Lithophyllum species from Brazilian coast: range extension of Lithophyllum margaritae and description of Lithophyllum atlanticum sp. nov. (Corallinales, Corallinophycidae, Rhodophyta)

Coralline algae (Corallinophycidae, Rhodophyta) are primarily characterized by the impregnation of calcium carbonate (calcite) in their cell walls. To determine the systematic position of genera and species, researchers are increasingly combining anatomical studies with comparisons of DNA sequences. In the present study we have described the main representatives of the genus Lithophyllum in intertidal and subtidal habitats from the southern coast of Brazil based on anatomical studies, supported by molecular markers. Analyses were conducted on specimens collected from five sites along the coast of southern Brazil. Anatomical studies were carried out by light and scanning electron microscopy. Molecular studies were based on DNA barcoding markers (cox1; UPA) and SSU rDNA. Using comparative anatomical features, corroborated by the molecular data, we identified two taxonomic entities from the Brazilian coast: Lithophyllum margaritae, and we propose the new species Lithophyllum atlanticum.

Given the morphological plasticity and the dependence of diagnostic characteristics based on the reproductive structures (Woelkerling et al. 1983, Riosmena-Rodriguez et al. 1999, alternative methods are necessary to investigate the systematics of Lithophyllum species and other non-geniculate coralline red algae. Therefore, a DNA-based approach in combination with detailed anatomical characterization is fundamental for accurate systematic evaluation of this group (Vidal et al. 2003). Herein, we used three markers to assess the diversity among Lithophyllum species in the Southern Brazilian coast: cox1 (the 5' region of the mitochondrial cytochrome c oxidase subunit I gene, Saunders 2005), UPA (the Universal Plastid Amplicon, Sherwood & Presting 2007, Sherwood et al. 2008, Sherwood et al. 2010) and the nuclear encoded SSU (Bailey & Chapman 1998, Bailey 1999. Our goal is to describe a new species of the genus Litphoyllum from the Brazilian coast and to extend the range distribution of Lithophyllum margaritae based on morphological, anatomical and molecular analyses.

Materials and methods
Collections. Specimens examined were collected from November 2009 to December 2010 along the subtropical western Atlantic coast located between the Paraná and Rio Grande do Sul states. In Paraná, samples were collected in the subtidal of Currais Archipelago. In Santa Catarina State, samples were collected from the intertidal rocky shore, at Ponta das Canas beach and in the subtidal from the rhodolith bed in Arvoredo Marine Biological Reserve and also from sandy bottom in Xavier Island. In the Rio Grande do Sul encrusting specimens were collected from the intertidal rocky shore in Guarita beach. Subtidal collections were taken by SCUBA dives. Collections have been incorporated in the Herbarium of University of Santa Catarina-FLOR (Thiers 2014) (Table 1). Light and scanning electron microscopy. All specimens used for both light and electron microscopy were previously fixed in 4% formaldehyde/seawater. Permanent slides for light microscopy were prepared following procedures adapted from Moura et al. (1997) and Horta (2002). Stubs for Scanning Electron Microscopy (SEM) were prepared following Chamberlain (1993) and analyzed using a SEM (JEOL JSM-6390LV). Voltage used ranged from 15-25 kV, seeking the best resolution. Conceptacle measurements follow Chamberlain (1983). Growth form terminology follows Woelkerling et al. (1993), and anatomical terminology follows Woelkerling (1988).

Molecular studies
Samples included in this study, including specimen voucher numbers and GenBank accession numbers for newly generated sequences, as well as for specimens from the referenced studies, are presented in Table 1. Sequences from GenBank were included in this study in order to provide phylogenetic context. Four sequences of Sporolithon sp. were included in the UPA analysis to test the distinction between Lithophyllum specimens and specimens with a similar thallus habit found along the Brazillian coast.
DNA Extraction and PCR amplification. Specimens were manually cleaned to eliminate epiphytes and other contaminants, dried in silica gel, and ground to a powder in liquid nitrogen. Total DNA was extracted using the kit "NucleoSpin Plant II" (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. Mitochondrial cox1 was amplified and sequenced using the primers GazF1 and GazR1 and protocols as described by Saunders (2005), plastidial UPA was amplified and sequenced using the primers p23Sv_f1 and p23Sv_r1 and protocols as described by Sherwood & Presting (2007) and nuclear SSU was amplified and sequenced using the primers and protocols as described by Bailey and Champan (1998). Alignment. Sequences were assembled and edited in BioEdit version 5.0.6 (Hall 1999), and chromatograms were checked to confirm the validity of ambiguous nucleotides. UPA and cox1 sequences were aligned manually with MacClade 4.08 (Maddison & Maddison 2000) and SSU sequences were initially aligned in MacClade 4.08 then exported and aligned using the CLUSTAL W (Thompson et al. 1994) program in MEGA 5.2.2 (Tamura et al. 2011). Ambiguous regions in the SSU alignment were cropped to the nearest conserved region. A UPA matrix was constructed with 20 sequences and was 370 bp in length. A cox1 matrix of 23 sequences and 664 bp in length was constructed. The SSU rDNA matrix was 1612 bp in length and included 17 sequences, with sequences for Mesophyllum erubescens (Foslie) Me. Lemoine (1928: 252) and Pneophyllum cf. conicum (Dawson) Keats, Y.M. Chamberlain & Baba (1997: 264), used as outgroups.
Phylogenetic Analysis. Neighbor-joining analyses (NJ) for UPA and cox1 data were conducted in MEGA 5.2.2 (Tamura et al. 2011) using the Maximum Composite Likelihood model with 2,000 bootstrap replicates to assess branch support. Bayesian analysis was conducted on SSU rDNA using the MrBayes 3.2.1 program (Huelsenbeck and Ronquist 2001). The appropriate evolutionary model was selected in MrModeltest 2.2 (Nylander 2004). Two parallel runs of four MCMC chains consisting of three heated chains and one cool chain were carried out with 4 x 10 6 generations and resampling every 1,000 generations resulting in a total of 80,002 trees. We discarded the first 50,000 generations as "burn-in" and a consensus tree was built using the remaining data. Sequences generated on this study were deposited on Genbank (for more details see Table 1).

Results
In this study two species were identified using the modern anatomical concepts following Woelkerling and Campbell (1992) that circumscribe the genus Lithophyllum, as follows: (1) cells of contiguous vegetative filaments joined by secondary pit-connections, (2) fusions between vegetative cells absent or not observed, (3) lack of genicula and haustoria, (4) lack of isobilateral organization, (5) tetrasporangial conceptacles uniporate. Also, molecular data were used as further evidence to distinguish the specimens in this study from the previously reported molecular entities. We have found Lithophyllum margaritae (Figs. 1-10; Table 2), which has had its distributional range extended from the Mexican Pacific to the Atlantic, and Lithophyllum atlanticum sp. nov.  Table 2).
Reproductive features: Tetrasporangia born in uniporate conceptacles (Fig. 7) flat or slightly sunken in relation to the thallus surface (Fig. 8). Chambers of these conceptacles are 180-260 μm in diameter and 70-95 µm in height (Fig. 9). The filaments of the conceptacle roof are 3-5 cells thick (including the epithallial cell) and the filaments surrounding the conceptacle are 10-14 cell layers thick from the epithallial cell to the chamber base ( Fig.  9 and 10). Chambers often presenting calcified central columella which, in some conceptacles, project into the pore channels (Fig. 9). Buried conceptacles were observed in older portions of the thallus. Pore channels delimited by cells, but in SEM view not occluded by them; zonate tetrasporangia 40-60 μm in height and 15-35 μm in diameter (Fig. 10). Gametophytes not observed.   Ecological observations. The specimens were found only as free-living rhodoliths in a rhodolith bed in the Arvoredo's Marine Biological Reserve (Florianópolis, Santa Catarina) at depths ranging from 10-15 m; no differences were observed in the samples during winter and summer seasons.

Description
Vegetative features: Plants non-geniculate,can grow as rhodoliths, on sandy bottom, or as encrusting forms, slightly or fully attached to the substratum, found on rocky shores. Surface smooth or warty to lumpy, protuberances 3-5 mm in diameter and 5-10 mm in height (Figs. 11-13). Thallus pseudoparenchymatous with monomerous organization in older and younger portions (Fig. 14), and 1-2 layers of epithallial cells, 3-6μm in height and 6-11μm in diameter with flattened or concave distal walls. Subepithallial cells 6.5-13 µm in height and 4.5 to 10 µm in diameter. Perithallial filaments more-or-less perpendicular to hypothallial cells; perithallial cells 8 to 17μm in height and 5.5 to 11 μm in diameter. Filaments are linked by secondary pit-connections (Fig. 15). Trichocytes were not observed.
Reproductive features: Tetrasporangia born in uniporate conceptacles, flush, slightly elevated or sunken in relation to the thallus surface (Fig. 16). Chambers of these conceptacles are 315-345 µm in diameter and 90-130 μm in height, the filaments of the conceptacle roof are 4-7 cell layers thick (including the epithallial cell) and the filaments surrounding the conceptacle are 9-13 cell layers thick from the epithallial cell to the chamber base . Non-calcified central columella are frequently present (Fig. 19), zonate tetrasporangia are 46-68 µm in height and 15-33 µm in diameter (Fig. 19-t). Gametophytes not observed.
Ecological observations. This species was found forming rhodolith beds in the Marine Biological Reserve at 5-15 m depth. Encrusting forms found growing on rocky shores in epilithic and occasionally epizoic (mollusks) forms. In the intertidal region, specimens were found on the rocky shores and beaches located in Florianópolis city and surrounding islands off the coast, in Torres at Guartia beach, and in the Currais Archipelago at 5-10 m depth.

UPA
The UPA NJ analysis resulted in 15 newly generated sequences (Fig. 20). The NJ phylogram shows two groups with strong bootstrap support: the Lithophylloidae, collected in southeastern Brazil, represented by Lithophyllum and Amphiroa, and Sporolithon (Sporolithales) collected in northeastern Brazil. In the Lithophylloideae, encrusting and rhodolith forming specimens from Santa Catarina and Rio Grande do Sul of L. atlanticum (Arvoredo Island and Guarita Beach respectively) grouped with strong support. Another strongly supported clade includes 4 specimens of L. margaritae (Santa Catarina). Specimens of L. insipidum Adey, Townsend & Boykins (1982) from Hawaii formed another strongly supported group. UPA sequences of L. atlanticum and L. margaritae showed no intraspecific variation and the interspecific variation among Lithophylum species ranged from 23-31 bp (6.2-8.4 %).

Cox1
The cox1 NJ analysis resulted in 6 newly determined sequences (Fig. 21). Individuals of L. atlanticum (Santa Catarina) formed a clade with full support, including rhodolith forming and epilithic specimens. L. margaritae (Santa Catarina) sequences formed a clade with full support. None of the sequences of Lithophylloideae species from different locations of the world grouped with Brazilian specimens. Intraspecific variation of cox1 sequences of L. atlanticum ranged from 0-1 bp (0-0.15%) while intraspecific variation of the 2 sequences of L. margaritae was 2 bp (0.3%). Interspecific variation among Lithophyllum species included in the analysis ranged from 64-118 bp (9.6-17.7%).

SSU rDNA
The results of the SSU rDNA analyses revealed seven sequences (1612 bp) of newly generated nuclear SSU rDNA (Fig. 22). The SSU rDNA phylogram shows results similar to the analyses of the other two markers. Lithophyllum atlanticum specimens grouped in a strongly supported clade separate from specimens identified as L. margaritae. Although the branching order of the major clades was only moderately supported (posterior probability (PP) = 0.6-0.68), several well supported lineages were observed. Lithophyllum atlanticum specimens comprised a monophyletic lineage. Sequences of L. stictaeforme and "Lithophyllum sp." from New Zealand were sister to each other with full support, and did not group with L. atlanticum specimens from southern Brazil described in this study, indicating that this species is not conspecific with the specimens from the Brazilian coast. L. incrustans and L. insipidium (United Kingdom and Hawaii) comprised a clade sister to L. stictaeforme and L. sp. with moderate support (PP= 0.6). A moderately supported clade (PP= 0.85) within the Lithophylloideae included L. margaritae from Brazil and L. kotschyanum Unger (1858:22) from Hawaii as well as nested lineages comprised of Titanoderma pustulatum (Lamoroux) Nägeli (1858: 532) (United Kingdom), Amphiroa sp. (Australia) and Amphiroa fragilisima (Australia). Intraspecific variation of SSU rDNA sequences of L. atlanticum ranged from 0-3 bp (0-0.2%), while L. margaritae showed none. Among Lithophylum species included in the analysis, interspecific variation ranged from 7-37 bp (0.4-2.3%), with 23-25 bp (1.4-1.55%) differences between L. stictarforme and L. atlanticum.

Discussion
Based on the results of the anatomical (Table 2) and molecular  analyses, we propose the newly described species Lithophyllum atlanticum sp. nov. and report the presence of L. margaritae along the Brazilian coast. Our specimens were compared with similar taxa recently studied from Brazil and other parts of the world (Chamberlain et al. 1991, Woelkerling & Campbell 1992, Chamberlain 1996, Furnari et al. 1996, Woelkerling 1996, Chamberlain 1997, Keats 1997, Athanasiadis 1999, Riosmena-Rodriguez et al. 1999, Harvey et al. 2005, Farr et al. 2009, Harvey et al. 2009, Villas-Boas et al. 2009). DNA sequences are available for few species of this genus (Bailey 1999, Bittner et al. 2011, and for Corallinales in general, reinforcing that more molecular data are needed for a better characterization of this group. However, in this study, it was possible to separate the two species and also to clearly segregate Lithophyllum stictaeforme from the new species we propose in this study.  Lithophyllum atlanticum was found all along the southern Brazilian coast and, considering anatomical features and molecular data, it is proposed as a new species. Considering the species described so far for the Brazilian coast, Lithophyllum stictaeforme is one that most closely resembles L. atlanticum. L. stictaeforme (type locality Mediterranean Sea), is reported to have a widespread distribution and the main features which distinguish this species from the new species described here are shown in Table 2. Also, the divergence values (1.4-1.55%) between L. stictaeforme (specimen from NZ) and L. atlanticum in the SSU rDNA analyses provide further evidence to distinguish L. atlanticum as a new species. For example, Bailey (1999) reported 1.72% sequence dissimilarity between Amphiroa fragilissima and Amphiroa sp., and congeneric sequence dissimilarities as low as 0.62% in other groups within the Corallinaceae. Furthermore, based on the geographical proximity and anatomical similarities with L. atlanticum, the specimens identified as L. stictaeforme found in other localities of Brazil (Nunes et al. 2008, Villas-Boas et al. 2009), on the Northwest and Southwest Brazilian coast respectively, should be reexamined with a DNA-based approach to determine the genetic affinities among these two species. The results presented in this study reinforce that the use of molecular tools, especially DNA sequencing and barcoding, in combination with the morphological and anatomical characterization will be essential to understand the diversity of Corallinophycidae in Brazil and to unveil new species in this highly diverse group of red algae.
In the case of Lithophyllum margaritae, our findings corroborate that this species is widely distributed (Riosmena-Rodriguez et al. 1999) and that anatomical characteristics of the population from Baja California (Mexico) are very similar to those observed in the specimens analyzed in this study (as seen in Table 1). However, some differences were observed. For example, the dimerous organization observed for specimens in this study, especially at margins, is not described in the type specimen nor was observed in specimens from Baja California. In addition, these two populations differ in some aspects of the tetrasporangial conceptacles, which are quite smaller and present more cells from the thallus floor to the surface in the specimens from Brazil (Table 2). Therefore, DNA sequencing of type specimen of Lithophyllum margaritae is desirable in order to clarify the species boundaries between these closely related populations.
Overall, in this study we conclude that both the molecular and morphological evidence distinguishes L. atlanticum sp. nov. from other closely related species. However, to achieve a better understanding of species diversity within the Lithophylloideae from Brazil and throughout the world, ongoing DNA sequencing, especially for type specimens and of newly collected specimens from other parts of Brazil, is highly recommended.