A reassessment of the foliose Bangiales (Rhodophyta) in the Balearic Islands including the proposed synonymy of Pyropia olivii with Pyropia koreana

Abstract A taxonomic reevaluation of the foliose Bangiales placed in the genus Porphyra from the Balearic Islands, based on the historical and recent collections, has resulted in a new floristic composition of this group. The molecular, morphological, and karyological analysis reveals that for these islands, there are no Porphyra species, and only two members of the genus Pyropia are present: Pyropia elongata and Pyropia koreana. The sequence data of the rbcL plastid gene indicate that Pyropia olivii and Pyropia koreana are conspecific. We, therefore, propose the synonymy of Pyropia olivii with Pyropia koreana, which is the taxon with nomenclatural priority. The study with the fresh and old material has revealed that neither Porphyra umbilicalis nor Pyropia leucosticta are present in this area and that most of the old herbarium material that has gone under this name belongs to Pyropia elongata.


Introduction
The order Bangiales has been the subject of much taxonomic change during the last decade due to the several local floristic studies in the different parts of the world (Broom et al. 1999, 2004, Brodie and Irvine 2003, Klein et al. 2003, Lindstrom and Fredericq 2003, Brodie et al. 2007, Mols -Mortensen et al. 2012, Verg é s et al. 2013 ).In addition to the traditional morphological studies, the use of the molecular tools has enabled significant changes to be made in the classification of this group of red seaweeds provide a taxonomic revision of this group in the Balearic Islands using both the molecular and morphological data obtained from the specimens collected in this area by us and the herbarium samples.

Collections and sampling
The sampling started in December 2009 and was finished in August 2010 in order to establish the seasonality of the bladed Bangiales.The study area was the coast of Majorca Island (Balearic Islands, Spain; 43 ° 27 ′ 24 ″ , 36 ° 00 ′ 03 ″ ) where 14 localities were sampled (Table 1 ).The specimens were collected from the rocky intertidal zone and the upper sublittoral (Table 1).The vouchers were pressed on the day they were collected, and a subsample of each was stored in the silica gel for the molecular analysis (Table 2 ).The herbarium sheets of the dried specimens are housed at the Herbarium of the University of Girona (HGI) and at the personal herbarium of Nat à lia Comalada.In addition to our collections, all the specimens referred to the genus Porphyra located in the 14 herbaria of the Iberian Peninsula [BCN-Phycophyta, BIO-Cryp, FCO, HGI-A, MA-Algae, MACB, MAF-Algae, MGC-Phyc., MUB, PO, SANT, UALG, VAL, and J.J. Rodr í guez y Femen í as herbarium housed at l ' Ateneu de Ma ó (Minorca)] were examined with the aim of detecting any earlier samples of the bladed Bangiales species in the study area.The herbarium abbreviations follow that of Thiers (2012) .

Molecular data analysis
The samples for use in the molecular analyses were selected based on the preliminary morphological identification (Tables 1 and 2).The total DNA was extracted using a modified CTAB microextraction protocol (modified from Rogers and Bendich 1994 ).The standard polymerase chain reaction (PCR) procedures were applied to amplify the rbc L fragment using a combination of the four primer pairs: F67 (TAC GCT AAA ATG GGT TAC TG), R502 (TAT CCA TAC GCT CAC GTT CTA CAA), F461 (GTC CTG CAA CTG GAT TGA TTG T) R901 (TAC CAG CTC TAT GTA AAT GTA AAA), F870 (TGA CAT GAT TTT ACA TTT ACA TAG AC) R1312 (3 ′ GGC CTT CAT TTC TTG CCA TAA C 3 ′ ), 5rc (GTG GTA TTC ATG CTG GTC AAA) rbcspc (CAC TAT TCT ATG CTC CTT ATT KTT AT) (Teasdale et al. 2002 ;A. Mols-Mortensen, personal communication).The PCR protocols used were one initial denaturation cycle at 93 ° C for 3 min followed by 29 cycles at 93 ° C for 30 s, 45 ° C for 1 min and 72 ° C for 1.5 min, and a final extension cycle at 72 ° C for 10 min as described in Teasdale et al. (2002) .The sequence reactions were run on an ABI 377 DNA sequencer (Perkin Elmer, Waltham, MA, USA).
The sequences were edited and assembled using the Sequencher ® version 5.0 (Gene Codes Corporation, Ann Arbor, MI, USA) and then aligned using the Mac Clade v4.8 (Maddison and Maddison 2005 ).The identity of the new sequences was checked by BLAST (Altschul et al. 1990 , http://blast.ncbi.nlm.nih.gov).To compare our specimens with the previous identifications, we downloaded representative rbc L sequences from Genbank of the most similar species obtained after the extensive BLAST search ( Pyropia elongata , Pyropia olivii , and Pyropia koreana ) together with the rbc L sequences from the traditionally recorded species in this area ( Pyropia leucosticta and Porphyra umbilicalis ) and the sequences from the other morphologically similar species that have been found recently from the areas close by including the Iberian Mediterranean coast ( Pyropia suborbiculata ; Verg é s et al. 2013 ).We analyzed 1333 nucleotides for each isolate.The identification of the most appropriate model of the evolution, the number of nucleotide differences, and the pairwise distances between the sequences were calculated using MEGA version 5 (MEGA, Tempe, AZ, USA) following Tamura et al. (2011) .The pairwise distances were conducted using the Tamura-Nei model, and the rate variation among the sites was modeled with a gamma distribution (shape parameter = 0.23) (Tamura and Nei 1993 ).

Morphological study
The morphological observations were made by rehydrating the selected fragments from the herbarium sheets with seawater.The sections were made by hand with a razor blade, and the morphological characters most frequently used to distinguish the foliose Bangiales (gross morphology, plus anatomical and reproductive structures; Brodie andIrvine 2003 , Brodie et al. 2007 ) were examined.The photomicrographs were taken with a Zeiss AxioCam MRc3 camera attached to an Axioskop 2 plus microscope (Carl Zeiss, Berlin, Germany).The terminology for the reproductive structures follows that of Nelson et al. (1999) .

Karyological methods
The chromosome number was obtained using the herbarium specimens that were stained with Wittmann ' s (1965) aceto-iron-hematoxylin-chloral hydrate technique following the protocol in Holmes and Brodie (2005) .The specimens used for the molecular studies.

Diversity of the genus Pyropia in the Balearic Islands
The molecular and morphological data of the representative specimens from the Balearic Islands led us to recognize the two members of the foliose Bangiales occurring in this area: Pyropia elongata and Pyropia koreana .

Molecular results
The molecular rbc L gene characterization based on the calculations of the nucleotide differences and the pairwise distances between the sequences (Table 3) indicated that the closest sequences to that of the specimen HGI-A 9565 collected in Majorca were those identified as Pyropia elongata samples (including its lectotype sequence FJ817088).The differences between the sequences ranged from 0.1% to 0.2% (one to three nucleotides).Accordingly, we identified the specimen from which the HGI-A 9565 sequence had been obtained as Py.elongata .The analysis for the sequence of the specimen labeled as HGI-A 9587 indicated that it was very similar to Pyropia olivii (including its holotype sequence DQ807007) and Pyropia koreana (HQ728198) sequences, with the differences between them ranging from 0% to 0.2% (zero to two nucleotides).Furthermore, the sequences from all these specimens ( Py. olivii and Py.koreana ), DQ807007 (holotype), DQ813625, HQ728198, and HGI-A 9587 from Greece, USA, Korea, and the Balearic Islands, respectively, indicated that they were strongly related to each other as the differences between their sequences only ranged from 0% to 0.2% (zero to two nucleotides).Given these small molecular differences, together with the very similar morphological, reproductive, and ecological features  (Table 4 ), we concluded that all these taxa are the same species.Py. koreana has nomenclatural priority over Py. olivii , which is, therefore, considered to be a synonym.
To corroborate these identifications and to be able to provide evidence to confirm their possible misidentification with the other traditionally reported species, we also compared HGI-A 9565 ( Pyropia elongata ) and HGI-A 9587 ( Pyropia koreana ) sequences with Pyropia leucosticta and Porphyra umbilicalis (Table 3).We observed that the differences between HGI-A 9565 and P. leucosticta sequences were approximately 5.3% (52 nucleotides), while the differences between HGI-A 9565 and P. umbilicalis were much more marked, at approximately 20.3% (132 nucleotides).The pairwise distances and nucleotide differences with HGI-A 9587 showed divergences of 4.4% (46 nucleotides) with Py. leucosticta and of approximately 18.9% (130 nucleotides) with P. umbilicalis .A comparison of HGI-A 9565 and HGI-A 9587 sequences with those for Pyropia suborbiculata , a species which has recently been reported from the Iberian Mediterranean coast (Verg é s et al. 2013 ) shows differences of approximately 12.7 -13.2% (94 -96 nucleotides) for HGI-A 9565 and 12.6 -12.9% (97 -99 nucleotides) for HGI-A 9587.
Overall, these genetic comparisons within the rbc L gene between all the analyzed specimens are among the expected variation in the Bangiales.Hence, among our specimens, the intraspecific divergences ranged from 0% to 0.2%, while the divergences between the species ranged from 4.4% to 13.2%, whereas the divergences between the genera ranged from 15.1% to 24% (Klein et al. 2003, Lindstrom and Fredericq , 2003, Brodie et al. 2007, Lindstrom 2008, Kikuchi et al. 2010, Sutherland et al. 2011, Kucera and Saunders 2012 ).

Pyropia elongata (Kylin) Neefus and J. Brodie (Figures 1 -22)
The Balearic specimens present foliose thalli, up to 22 cm long and 11 cm broad, with minute discoid holdfasts and sometimes a short stipe that expands into a single or few elongated, rounded, and sometimes lobed or laciniate blades (Figures 1 -9); when fresh, the specimens were brown with gray or greenish spots; when dried, they were brownish or slightly blue.The blades were monostromatic, with an entire margin, folded or ruffled.The vegetative cells were easily recognizable as they were polygonal in the surface view (SV) and isodiametric in the TS (38 -57 μ m).The thalli were monoecious; the male gametangia were

References
This study Huang and Lee 1994Brodie et al. 2007Brodie et al. 2007 scattered in patches in the upper margin of the blade forming narrow streaks elongated in the direction of the blade expansion and surrounded by a marginal zygotosporangial zone (Figures 1 -5, 7 -8).In the SV, the male gametangia occurred in 8 packets of 16 each (Figure 10) divided periclinally to give 8 cells in the TS , giving 1024 male gametes in each packet, and with n = 4 chromosomes in each male gamete and, occasionally, n = 3 chromosomes (Figure 12).The female gametangia developed a small protuberance, the trichogyne ; the cell walls of the blade bulged when the trichogynes protruded beyond them; the cell wall bulge remained even after the zygotospores were formed.The zygotosporangia (Figure 19) occurred in 4 packets of 4 (sometimes eight) each divided periclinally to give 4 cells in the TS (Figures 20 -22), giving 64 zygotospores in each packet with each spore 14 -16 μ m in diameter, which were distributed in scattered streaky areas along the blade margin.The conchocelis phase was not recorded in the field.
The Pyropia elongata thalli were found in the intertidal, up to 20 cm above the sea level, growing on the rocks or limpets, and occasionally epiphytic on the other algae or artificial substrata such as the ropes, and formed a clear brown band covering the substrata.They occurred from winter to spring.The individuals appeared in March, after which they began to decline, and they had completely disappeared in May.The study of the anatomical and reproductive structures characterize this species in the Mediterranean as having a small foliose thallus, 0.4 -4 cm long and 0.5 -1 cm broad, attached with a rhizoidal holdfast and short stipe, often not located in the center of the blade, which expands into a single or usually several, ovate, elongate, or sometimes reniform blades .When fresh, the specimens were brown to brownishred or pink, translucent and delicate, and when dried, the thalli were purple or pinkish in color.The blades were monostromatic, with an entire margin, sometimes ruffled or slightly folded.The vegetative cells were mixed with rhizoidal filaments at the base of the blade (Figure 39), in the rest of the thallus, they were polygonal in the SV and isodiametric in the TS, up to 16 μ m in diameter (Figure 40).The thalli were monoecious; the male gametangia were scattered in small patches (Figure 41) in the middle-upper part of the blade.In the SV, the male gametangia occurred in 4 packets of 8 (sometimes 16; Figures 42 and 43) each divided periclinally to give two cells in the TS (Figure 45), giving 64 male gametes in each packet, with two chromosomes in each male gamete (Figure 44).The female gametangia developed a small protuberance, the trichogyne (Figures 45 and 46); the cell walls of the blade bulged when the trichogynes protruded beyond them; the cell wall bulges and fertilization canals remained even after the zygotospores were formed (Figure 47).

Synonymy of Pyropia olivii
The zygotosporangia consist of 2 packets with one spore each, which are divided periclinally to give 2 cells in the TS (Figure 48), so that each zygotosporangium contains four zygotospores, 10 -12 μ m in diameter, which were formed adjacent to the male sori.The conchocelis phase was not recorded in the field.
The Pyropia koreana thalli were found in the upper subtidal level growing mainly epiphytically on the Corallina elongata and also on the other seaweeds of the upper sublittoral (e.g., Pterocladiella capillacea , Gelidium sp.) or  on the artificial substrata such as the ropes.This species was found from winter to spring.The individuals appeared from the end of February until mid-April and completely disappeared in May.

Discussion
The molecular data, morphology, karyology, and ecology of Pyropia elongata matched with the other previously published surveys (Brodie et al. 2007 , Neefus andBrodie 2009 ) and allowed us to establish that this species is very common in the Balearic Islands.This report confirms that this species is widespread in the North Atlantic (Brodie et al. 2007, Mols -Mortensen et al. 2012 ), and its distribution in the Mediterranean has been found to be more widespread as more detailed studies were done.The revision of the old sheets collected since 1886, located in the J.J. Rodr í guez y Femen í as Herbarium and misidentified as Porphyra leucosticta (currently Pyropia leucosticta ) led us to hypothesize that the identity of Py. elongata could have been mistaken for a long time.This highlights the possibility that Py. leucosticta was never present in the Balearic Islands and has been confused with Py. elongata , as demonstrated in the other parts of the Mediterranean (Brodie et al. 2007 ).
The other Balearic species of the genus Pyropia , Pyropia koreana , is easy to differentiate from Pyropia elongata as it is much smaller (up to 4 cm), occurs in the sublittoral, and has male sori that are located in very small patches.The molecular analysis demonstrated that the rbc L sequences were very similar among the Balearic specimen (HGI-A 9587), Pyropia olivii (from Greece, DQ807007, and Atlantic USA, DQ813625) and Py.koreana (from Korea, HQ728198), differing by a maximum of two nucleotides (Table 2).This similarity led us to the conclusion that these three entities are not distinct.Moreover, the low level of the genetic distances between them (maximum 0.2%), falls within the limits for intraspecific variation (0% to 1% up to 2% in some taxa, Klein et al. 2003, Lindstrom and Fredericq 2003, Lindstrom 2008, Kucera and Saunders 2012 ).The anatomical and reproductive structures of Py. koreana from the Balearic Islands were compared (Table 4) with the material from the type locality (Korea) and the Mediterranean record that belong to the holotype of Py. olivii .No significant differences among these three entities were found (Table 4).As there are molecular and morphological similarities among all these specimens (HGI-A 9587, DQ807007, DQ813625, and HQ728198), we can consider them synonymous, and because Py. koreana has nomenclatural priority, we propose the synonymy of Py. olivii with Py. koreana .However, it is of note that after all the sheets, located at the main herbaria for the bladed Bangiales of the Balearic Islands, have been revised, Py. koreana has only been detected in a unique sample (SANT-A 1279, collected in April 1987), indicating that this species is less abundant than Py.elongata , at least for the islands (Table 1).Although the existence of Py. elongata and Py.koreana in the Mediterranean has only been elucidated recently, the evidence from the herbarium specimens indicates that these species have been present in this area under different names since the nineteenth century (Brodie et al. 2007 ;this study).As Py. koreana had until now only been recorded from Korea (Lee andKang 2001 , Kim andKim 2011 ), our results indicate that the known worldwide distribution of this species is significantly greater than previously documented, as this species as Py.olivii had been reported from the coasts of Greece and Italy, and in the Atlantic Ocean on the coasts of the United States of America.
It was not possible to check the reports of Porphyra laciniata and Porphyra vulgaris (Colmeiro 1868) as we were not able to locate those sheets in any herbarium collection.However, considering the abundance of Pyropia elongata populations and that it is the only foliose bangiophyte that has been reported in the intertidal of the Balearic islands, so far, we have concluded that these species were almost certainly misidentifications of Py. elongata as the gross morphology is very confusing between some members of this group.Our confirmation that the oldest sheets located at the J.J. Rodr í guez y Femen í as Herbarium were identified as Porphyra leucosticta but belong to Py. elongata , again demonstrates the confusion between these two entities, and the importance of revising the historical reports with the aim of clarifying the real diversity of this group of algae.
In conclusion, this study provides new data on the foliose Bangiales in the Mediterranean and represents a new contribution to the global knowledge of the biodiversity of the Bangiales.Furthermore, it emphasizes the need to use the molecular tools in conjunction with the old reports and associated collections in order to elucidate the diversity of these organisms.within the framework of the project Ref. CGL2008-00932/ BOS (Ministerio de Educaci ó n y Ciencia, FEDER) funded by the Spanish government and by a Jose Castillejo postdoctoral fellowship from the Spanish government, a postdoctoral mobility grant from the University of Girona, and a postdoctoral scholarship for research abroad from the Catalan government.We acknowledge the Natural History Museum Molecular Laboratory and all the people who have contributed in many ways to this work.We also thank the Porphyra global network and especially J. Sutherland, A.M. Mortensen, M.J. Holmes, R. Walker, and M. Lynch for their help and advice.We also thank the Instituto Espa ñ ol de Oceanograf í a de Palma de Mallorca for their support and for hosting a large component of the field work for the study, with special mention for the director of the center, Dr. Enric Massut í , and our colleagues there, and also to Dr. Biel Moy à from the University of Balearic Islands, for managing this hosting in Mallorca, as well.

FJ817088
of nucleotide substitutions are below the diagonal and pairwise genetic distance above ( a Brodie et al. 2007; b This work; c Sutherland et al. 2011; d Verg é s et al. 2013).

Table 1
The collection information for Pyropia elongata and Pyropia koreana records in the Balearic Islands.

Table 2
The samples used in calculated genetic distances, voucher or isolate number, locality, Genbank accession number and reference are indicated.

Table 3
Genetic differences for rbc L gene between Pyropia elongata , Pyropia olivii , and Pyropia koreana in the Balearic Islands and the recently found species in the neighboring areas (Iberian Mediterranean coast).

Table 4
A comparison of Pyropia koreana from the Mediterranean Iberian Peninsula with the original description from Korea (holotype), the original description of Pyropia olivii from Greece (holotype), and the specimens of P. olivii from New England (ND = no data).