshida, J.A. Shin et M. Miyata, Lysithea WA Nelson, Porphyra C. Agardh, Pyropia J.

Agardh, Wildemania De Toni and Neothemis A.Sánchez, A.Vergés, C.Peteiro, J.Sutherland & J.Brodie. Of particular note, most of the "nori" species cultivated in Asia nowadays belong to the genus Pyropia [START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF][START_REF] Zuccarello | The greatest contribution to PC1 is from the thallus length and thallus width and allows separating Porphyra sp. CHC (with the longest and thinnest blades) from the other three species. Red -Porphyra sp. CHC; pink -Porphyra sp. CHE; green -Pyropia sp. CHK and brown -Pyropia orbicularis. 108x53mm (600 x 600 DPI) Boxplot of the sizes of two out of eight microscopic traits (all in Log µm) and two out of two macroscopic traits (in Log cm) measured in the four-dominant foliose Bangiales species observed at Maitencillo beach. Different letters indicate statistically significant differences between species in Tukey multiple comparisons test. Po. CHC = Porphyra sp. CHC; Po. CHE = Porphyra sp[END_REF]. At the genus level, the delimitation of species was undertaken recently using molecular tools, an advent that has redefined the frontiers of former taxa over the last ten years (e.g. [START_REF] Broom | A reassessment of the taxonomic status of Porphyra suborbiculata, Porphyra carolinensis and Porphyra lilliputiana (Bangiales, Rhodophyta) based on molecular and morphological data[END_REF][START_REF] Brodie | New insights into the cryptic diversity of the North Atlantic-Mediterranean Porphyra leucosticta complex: P. olivii sp. nov. and P. rosengurttii (Bangiales, Rhodophyta)[END_REF][START_REF] Neefus | The distribution, morphology, and ecology of three introduced asiatic species of Porphyra (Bangiales, Rhodophyta) in the Northwestern Atlantic[END_REF][START_REF] Nelson | Pyropia plicata sp. nov. (Bangiales, Rhodophyta): naming a common intertidal alga from New Zealand[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF][START_REF] Dumilag | Morphological and molecular confirmation of the occurrence of Pyropia tanegashimensis (Bangiales, Rhodophyta) from Palaui Is[END_REF][START_REF] Reddy | A rosette by any other name: species diversity in the Bangiales (Rhodophyta) along the South African coast[END_REF][START_REF] Yang | A molecular phylogeny of the bladed Bangiales (Rhodophyta) in China provides insights into biodiversity and biogeography of the genus Pyropia[END_REF]. A high level of cryptic genetic diversity was found within the two genera Porphyra and Pyropia [START_REF] Brodie | New insights into the cryptic diversity of the North Atlantic-Mediterranean Porphyra leucosticta complex: P. olivii sp. nov. and P. rosengurttii (Bangiales, Rhodophyta)[END_REF][START_REF] Niwa | Genetic diversity and introgression in two cultivated species (Porphyra yezoensis and Porphyra tenera) and closely related wild species of Porphyra (Bangiales, Rhodophyta)[END_REF][START_REF] Nelson | Porphyra columbina (Bangiales, Rhodophyta): originally described from the New Zealand subantarctic islands[END_REF][START_REF] Lindstrom | Four new species of Pyropia (Bangiales, Rhodophyta) from the west coast of North America: the Pyropia lanceolata species complex updated[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF][START_REF] Koh | DNA barcoding reveals cryptic diversity of economic red algae, Pyropia (Bangiales, Rhodophyta): description of novel species from Korea[END_REF][START_REF] Reddy | A rosette by any other name: species diversity in the Bangiales (Rhodophyta) along the South African coast[END_REF][START_REF] Yang | A molecular phylogeny of the bladed Bangiales (Rhodophyta) in China provides insights into biodiversity and biogeography of the genus Pyropia[END_REF]. According to [START_REF] Zuccarello | The greatest contribution to PC1 is from the thallus length and thallus width and allows separating Porphyra sp. CHC (with the longest and thinnest blades) from the other three species. Red -Porphyra sp. CHC; pink -Porphyra sp. CHE; green -Pyropia sp. CHK and brown -Pyropia orbicularis. 108x53mm (600 x 600 DPI) Boxplot of the sizes of two out of eight microscopic traits (all in Log µm) and two out of two macroscopic traits (in Log cm) measured in the four-dominant foliose Bangiales species observed at Maitencillo beach. Different letters indicate statistically significant differences between species in Tukey multiple comparisons test. Po. CHC = Porphyra sp. CHC; Po. CHE = Porphyra sp[END_REF], the discovery of these new taxa, that cannot be identified based on morphology, could drive field ecology studies to misleaded conclusions and impair our understanding of the evolutionary processes that generated this diversity. Hence, to aid in the rapid, cheap and reliable identification of some Asiatic bladed Bangiales, a number of studies carried by Niwa and collaborators [START_REF] Teasdale | A simple Restriction Fragment Length Polymorphism (RFLP) Assay to discriminate common Porphyra (Bangiophyceae, Rhodophyta) taxa from the Northwest Atlantic[END_REF][START_REF] Saunders | Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications[END_REF], 2009, 2010a, b, and 2014[START_REF] Niwa | Identification of currently cultivated Poprhyra species by PCR-RFLP analysis[END_REF] developed molecular markers based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses of nuclear and cytoplasmic genes.

Considering the economical, biotechnological, ecophysiological and evolutionary research potentials of species of bladed Bangiales, there is clearly a need to better define their taxonomic frontiers [START_REF] Hurd | Seaweed Ecology and Physiology[END_REF]. Moreover, this new appraisal of the taxonomic and biochemical diversity within the Bangiales could also unveil ecological patterns unseen under the old paradigm of identification based exclusively on morphology. Contradictory results have been published concerning intertidal zonation or habitat partitioning of Porphyra and Pyropia species living in sympatry (see, for example, [START_REF] West | Molecular ecological studies of New England species of Porphyra (Rhodophyta, Bangiales)[END_REF], for a positive answer, and [START_REF] Schweikert | Analysis of spatial and temporal diversity and distribution of Porphyra (Rhodophyta) in southeastern New Zealand supported by the use of molecular tools[END_REF], for a negative one). Nevertheless, studies in other groups of red and brown algae showed that non-random small-scale distribution patterns occur locally between related algal species (e.g. [START_REF] Billard | Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level[END_REF][START_REF] Couceiro | Evolution and maintenance of haploid-diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus[END_REF][START_REF] Muangmai | Cryptic species in sympatry: nonrandom small-scale distribution patterns in Bostrychia intricata (Ceramiales, Rhodophyta)[END_REF][START_REF] Montecinos | Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae)[END_REF].

In Chile, a recent study using molecular methods and samples spanning most of the coast (18°S-53°S), identified 18 species of bladed Bangiales belonging to the genus Porphyra, Pyropia, Fuscifolium and Wildemania [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF]. Of the 18 species encountered in Chile only two are named and were characterized morphologically [START_REF] Ramírez | Pyropia orbicularis sp. nov. (Rhodophyta, Bangiaceae) based on a population previously known as Porphyra columbina from the central coast of Chile[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF]. A group of closely related species (namely Pyropia sp. CHJ, Pyropia sp. CHK and Pyropia orbicularis Ramírez, Contreras-Porcia & Guillemin [START_REF] Ramírez | Pyropia orbicularis sp. nov. (Rhodophyta, Bangiaceae) based on a population previously known as Porphyra columbina from the central coast of Chile[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF] co-occur in central Chile. Pyropia orbicularis and Pyropia sp CHK were even observed in sympatry in the locality of Maitencillo. It is well known that adaptation to different ecological micro-niches could facilitate the coexistence of closely by related species in the same locality [START_REF] Billard | Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level[END_REF][START_REF] Muangmai | Cryptic species in sympatry: nonrandom small-scale distribution patterns in Bostrychia intricata (Ceramiales, Rhodophyta)[END_REF]), but differences in intertidal zonation or habitat between Pyropia sp. CHJ, Pyropia sp. CHK and Pyropia orbicularis have not yet been studied.

The main aims of the present study was to (i) determine how many species of bladed Bangiales are present in the locality of Maitencillo when using a sampling method that takes into account the diversity of habitats present in the site, (ii) determine their distribution along the tidal gradient and in the different habitats sampled, (iii) develop simple and rapid species-diagnostic markers using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) on rbcL gene amplicons for the discrimination of these bladed Bangiales species, and (iv) describe the unnamed species living at the study site using a combination of molecular tools and statistical analyses of morphological characters.

MATERIALS AND METHODS

Sampling of foliose Bangiales in Maitencillo beach

Sampling was performed in the rocky intertidal shore of Maitencillo, Valparaíso (32° 39´S, 71° 26´ W, Fig. S1). This site is characterized by a rocky platform that gently slopes into the sea with small areas characterized by boulders and small, but steep rocky walls. Foliose Bangiales are prevalent in the upper intertidal zone while Mazzaella laminaroides (Rhodophyta) dominate the middle intertidal [START_REF] Betancourtt | Spatio-temporal variation in the composition of the macroalgae assemblage of the intertidal rocky zone from Maitencillo, Valparaíso, central coast of Chile[END_REF]. The lower intertidal zone is characterized by a sandy bottom. Two sampling schemes were used during the study. First, to characterize species diversity and small-scale distribution in Maitencillo we sampled 66 specimens of bladed Bangiales from July to December 2013 and 2014 (i.e. winter-spring) in different intertidal zones and habitats. Sampling took place along three transects extending 20 m from the upper to lower intertidal zone of the rocky platform.

Twenty-two specimens of Bangiales were sampled within three areas of 5-6 meters long per 0.5 meters wide along each transect. These three areas were defined as upper, middle and lower intertidal, respectively, according to animal and seaweed limits previously described in central Chile (Hoffmann & Santelices 1987). This sampling was completed by seventeen samples taken from two quadrants of 0.5 m 2 placed on a steep wall adjacent to the upper intertidal zone; and by 24 samples taken from four quadrants of 0.5 m 2 placed in two boulder zones (two quadrats in each zone, 12 samples per boulder zone). The boulder zones were located 150 m from the main transect. One was surrounded by rocky pools while the other was surrounded by pools of sandy bottoms. Some representative samples were chosen as voucher specimens that are housed in the herbarium of the National Museum of Natural History, Chile, under the SGO herbarium numbering system (see voucher numbers in Table S1; Museo Nacional de Historia Natural, herbarium code: SGO, http://www.mnhn.cl). To study the morphology of the four-dominant species encountered in Maitencillo (see below for more information about species determination), a second sampling was performed from August to December 2014 (i.e. winter-spring) where 87 specimens of bladed Bangiales were sampled in different intertidal habitats.

DNA extraction, amplification, sequencing and species delimitation by molecular tools

DNA extraction. Total genomic DNA was extracted from dried algal tissue grounded in liquid nitrogen following the protocol originally described by [START_REF] Saunders | Gel purification of red algal genomic DNA: an inexpensive and rapid method for the isolation of polymerase-chain reaction-friendly DNA[END_REF], with modifications by [START_REF] Faugeron | Hierarchical spatial structure and discriminant analysis of genetic diversity in the red alga Mazzaella laminarioides (Gigartinales, Rohodophyta)[END_REF].

8

COI and rbcL amplification and sequencing. A partial sequence of the COI was obtained using a newly defined forward primer COIF3 (5'-AATTAGRATGGAAYTAGCKCAACC-3') and the GazR1 primer, following the amplification protocols of [START_REF] Saunders | Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications[END_REF]. A partial sequence of the rbcL was obtained using the primers F-rbcL and R-rbcS [START_REF] Hommersand | Phylogenetic systematics and biogeography of the Gigartinaceae (Gigartinales, Rodophyta) based on the sequence analysis of rbcL[END_REF], following the previously published amplification protocols [START_REF] Hommersand | Phylogenetic systematics and biogeography of the Gigartinaceae (Gigartinales, Rodophyta) based on the sequence analysis of rbcL[END_REF]López-Bautista 2003). All PCR products were purified using UltraClean TM DNA Purification kits (MO BIO Laboratories, Carlsbad, CA, USA) and sequenced using the forward and reverse primers used for amplification by Macrogen Inc. (Seoul, South Korea). Sequences were aligned and edited using Bioedit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Sixty-six specimens from the first sampling scheme and 24 from the second sampling scheme (for morphological analyses) were sequenced. During this study, 90 COI sequences (603 bp) and 46 rbcL sequences (873 bp) were obtained and deposited in GENBANK. Details about specimen collection information and GENBANK accession numbers are given in Table S1.

Species delimitation and phylogenetic relationship reconstruction. For the rbcL, in addition to the 46 sequences newly obtained from Maitencillo, 174 sequences of Chilean Bangiales specimens (including 20 sequences from Maitencillo, [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF]) and 157 sequences retrieved from GENBANK were included in our data set. For the COI, in addition to the 90 sequences newly obtained (i.e. first and second sampling scheme) from Maitencillo, 159 sequences of Chilean Bangiales (including 20 from sequences from Maitencillo, [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF] as well as 39 sequences retrieved from GENBANK were included in our data set. The complete list of specimens used in molecular analyses is available in Tables S1 andS2.

The monophyly of each Pyropia and Porphyra species, previously determined by [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF], was examined using a maximum likelihood (ML) phylogenetic relationship reconstruction performed using IQ-TREE online server [START_REF] Trifinopoulos | W-IQ-TREE: a fastonline phylogenetic tool for maximum likelihood analysis[END_REF]. We selected the best-fit substitution model using the Akaike information criterion implemented in IQ-TREE [START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF][START_REF] Trifinopoulos | W-IQ-TREE: a fastonline phylogenetic tool for maximum likelihood analysis[END_REF]. The selected models were TIM3+F+G4 and TN+F+I+G4 for the COI (Pyropia and Porphyra, respectively) and TIM+I+G4 for the rbcL. Statistical support was estimated using 1,000 ultrafast bootstrap replicates (Nguyen et al. 2014). Minerva aenigmata W.A. Nelson (EU570053) and Dione arcuata W.A. Nelson (EU570052) were used as outgroups in the phylogenetic analysis of rbcL. For the COI [START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF], tree reconstructions were done separately for Pyropia and Porphyra species, using as outgroups Boreophylum birdiae and Bangia fuscopurpurea, respectively, following [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF] and [START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF].

For each of the 90 specimens sequenced, affiliation to one of the 18 cryptic species observed in Chile [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF] or to a newly encountered species was determined using the ML tree reconstruction. For the more problematic groups of sequences (i.e. corresponding to the group of closely related species Pyropia sp. CHJ, Pyropia sp. CHK and Pyropia orbicularis), we followed the grouping proposed by [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF] and a COI phylogeographic network was reconstructed using the median-joining algorithm implemented in HapView version Beta [START_REF] Salzburger | The performance of phylogenetic algorithms in estimating haplotype genealogies with migration[END_REF]. For Pyropia sp. CHJ, Pyropia sp. CHK and Pyropia orbicularis, we calculated mean genetic distances withinand between-species for the COI in Mega6 [START_REF] Tamura | Molecular evolutionary genetics analysis version 6.0[END_REF]. Distances were calculated using the Maximum Composite Likelihood method and variation rate among sites was modeled with a gamma distribution; codon positions included were 1st + 2nd + 3rd.

10

Development of PCR-RFLP to rapidly assess putative species identity of Maitencillo foliose

Bangiales

Using the 44 sequences of rbcL available for the four more common species of foliose Bangiales encountered in Maintecillo (this study, [START_REF] Ramírez | Pyropia orbicularis sp. nov. (Rhodophyta, Bangiaceae) based on a population previously known as Porphyra columbina from the central coast of Chile[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF], see results for more information), we developed a method of species identification using polymerase chain reaction restriction fragment length polymorphism analyses (PCR-RFLP) of plastid DNA.

Differences in pattern of restriction digestion between species were examined insilico using the program Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2/copyright 1997 Max Heiman). Amplification protocol of the rbcL used in the PCR-RFLP analyses was the same as described above. Nonetheless, PCRs were done using a different forward primer than previously mentioned. Our newly developed primer rbcL-Pyr-F (5'-AGGTGTTGACCCGATTGAAG-3'), producing a longer fragment of 1230 bp instead of 873 bp, was used instead of F-rbcL [START_REF] Hommersand | Phylogenetic systematics and biogeography of the Gigartinaceae (Gigartinales, Rodophyta) based on the sequence analysis of rbcL[END_REF] in the PCR mix. The digestion of 0.5-1 µg of rbcL PCR product with 1.5 U AfaI (Thermo Scientific, Pittsburgh, PA) was performed following manufacturer instructions, with slight modifications. After 16 h at 37 °C, the reaction was stopped using a heat denaturation of 20 min at 65 °C. The fragments obtained after digestion with AfaI were separated on a 2 % TBE-agarose electrophoresis. Because the rbcL PCR-RFLP patterns obtained were extremely similar between the two Pyropia species (see results for more details), a partial sequence of the COI was obtained for 24 Pyropia samples using the same protocol as described above to confirm the species identification obtained by PCR-RFLP.

Morphological measurements and statistical analyses

For classical descriptions of foliose Bangiales species found at Maitencillo, the thallus shape, color, texture, and rhizoid position were described in 87 specimens previously identified at the species level using the newly developed PCR-RFLP method and COI sequences (see above). Microscopic observations of superficial and hand-cut transverse sections were used to establish tissue thickness and the number of cell layers, as well as to identify vegetative (generally at the thallus center) and reproductive structures (generally at the thallus border). More precisely, eight microscopic features of the gametophytic thallus were recorded, measuring three cells in three visual fields per photo, in a total of three photos per specimen. These morphological microscopic traits were the length and width of rhizoidal, vegetative, and zygotosporangial cells (surface views) and the thickness of vegetative and zygostosporangial lamina (hand-cut transverse sections). Moreover, the maximum length and maximum width of the thallus were also measured and considered in statistical analyses as macroscopic characteristics. Images were captured on an upright Leica ICC50 HD microscope (Wetzlar, Germany) using the Leica Application Suite Advanced Fluorescence EZ Imaging Software v.3.0 (Wetzlar, Germany).

To describe in multivariate space and search for morphological differences between foliose Bangiales species present at Maitencillo, principal component analysis (PCA) was applied to microscopic and macroscopic measurements using the software PAST specifying a variance-covariance matrix [START_REF] Hammer | PAST: Paleontological Statistics Software Package for Education and Data Analysis[END_REF]. All statistical analyses were done with normalized data, using the Box-Cox transformation option [START_REF] Box | An analysis of transformations (with discussion)[END_REF] available in the statistical software R (R core team, 2017). Statistical differences between species for all traits were assessed through multivariate nested, permutational ANOVA using the Adonis function in R [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. The "plant" (i.e. individual) level was treated as nested factor within the main factor "species". Moreover, data for each morphological trait was subjected to univariate post-hoc Tukey multiple comparisons in R (Abdi and Williams 2010) (after univariate ANOVA were done) to detect specific treatment differences between species. the 90 Bangiales samples from Maitencillo were assigned to 5 genetic species of bladed Bangiales (i.e. represented as black triangles in the tree reconstructions). All five-delimited species correspond to genetic entities previously identified by [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF]. Among these five species, four were dominant at Maitencillo: Pyropia orbicularis (N = 14), Pyropia sp. CHK (N = 47), Porphyra sp. CHE (N = 16) and Porphyra sp. CHC (N = 12). Their habits are shown in Fig. 2. A fifth species, Porphyra sp. CHF, was also present but in low frequency (N = 2, Table 1 andTable S1). Porphyra sp. CHE and Porphyra sp.

RESULTS

Genetic species present at

CHF were recovered as monophyletic sister groups both in the rbcL and COI phylogenetic trees (Fig. 1). Porphyra sp. CHC was also retrieved as a well-supported monophyletic group in the two phylogenetic trees (Fig. 1 and Fig. S2). On the other hand, phylogenetic trees were less clear concerning the number of distinct species that could be defined within the Pyropia orbicularis / Pyropia sp. CHK / Pyropia sp. CHJ species complex. Mean sequence divergence between species pairs was higher than 6% between Pyropia orbicularis and the other two species, whereas the distance between Pyropia sp. CHK and Pyropia sp. CHJ was 3.7% (Table S3). All mean sequence divergences calculated within species were, at least, five times lower than the ones calculated between species (Table S3).

Moreover, three groups of highly differentiated haplotypes (Pyropia orbicularis, Pyropia sp. CHK, and Pyropia sp. CHJ) were clearly shown by the reconstruction of the COI network for these recently diverged species (Supplementary material 4; [START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF]), all our newly acquired sequences being part of the Pyropia orbicularis and Pyropia sp. CHK groups (Fig. S3).

Microhabitats and intertidal distribution of genetic species. Species distribution of foliose

Bangiales reported here is based on the information obtained during our first sampling scheme (66 specimens sequenced for the COI and 46 for rbcL). Foliose Bangiales were never observed in medium or low intertidal zones of the rocky platform (Table 1). From twenty-five foliose Bangiales sampled at the high intertidal zone on the rocky platform, 13 were assigned to Pyropia sp. CHK, nine to Porphyra sp. CHE, and three to Pyropia orbicularis (Table 1). On the rocky wall, 15 of the 17-sampled foliose Bangiales were identified as Pyropia sp. CHK, one as Porphyra sp. CHE, and one as Pyropia orbicularis (Table 1). In the boulder zone surrounded by rocky pools, six of the 12 samples were classified as Porphyra sp. CHE, four as Pyropia sp. CHK, and two as Porphyra sp. CHF (Table 1). In the boulder zone surrounded by pools of sandy bottom, all 12 samples were assigned to the genetic species Porphyra sp. CHC (Table 1). Fig. 2 shows the habits of the four dominant genetic species observed at Maitencillo beach.

PCR-RFLP development and analysis for the rbcL. Among the 1230 bp fragment amplified by PCR for the rbcL, 94 polymorphic sites can be observed between aligned sequences of the four Bangiales species dominating the intertidal of Maitencillo. The sequence positions (i.e. variable sites) corresponding to the restriction site of AFA I in the rbcL-amplified fragment are given in Table 2. As shown in Fig. 3, PCR-RFLP patterns of the rbcL allowed us to easily distinguish Porphyra sp. CHE from Porphyra sp. CHC and from the two species of Pyropia. One very intense fragment located at approximately 900 bp was observed in Porphyra sp. CHC, whereas two fragments of approximately 580 and 430 bp were the most visible and characteristic of Porphyra sp. CHE (Fig. 3). On the other hand, only the presence of two fragments of approximately 600 and 700 bp in Pyropia orbicularis and only one in Pyropia sp. CHK in the same size range, allowed differentiating the two closes by Pyropia species (Fig. 3). Restriction patterns were extremely similar between the two Pyropia species and generate doubts about species determination for most samples of Pyropia. Indeed, as visible in Fig. 3, the two Pyropia orbicularis bands of roughly similar size are not easily separated and generate a very intense band/smear around 600-700 bp. This pattern can easily be confounded with Pyropia sp. CHK for which PCR-RFLP products are overloaded. In order to complement our PCR-RFLP analysis, 24 samples of Pyropia were then sequenced for the COI gene. Fifteen samples corresponded to Pyropia sp. CHK and 9 to Pyropia orbicularis. Only these 24 sequenced individuals were used for morphological analyses when considering Pyropia samples.

Variation in morphological characters. Results of the PCA based on morphological characters are shown in Fig. 4. According to the eigenvalues, the cumulative proportion of the first two principal components explains 81% of the total variation in the data.

Component 1 was represented mainly by thallus width and component 2 by thallus length.

If we do not take into account the Pyropia sp. CHK data, specimens of Porphyra sp. CHE and Pyropia orbicularis would appear as discrete morphological clusters (data not shown).

Nevertheless, both species show a high level of overlap with Pyropia sp. CHK in the PCA (Fig. 4). Conversely, Porphyra sp. CHC formed a discrete and distinctive morphological cluster in the PCA. Multivariate nested, permutational ANOVA (permanova) realized using the ten morphological traits measured, indicated that statistical differences exist between species and between plants (i.e. specimens) within species (Table 3). Indeed, post-hoc Tukey test for multiple comparisons identified significant differences (p < 0.05) between all pairs of species for four out of ten traits (Fig. 5 4).

For the P. orbicularis description, see Fig. S4 and [START_REF] Ramírez | Pyropia orbicularis sp. nov. (Rhodophyta, Bangiaceae) based on a population previously known as Porphyra columbina from the central coast of Chile[END_REF]. Habitat: Thalli of the GM morphotype grow on shady and humid steep rock faces (Fig. 2D). This morphotype is present mainly during the winter and spring seasons and is almost absent in summer. Thalli of the LM morphotype grow on the sunnier and drier platforms of the high intertidal (Fig. 2E). It is present mainly during the winter and spring seasons [START_REF] Zapata | Diferenciación ecológica entre ecotipos de dos especies hermanas de Pyropia (Bangiales, Rhodophyta) mediado por estrés ambiental[END_REF].

Etymology: variabilis-variable in shape. The name variabilis aims to describe the very variable morphology of the gametophytic habit. andS3). Incongruence between rbcL and COI results could be the result of the lower basesubstitution mutation rate of the rbcL gene for which incomplete lineage sorting and lack of monophyly has been observed in macroalgae species complex [START_REF] Tellier | Phylogeographic analysis of the 30°S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: Vicariance or parapatry?[END_REF][START_REF] Montecinos | Species replacement along a linear coastal habitat: phylogeography and speciation in the red alga Mazzaella laminaroides along the south east pacific[END_REF]. COI sequences, in contrast, allowed to determine genetic species in these recently diverged species complex. In red macroalgae, hybridization and introgression have also been linked to the existence of incongruence between mitochondrial and chloroplast markers [START_REF] Destombe | Diversity and natural hybridization in two related red algae species: Gracilaria gracilis and Gracilaria dura using multi DNA markers: resurrection of the species G. dura previously described in the Northern Atlantic 200 years ago[END_REF]. We cannot discard the possibility of past and/or ongoing gene flow between the three Pyropia sp. CHJ, Pyropia orbicularis and Pyropia sp. CHK, especially in central Chile where they co-occur (see [START_REF] Niwa | Genetic diversity and introgression in two cultivated species (Porphyra yezoensis and Porphyra tenera) and closely related wild species of Porphyra (Bangiales, Rhodophyta)[END_REF] for an example of introgression in Bangiales, between Py. Yezoensis and Py. tenera).

In spite of significant differences between all pairs of species for four morphological characters, the huge variance in traits observed for Pyropia sp. Nevertheless, even if the three species were generally observed sympatrically, some habitats seem mostly dominated by one species (e.g. Pyropia sp. CHK along the rocky wall). As previously reported in distributional studies supported by molecular tools for intertidal sites in New England, USA [START_REF] West | Molecular ecological studies of New England species of Porphyra (Rhodophyta, Bangiales)[END_REF] and in the southern West Cape, South Africa [START_REF] Griffin | Porphyra aeodis sp. nov. (Bangiales, Rhodophyta), an epiphyte of Aeodes orbitosa from South Africa[END_REF], we expected that the abundance and occurrence of specific intertidal zones by bladed Bangiales would differ. Indeed, in a highly heterogeneous environment, in terms of landscape complexity, physical and chemical variables and biotic interactions, differences in microhabitats could be expected among related species [START_REF] Billard | Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level[END_REF][START_REF] Couceiro | Evolution and maintenance of haploid-diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus[END_REF][START_REF] Muangmai | Cryptic species in sympatry: nonrandom small-scale distribution patterns in Bostrychia intricata (Ceramiales, Rhodophyta)[END_REF][START_REF] Montecinos | Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae)[END_REF]. Nonetheless, in our study, most specimens occurred in the high intertidal, probably experiencing relatively homogeneous abiotic or biotic conditions during late 2015) revealed that differences in micro-niches existed between species and phases (i.e. gametophytes and sporophytes). Along the European coast, the Fucus species complex (Fucales, Ochrophyta) was observed as the succession of different ecological species along a tidal gradient of contrasting habitats with a segregation between species in less than 50 cm on the vertical shore gradient [START_REF] Billard | Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level[END_REF]. As in the marine gastropod Littorina saxatilis (Olivi), where ecotypes are segregated in different habitats along the shore [START_REF] Butlin | Sympatric, parapatric or allopatric: the most important way to classify speciation? Transt[END_REF], divergence and speciation has been deemed to be driven by slight differences in ecological selective pressures as desiccation stress in Fucus [START_REF] Billard | Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level[END_REF]. It is possible that our sampling was too coarse to detect microhabitat differences between Pyropia orbicularis, Porphyra sp. CHE and Pyropia sp. CHK in Maitencillo, and new field observation along a fine vertical shore gradient linked with the acquisition of data on physical environmental factors and biotic local interaction should now be performed.

However, in our study, we observed what seems to be a variable or specialized ecotype of Pyropia sp. CHK (see Figs. and Figs. 8A and 9A). Indeed, gametophytes of the GM morphotype of Pyropia sp. CHK were dominant on rocky walls, with very low presence of gametophytes of the LM morphotype of Pyropia sp. CHK or other bladed Bangiales species therein [START_REF] Zapata | Diferenciación ecológica entre ecotipos de dos especies hermanas de Pyropia (Bangiales, Rhodophyta) mediado por estrés ambiental[END_REF], Betancourtt et al. 2018, this study). Interestingly, in the study of Coucerio et al. (2015), two ecotypes were observed among the E. siliculosus sporophytes sampled in North West France with one ecotype encountered as epiphyte on several different algal hosts and the other attached to abiotic substrates. The authors proposed that these two ecotypes probably arose due to phenotypic plasticity in the case of E. siliculosus. Our molecular data sets include only sequences of the COI and rbcL genes and these were not sufficiently variable to detect genetic difference between our two Pyropia sp. CHK ecotypes. However, these two molecular markers are characterized by low mutational rates in comparison to microsatellites [START_REF] Jarne | Microsatellites, from molecules to populations and back[END_REF], and we cannot discard the hypothesis of possible genetic adaptation of the GM morphotype in Pyropia sp. CHK to the conditions encountered on the step walls of Maitencillo. To determine if the two ecotypes within Pyropia sp. CHK are due to phenotypic plasticity or represent traits related to intraspecific genetic differentiation and adaptation, samples should be genotyped using more variables markers (i.e. microsatellites or SNP's) that allow testing for signatures of selection (e.g. Schlotterer 2000[START_REF] Haasl | The effects of microsatellite selection on linked sequences diversity[END_REF]. We also propose that transplant experiments and/or common garden experiments should be considered to complement the population genetic analyses and to test for adaptation (e.g. de [START_REF] De Villemereuil | Common garden experiments in the genomic era: new perspectives and opportunities[END_REF].

CONCLUSIONS

As pointed out by [START_REF] Sutherland | A new look at an ancient order: generic revision of the Bangiales[END_REF](following Matsuyama-Serisawa et al. 2004) the very simple and at the same time highly variable morphology of foliose Bangiales make taxonomic identification using key morphological traits an unresolved problem in this group. On the other hand, phylogenetic reconstructions using molecular markers have revealed cryptic diversity at the genus and species level in Porphyra and Pyropia (e.g. Guillemin et al. 2016 in Chile), and allowed the accurate reassignment of these organisms (e.g. Nelson and Broom 2010 in New Zealand). In our study, in spite of substantial sample sizes and significant differences between all pairs of species for various morphological characters, the overlap observed in morphological trait values between Porphyra and Pyropia species suggests that morphology is not sufficient to delineate genetic species in this group. In spite of this, some clear morphological differences were observed among the four species of foliose Bangiales analyzed, such as the differential thickness of the blade. This trait was associated with a differential degree of palatability [START_REF] Niwa | Changes of growth characteristics and free amino acid content of cultivated Porhyra yezoensis Ueda (Bangiales, Rhodophyta) blades with the progression of the number of harvest in a nori farm[END_REF]) and would have a potential utility in the selection of strains (each belonging to one or more species) for the food industry. Scweikert et al. (2012) proposed that the very similar morphology between some foliose Bangiales implies that they developed the most suited thallus shape for their gametophytes in their habitat. In this case, an optimal (and common) structure in phenotypic morphology in this group of algae would have been shaped by the particular genetic, developmental and environmental constraints operating on these algal taxa [START_REF] Rosen | Optimality Principles in Biology[END_REF]. The rapid and accurate identification of local genetic diversity in foliose Bangiales recently achieved using molecular techniques (Scweikert et al. 2012[START_REF] Nelson | Pyropia plicata sp. nov. (Bangiales, Rhodophyta): naming a common intertidal alga from New Zealand[END_REF][START_REF] Dumilag | Morphological and molecular confirmation of the occurrence of Pyropia tanegashimensis (Bangiales, Rhodophyta) from Palaui Is[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF][START_REF] Reddy | A rosette by any other name: species diversity in the Bangiales (Rhodophyta) along the South African coast[END_REF][START_REF] Yang | A molecular phylogeny of the bladed Bangiales (Rhodophyta) in China provides insights into biodiversity and biogeography of the genus Pyropia[END_REF] will allow researchers to refine the knowledge about the intrinsic and environmental determinants of their distribution across tidal gradients of contrasting habitats and climates.

Figure

  Figure 2.tif Figure 6.tif Figure 7.tif Figure 8.tif Figure 9.tif

  Fig.1of[START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF]. As shown by the Maximum Likelihood phylogenetic trees

  ). The largest values in length and width of the thallus were observed in Porphyra sp. CHC and Porphyra sp. CHE, respectively, while the thinnest and thickest reproductive laminae characterized Porphyra sp. CHC and Pyropia orbicularis, respectively. Within Porphyra, Porphyra sp. CHE showed bigger vegetative and zygotosporangial cells than Porphyra sp. CHC. Within Pyropia, Pyropia sp. CHK reached a larger width of vegetative cells and a smaller length and width of zygotosporangial cells than Pyropia orbicularis. Even if statistical analyses indicate that the width of vegetative cells is different between all 4 species, the boxplots for this trait inFig. 5 show that the high variance observed in Pyropia sp. CHK overlap values measured in both Porphyra sp. CHC and Porphyra sp. CHE. Taxonomic treatment. Below we describe in detail three previously unnamed species: one new species of Pyropia and two new species of Porphyra (See Fig. 2, Figs. 6-9, Table

  1.-Porphyra luchensis Meynard, Ramírez, Contreras-Porcia sp. nov. Description: The gametangial blades are semi-translucent, 39.0-156.0 µm thick in transverse section, reniform, rhomboid to spear-shaped and asymmetrical when fully extended, 5.0-22.0 cm long and 0.5-9.0 cm wide (Fig.2Aand Fig.6A) with a loose, wrinkled surface. Blade margins are spiny to undulated, often folded. Color green on the center of the blade to reddish-brown on the margins. Blades are attached to rock substratum by a basal rhizoidal holdfast. Thalli are monostromatic and monoecious. In sectional view, blade vegetative portions are monostromatic and their thickness range from 68.0 to 156.0 µm (Fig.6 C-D). Sexual regions of the thalli are monoecious, 39.0-136.0 µm thick in sectional view (Fig.6 E-H), with contiguous groups of cells forming either spermatangia or zygotosporangia and divided into separate male and female sectors. Reproductive cells are formed as continuous areas along the margins of the blade. The division formula of spermatangia is 128 (a/4, b/4, c/8) whereas the division formula of zygotosporangia is 64 (a/2, b/4, c/8) or 128 (a/4, b/4, c/8) (Fig.6 E-H). Table4 summarizesthe detailed morphological features of 32 blades of Po. luchensis identified through molecular assisted methods (i.e. PCR-RFLP). Holotype: Voucher specimen = SGO168338, housed in the herbarium of the National Museum of Natural History, Chile; sample code = CHE0027; gametophytic blade (vegetative) collected from boulders in the upper intertidal zone from Maitencillo beach (Chile: Valparaíso), 32° 39´S, 71° 26´ W, coll. Zapata, Contreras-Porcia, 10 Oct 2013. (Fig. 6A). GenBank accession numbers: COI, MH123945 and rbcL, MH124031. Distribution: Chile, from Atacama, Chañaral de Aceituno (29°04´S, 71°29´W), to Valparaíso, Maitencillo beach, (32°39´S, 71°26´W). Habitat: Thalli present year-round but more abundant in winter and spring, growing on boulders surrounded by stony ground pools at low tide. The species is less abundant in summer in the high and intermediate intertidal zones of rock platforms, where Pyropia orbicularis is dominant. Etymology: The specific epithet luchensis refers to the word "luche", a traditional name used by fishermen communities along the Chilean coast and referring to the bladed Bangiales harvested and sold for food consumption. Comments: Porphyra luchensis Meynard, Ramírez, Contreras-Porcia sp. nov. corresponds to the genetic species Porphyra sp. CHE originally identified by Guillemin et al. (2016). 2.-Porphyra longissima Meynard, Ramírez, Contreras-Porcia sp. nov. Description: The gametangial blades are semi-translucent, 36.0-122.0 µm thick in transverse section, linear to lanceolate and very long in comparison to their width, 16.0-50.0 cm long and 0.3-3.5 cm wide, and having a soft, relatively mucilaginous and flexible surface (Fig. 2B, and Fig. 7A). Blade margins are entire to sinuate. Color rosy pink to brown. Blades are attached to rock substratum by a basal rhizoidal holdfast. Thalli are monostromatic and monoecious. In sectional view, vegetative portions of blades are monostromatic, and their thickness range from 38.0 to 122.0 µm (Figs. 7C-D). Sexual regions of the thalli are monoecious, 36.0-94.0 µm thick in transverse section and divided into separate male and female sectors by a vertical line. Spermatangia are light green while zygotosporangia are light reddish-brown (Figs. 7 E-H). Zygotosporangia are formed as continuous areas along most of the thallus, except the basal portion (vegetative and rhizoidal cells), whereas spermatangia develop along the margins of the blade. The division formula of spermatangia is 128 (a/4, b/4, c/8) whereas the division formula of zygotosporangia is 64 (a/4, b/4, c/4) (Figs. 7 E-H). Table 4 summarizes the detailed morphological features of 32 blades of Po. longissima identified through molecular assisted methods (i.e. PCR-RFLP). Holotype: Voucher specimen = SGO168348, housed in the herbarium of the National Museum of Natural History, Chile; sample code = CHC0010; gametophytic blade (reproductive) collected from boulders of the upper intertidal zone of Maitencillo beach (Chile: Valparaíso, 32°39´S, 71°26´W), coll. Zapata, Contreras-Porcia, 12 Aug 2014. (Fig. 7A). GenBank accession numbers: COI, MH123931 and rbcL, MH124021. Distribution: Chile-from Atacama, Los Burros (28°55´S/71°31´W), to Los Ríos, Playa Rosada (39°48´S/73°24´W).Habitat: Gametophytic thalli only registered in winter, growing on boulders surrounded by sandy bottom or even buried in sand (Fig.2B).Comments: Porphyra longissima Meynard,Ramírez, Contreras-Porcia, sp. nov. corresponds to the genetic species Porphyra sp. CHC, originally identified by[START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF].

  3.-Pyropia variabilis Zapata, Meynard, Ramírez, Contreras-Porcia sp. nov. Description: The gametangial blades are monostromatic and monoecious. Blades are 63.0-139.0 µm thick in transverse section, oblong to lanceolate, sometimes cuneate or acuminate, with wavy surface, 2.8-14.0 cm long and 1.1-7.0 cm wide, generally very flexible and relatively resistant (Figs. 2D-E, Figs. 8A and 9A). Blade margins are entire to undulate or lobate. Blades are attached to rock substratum by a basal or subcentral rhizoidal holdfast. This species shows two different gametophytic morphotypes, occurring in different intertidal microhabitats, and characterized principally by external morphology (see Figs. 2D-E, Figs. 8A and 9A). The Green Morph (GM), consists of a single blade, generally lanceolate and forest green to yellowish green in color (Figs. 2D and 8A). A second morphotype, the Long Morph (LM), generally forestgreen to light-brown in color, is characterized by a long central blade and one or two additional shorter blades arising from a subcentral disc (Figs. 2E and 9A). LM blades are highly variable, being oblong to lanceolate, cuneate or acuminate. In spite of their variable morphology, all fresh gametophytic blades of Py. variabilis mostly display shades of green and present wavy ruffled-margins (Figs. 2D-E). In sectional view, vegetative portions of blades are monostromatic, and their thickness range from 63.0 to 137.0 µm (Figs. 8C-D and 9C-D). Sexual regions of the thalli are monoecious, 68.0-139.0 µm thick (Figs. 8E-H and 9E-H), with contiguous groups of cells forming either spermatangia or zygotosporangia and divided into separate male and female sectors by a vertical line. Reproductive cells are formed as continuous areas along the margins of the blade. The division formula of spermatangia is 128 (a/4, b/4, c/8) whereas the division formula of zygotosporangia is 16 20 (a/2, b/2, c/4) or 32 (a/2, b/4, c/4) (Figs. 8E-H and 9E-H). Table 4 summarizes the detailed morphological features of 15 sequenced blades of Py. variabilis identified through molecular assisted methods (i.e. sequencing and PCR-RFLP). Holotype: Voucher specimen = SGO168333, housed in the herbarium of the National Museum of Natural History, Chile; sample code = CHK0025; gametophytic blade (reproductive) collected from a rocky platform of the upper intertidal zone of Maitencillo beach (Chile: Valparaíso), 32° 39´S, 71° 26´ W, coll. J. Zapata, L. Contreras-Porcia, 6 September 2013 (Fig. 9A). GenBank accession numbers: COI, MH123975 and rbcL, MH124050. Isotype: Voucher specimen = SGO168334, housed in the herbarium of the National Museum of Natural History, Chile; sample code = CHK0028; gametophytic blade (reproductive) collected from steep rock faces of the upper intertidal zone of Maitencillo beach (Chile: Valparaíso), 32° 39´S, 71° 26´ W, coll. C. Fierro, F. Castañeda, 10 October 2013 (Fig. 8A). GenBank accession numbers: COI, MH123978 and rbcL, MH124051. Distribution: Chile-from Antofagasta, Pan de Azúcar (26°10´S/70°38´W) to Coquimbo, Horcón (32°42´S/71°29´W).

Comments:

  Pyropia variabilisZapata, Meynard, Ramírez, Contreras-Porcia, sp. nov. corresponds to the genetic species Pyropia sp. CHK, originally identified by[START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF].DISCUSSIONFew molecular studies have characterized genetic and morphological local diversity of bladed Bangiales and considered different types of habitats of the intertidal to describe their distribution within this environment. We have confirmed the presence of five species of bladed Bangiales in the rocky intertidal of Maitencillo beach, four of them being dominant, during winter-spring 2013-2014. All correspond to species previously detected by Guillemin et al. (2016) along the coasts of Chile, but our study report for the first time the presence of Porphyra sp. CHF, Porphyra. sp. CHE and Porphyra. sp. CHC in Maitencillo. Porphyra. sp. CHE was reported by Guillemin et al. (2016) in only one site located at 29°S of latitude in Chile. It is clear that the species is probably more common and broadly distributed than previously described, extending at least to Central Chile (32°S of latitude). Phylogenetic analyses of rbcL and COI gene sequences (Figs. 1 and S2) were not fully concordant, only the COI allowing to clearly separate Pyropia sp. CHJ, Pyropia orbicularis and Pyropia sp. CHK as three highly supported monophyletic clades (Figs. 1

  CHK only allow to clearly separate the species Porphyra sp. CHC from the other three dominant bladed Bangiales of Maitencillo Beach using morphology. Indeed, Porphyra sp. CHC consistently shows extreme values of length and width of the thallus and thickness of the reproductive lamina (see Fig. 5). Porphyra sp. CHC is also the only species showing a specific habitat preference in the intertidal and was encountered only on boulders surrounded by pools of sandy bottom. This suggests that, unlike the other three dominant species present at the study site, Porphyra sp. CHC morphology and/or other associated characteristics (e.g. metabolites or physiological traits) could be central in explaining its local distribution. Interestingly, a recent study reported the presence of a morphologically very similar species of Porphyra with long thin blades, Po. mumfordii, on boulders surrounded by sand in central Chile (see Fig. 2 of Muñoz-Muga et al. 2018), supporting the possibility of morphs adapted to particular habitats. Our study suggests that only one out of the four-dominant foliose Bangiales species show specific distributional ranges in the intertidal at Maitencillo since the distribution of Pyropia orbicularis, Porphyra sp. CHE and Pyropia sp. CHK overlap at the high intertidal zone on the rocky platform and at the boulder zone surrounded by rocky pools.

  winter and early spring (i.e. study period). Accordingly, Scweikert et al. (2012) found no distinct intertidal zonation patterns for bladed Porphyra sp. at Brighton Beach, southeast New Zealand, with two dominant species showing a similar distributional pattern across intertidal zones and seasons and seven other species being present only sporadically. In the present study we have focused only on differences in zonation, but we did not, however, test if Pyropia orbicularis, Porphyra sp. CHE and Pyropia sp. CHK presented any differences in micro-ecological niches. Supporting this possibility, Scweikert et al. (2012) hypothesized that the differing distributional patterns within and between seasons observed in the generally overlapping Porphyra sp. in Brighton Beach could still be explained by their differing degrees of physiological adaptation to abiotic factors. Previous studies integrating finer sampling scale than in our study, or the one of Scweikert et al. (2012), and records of physical environmental factors in distinct micro-habitats, have revealed differences in micro-niche partitioning between related species of macroalgae. The study of Muangmai et al. (2016) describing the small-scale distribution of three sympatric cryptic species of the red alga Bostrychia intricata (Bory) Montagne (Ceramiales, Rhodophyta) along the shore of Moa Point, Wellington, New Zealand, selected sampling patches according to three a priori contrasting factors: tidal position, wave exposure, and sun exposure. Muangmai et al. (2016) demonstrated that the distribution of three cryptic species of Bostrychia in the intertidal was non-random, and highly influenced by tidal height and wave exposure. In the case of the brown algae Ectocarpus crouaniorum Thuret in Le Jolis and E. siliculosus (Dillwyn) Lyngbye, Coucerio et al. (

Figure 1 .

 1 Figure 1. Maximum likelihood (ML) trees of Pyropia and Porphyra using Boreophylum

Figure 2 .

 2 Figure 2. Habit of the four dominant genetic species of foliose Bangiales observed at

Figure 3 .

 3 Figure 3. PCR-RFLP profiles resulting from the digestion of the 1230 bp rbcL

Figure 4 .

 4 Figure 4. PCA plot carried out for two microscopic and two macroscopic traits (i.e.

Figure 5 .

 5 Figure 5. Boxplot of the sizes of two out of eight microscopic traits (all in Log µm) and two

Figure 6 .Figure 9 .

 69 Figure 6. Images of macro and micromorphology of Porphyra luchensis sp. nov.,

Figure 1 .

 1 Figure 1. Maximum likelihood (ML) trees of Pyropia and Porphyra using Boreophylum birdiae and Bangia fuscopurpurea as outgroups, respectively, and based on DNA sequences of the cytochrome oxidase I (COI) gene. a) Details of the Pyropia COI ML tree (outgroup not shown). b) Details of the Porphyra COI ML tree (outgroup not shown). For each node, ML bootstrap values are indicated. Only high support values (>80) are shown. For species for which two or more sequences were considered in the analyses branches have been collapsed for easier reading and are represented as triangles. Black triangles indicate species present at the study site, Maitencillo Beach. 231x163mm (96 x 96 DPI)

Figure S2 .

 S2 Figure S2. Details of the Maximum Likelihood (ML) rooted tree for rbcL sequences (873 bp) of Bangiales. The complete tree is not shown but is similar to the one presented in Fig. 1 in Guillemin et al. (2016), save for some difference in branch support. a) Details of the Porphyra clade of the Bangiales rbcL ML rooted tree. b) Details of the Pyropia clade of the Bangiales rbcL ML rooted tree. For each node, ML bootstrap values are indicated. Only high support values (>80) are shown. For species for which two or more sequences were considered in the analyses branches have been collapsed for easier reading and are represented as triangles. Grey triangles indicate species present at the study site, Maitencillo Beach.

Figure S4 .

 S4 Figure S4. Images of macro and micromorphology of Pyropia orbicularis Ramírez, Contreras-Porcia & Guillemin. A) Habit of the foliose gametophyte sampled from the intertidal zone in Maitencillo beach, Valparaíso, Chile (scale bar= 10 cm). B) Surface view of basal, rhizoidal cells. C) Surface view of vegetative region of the thallus. D) Crosssection of vegetative region of thallus. E) Surface view of zygotosporangia. F) Crosssection of zygotosporangial region of thallus. G) Surface view of spermatangia (smaller and colourless). H) Cross-section of spermatangial region of thallus. Scale bar B-H = 20 µm.

Table 1 .

 1 Distribution of the five taxa of foliose Bangiales sampled in the site of Maitencillo along different intertidal habitats: Pyropia sp. CHK, Pyropia orbicularis, Porphyra sp. CHE, Porphyra sp. CHC and Porphyra sp. CHF. Species delimitation was based on molecular criteria using sequences of COI (66 sequences) and rbcL (46 sequences).

				INTERTIDAL HABITATS
	Species	Rocky Wall High Medium Low	Boulders	Boulders
						(rocky pools) (sandy pools)
	Pyropia sp. CHK	15	13	0	0	4	0
	Pyropia orbicularis	1	3	0	0	0	0
	Porphyra sp. CHE	1	9	0	0	6	0
	Porphyra sp. CHC	0	0	0	0	0	12
	Porphyra sp. CHF	0	0	0	0	2	0

Table 2 .

 2 AFA I cut sites (base position) among the variable bases of the aligned rbcL sequences of Pyropia sp. CHK, Pyropia orbicularis, Porphyra sp. CHE and Porphyra sp. CHC. A total of 873 sites were analyzed using the program Webcutter 2.0. We show base positions going from the forward to the reverse primers and indicate as reference for base position the rbcL sequence AB818919.1 of Pyropia yezoensis retrieved from Genbank. It is worth mentioning that amplified fragments used for PCR-RFLP were larger (1230 bp) than our obtained sequences (873 bp). See Materials and Methods for more details.

	Base Position	582 765 996 1134
	Pyropia sp. CHK	T	C	T	T
	Pyropia orbicularis	T	C	T	A
	Porphyra sp. CHE	C	T	A	A
	Porphyra sp. CHC	T	T	A	A

Table 3 .

 3 Nested Permanova carried out on the eight microscopic and two macroscopic morphological characters measured for four dominant sympatric Pyropia and Porphyra species. Null hypothesis: no morphological differences between species.

	Source of Variation	df	Sums of Sqs Mean Sqs F. Model	R 2	Pr(>F)
	Species	3	1.10743	0.36914	1583.01	0.44084	0.001***
	Species/Plant	88	1.23304	0.01401	60.09	0.49084	0.001***
	Residuals	736	0.17163	0.00023		0.06832
	Total	827	2.51210			1.00000

Table 4 .

 4 Habits and morphological features of Pyropia variabilis sp. nov. (CHK), Porphyra 1 luchensis sp. nov. (CHE), Porphyra longissima sp. nov. (CHC), and Pyropia orbicularis

	2
	3	from Maitencillo beach, Valparaíso, Chile.
	4
	Feature		Pyropia orbicularis	Pyropia variabilis	Porphyra luchensis	Porphyra longissima
	Size blade	2.8-5.5 x 2.4-5.3	2.8-14.0 x 1.1-7.0	5.4-22.0 x 0.5-9.0	16.0-50.0 x 0.3-3.5
	(cm, length × width)
	Shape		Orbicular (Several	Oblong to lanceolate (one or	Reniform to spear-shaped	Linear to lanceolate,
			laminae interwined)	several laminae)	asymmetrical (One lamina)	very long (one lamina)
	Color		Green-brown	Forestgreen to light-brown	Redish-brown to green	Rosy pinky to brown
	Habitat		Upper-mid intertidal	Upper-mid intertidal zone	Boulders surrounded by stony	Boulders surrounded
			zone	and steep rock faces	ground and rocky platforms	by sand
	Seasonality	Year-round:	Year-round:	Year-round:	Present only in Winter
			More abundant in	More abundant in Winter	More abundant in Winter and
			Spring and Summer	and Spring	Spring
	Sexuality	Monoecious	Monoecious	Monoecious	Monoecious
	Vegetative thickness	68-128	63-137	68-156	38-122
	(µm)
	Reproductive	72-130	68-139	39-136	36-94
	thickness (µm)
	Vegetative cells	16-30 x 10-22	12-35 x 8-27	17-43 x 12-29	9-42 x 6-30
	(µm, length × width)
	Rhizoidal cells	17-46 x 15-41	16-55 x 14-48	24-59 x 21-48	13-49 x 4-40
	(µm, length × width)
	Spermatangium	a4/b4/c4	a4/b4/c8	a4/b4/c8	a4/b4/c8
	Zygotosporangium	a2/b2/c4	a2/b2/c4 or a2/b4/c4	a2/b4/c8 or a4/b4/c8	a4/b4/c4
	5
	6

Table S3 .

 S3 Mean genetic distances calculated within-and between species for Pyropia sp. CHJ, Pyropia sp. CHK and Pyropia orbicularis for the COI. Genetic distances calculated within species are noted in bold along the diagonal. Genetic distances calculated between species pairs are noted below the diagonal. Sequences obtained in Maitencillo during this work were completed by the ones already available in[START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF].

		Pyropia sp. CHJ Pyropia orbicularis Pyropia sp. CHK
	Pyropia sp. CHJ	0.008
	Pyropia orbicularis	0.073	0.006
	Pyropia sp. CHK	0.038	0.063	0.001

Journal of Phycology

ACKNOWLEDGEMENTS

This work was supported by FONDECYT 1120117, 1170881, and DI-501-14/R & DI-1245-16/R (Universidad Andrés Bello) awarded to LC-P. We would like to thank two anonymous reviewers for their insightful and constructive comments. Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. CHK Pyropia sp. FIA Pyropia sp. FIA Pyropia sp. FIA Región de Atacama, Chañaral de Aceituno, Huasco, Chile Región de Coquimbo, Elqui, Abalonera Chica, Chile Región de Coquimbo, Puerto Aldea, Elqui, Chile Región de Coquimbo, Mina Talca, Limarí, Chile Región de Coquimbo, Choapa, Puerto Oscuro, Chile Región de Valparaíso, Petorca, Los Molles, Chile Región de Valparaíso, Valparaíso, Maitencillo, Chile Región de Valparaíso, Petorca, Salinas de Pullay, Chile Región de Magallanes, Punta Arenas, Chile Región de Magallanes, Fuerte Bulnes, Chile Región de Magallanes, Agua Fresca, Chile KP781612/ KP781613/ KP781614/ KP781615/ KP781616/ KP781617/ KP781618 KP781619/ KP781623/ KP781624 KP781625 KP781626/ KP781627/ KP781628 KP781629/ KP781630/ KP781632 KP781633/ KP781634/ KP781635 KP781604/ KP781605/ KP781606/ KP781607/ KP781608/ KP781609/ KP781610 KP781636 KP781603 KP781668 KP781669 Pyropia sp. HK-2011d Rhode Island, Governor Sprague Bridge 17, Narragansett, USA JN028798 Pyropia spiralis Espírito Santo, Vila Velha, Brazil JN222754 Pyropia tanegashimensis Sao Paulo, Praia Dura, Ubatuba, Brazil JN222752 Pyropia thuretii British Columbia, Island south of Clotchman I., Spanish Pilot Group, Tahsis, Canada JN028801 Pyropia torta British Columbia, Alder Island, Gwaii Haanas, Canada JN028802 Pyropia vietnamensis Parnaíba, Pedra do Sal, Piauí, Brazil JN222751 Wildemania amplissima British Columbia, Murchison Island Lagoon, Gwaii Haanas, Canada HQ969863 Wildemania miniata Quebec, Escoumins, Canada JN028874 Wildemania occidentalis Wildemania sp. FII British Columbia, Tahsis Nuchatliz Island, Canada Región de Magallanes, Buque Quemado, San Gregorio Chile JN028923 KP781664

. FIH Porphyra sp. FIH Porphyra sp. FIH Porphyra sp. FIH Porphyra sp. FIH Porphyra sp. FIH Porphyra sp. FIH KP781677KF479515/ KF479507/ KF479508/ KF479509/ KF479512/ KF479513/ KF479514 KP781576/ KP781577/ KP781578/ KP781579 KP781580/ KP781581/ KP781582/ KP781583 KP781587 KP781589 KP781591 KP781602 KP781652 KP781685/ KP781697 KP781665/ KP781774

Surface view of zygotosporangia. F) Cross-section of zygotosporangial region of thallus. G) Surface view of spermatangia (smaller and colourless). H) Cross-section of spermatangial region of thallus. Scale bar B-H = 20 µm.

Figure 7. Images of macro and micromorphology of Porphyra longissima sp. nov., SGO168348, holotype, Museo Nacional de Historia Natural, Santiago, Chile (Table S1). A) Habit of the foliose gametophyte sampled from the intertidal zone in Maitencillo beach, Valparaíso, Chile (scale bar = 10 cm). B) Surface view of basal, rhizoidal cells. C) Surface view of vegetative region of the thallus. D) Cross-section of vegetative region of thallus. E) Surface view of zygotosporangia. F) Cross-section of zygotosporangial region of thallus. G) Surface view of spermatangia (smaller and colourless). H) Cross-section of spermatangial region of thallus. Scale bar B-H = 20 µm. Green Morph (GM), SGO168334, isotype, Museo Nacional de Historia Natural, Santiago, Chile (Table S1). A) Habit of the foliose gametophyte sampled from the intertidal zone in Maitencillo beach, Valparaíso, Chile (scale bar = 10 cm). B) Surface view of basal, rhizoidal cells. C) Surface view of vegetative region of the thallus. D) Cross-section of vegetative region of thallus. E) Surface view of zygotosporangia. F) Cross-section of zygotosporangial region of thallus. G) Surface view of spermatangia (smaller and colourless). H) Cross-section of spermatangial region of thallus. Scale bar B-H = 20 µm.