Morphological and Molecular Characterization of Neoralfsia Hancockii Comb

DOI 10.1515/bot-2013-0095 Botanica Marina 2014; 57(2): 139–146

Daniel León-Álvarez*, Maria L. Núñez-Resendiz and Maria E. Pónce-Márquez Morphological and molecular characterization of Neoralfsia hancockii comb. nov. (Ralfsiales, Phaeophyceae) from topotype of San José del Cabo, Baja California, Mexico

Abstract: To resolve the taxonomic status of Ralfsia han- fig. 7) surrounded by paraphyses. Under these criteria, dif- cockii and its relationship to related species, we have car- ferent specimens were recorded as R. hancockii along the ried out a morphological and molecular study (rbcL) on tropical Pacific coast of Mexico (Dawson 1944, 1953, 1954, the topotype material of the species. Our analyses, using León-Álvarez and González-González 1993, Morph A in maximum parsimony and Bayesian posterior probability, León-Álvarez and González-González 1995, León-Álvarez located this species in a clade that is distinct and distant and González-González 2003) and the Gulf of California from other species of the genus Ralfsia and other genera (León-Álvarez and Norris 2010). of the family Ralfsiaceae, but is shared with the family A very similar crustose species, Neoralfsia expansa Neoralfsiaceae. Morphological data confirmed that the (J. Agardh) P.-E. Lim et H. Kawai ex Cormaci et G. Furnari, presence of unangia with (2–) 3–6 stalk cells and mostly was originally recorded from Veracruz on the Atlantic unilateral symmetry with partial bilateral development coast of Mexico [as Myrionema (?) expansum J. Agardh is characteristic of this species and distinguishes it from 1847]. On the basis of a morphological study of specimens the morphologically similar Neoralfsia expansa sensu from Veracruz and various localities from the Mexican Børgesen. Similarities to N. expansa sensu Tanaka and Pacific coast, León-Álvarez and González-González (1995, Chihara, from which the latter species is distinguished by 2003) found that sessile unangia (or unangia with a single having paraphyses with thinner and larger basal cells, are stalk cell) and mainly bilateral symmetry are characteris- discussed. We propose Neoralfsia hancockii comb. nov. tic of N. expansa [as R. expansa (J. Agardh) J. Agardh sensu Børgesen 1912 non sensu Tanaka and Chihara 1980], while Keywords: morphology; Neoralfsiaceae; nucleotide diver- the presence of two or more unangial stalk cells and mainly gence; Ralfsiaceae; rbcL. unilateral symmetry characterizes R. hancockii (Dawson 1944, León-Álvarez and González-González 2003: holotype D640, HAHF9 in LAM500460, now in UC). Lim et al. *Corresponding author: Daniel León-Álvarez, Laboratorio de (2007) considered the circumscription of N. expansa (as Ficología y Sección de algas del Herbario de la Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico 03510, D.F., R. expansa) based on these characters to be insufficient. Mexico, e-mail: [email protected] On the basis of rbcL data, they placed N. expansa in a new Maria L. Núñez-Resendiz and Maria E. Pónce-Márquez: Laboratorio genus, Neoralfsia Lim et Kawai, in a clade distinct from de Ficología y Sección de algas del Herbario de la Facultad de the Ralfsiaceae W.G. Farlow, and now in Neoralfsiaceae Ciencias, Universidad Nacional Autónoma de México, Mexico 03510, Lim et Kawai. However, they did not include specimens of D.F., Mexico R. hancockii in their study. In the present investigation, we used rbcL to resolve the taxonomic status of R. hancockii and its relationship to other closely related species. Introduction

Ralfsia hancockii E.Y. Dawson (1944) is a brown crustose alga that is common on the Pacific coast of Mexico, and Materials and methods was originally described from specimens from San Jose del Cabo, Baja California, Mexico, as having “…cells Sampling of basalmost 2–3 layers elongated horizontally, these leading into branching, assurgent rows in the direction of Two collections of Ralfsia hancockii from the type locality, the margins…” (sic. p. 223) and unangia “…with a narrow San Jose del Cabo, BC [PTM9165 and PTM9167 in Her- basal stalk…” (sic., p. 223; drawn with a three-celled stalk, bario de la Facultad de Ciencias, Universidad Nacional

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM 140 D. León-Álvarez et al.: Characterization of Neoralfsia hancockii comb. nov. from topotype

Autónoma de México (FCME)], were collected in trip- of cells, presence or absence of reproductive structures, licate (for molecular analysis, a morphological study, paraphyses, number and size of the cells in the paraphy- and deposit in the Herbarium FCME). Twenty-six rbcL ses, shape, and number and sizes of the cells in the stalk sequences from related species from GenBank [National of unangia. The specimens were identified on the basis Center for Biotechnology Information (NCBI)] were of the approach taken by Dawson (1944) and León-Álva- included in the analyses (Table 1). rez and González-González (2003) and the nomencla- ture recommended by León-Álvarez and Norris (2005). Because the exact nature of the reproductive cells (i.e., Morphological observations gametes or spores) produced by unilocular or plurilocular structures in Ralfsia and related genera could not be Observations were made on the shape, color, and texture established with certainty, we use the morphological of the crust, form of the margins, adhesion to the sub- terms unangium and plurangium instead of unilocular strate, and external measurements. Sections were cut in or plurilocular sporangia. The terms monomerous and a longitudinal-radial plane under a stereomicroscope, dimerous are used in an analogous way to their use by and observations were made of medullary and cortical Woelkerling (1988) to describe the crustose coralline red filaments, the number of cells that compose them, size algae.

Table 1 Molecular sequence data (NCBI 2013 and this study) and herbarium references.

Species Accession no. Locality Author

Ralfsia hancockii KF977828 La Palmilla, San José del Cabo, B.C., Mexico (PTM9165 This study FCME, 09/30/2009) KF977827 Country club, San José del Cabo, B.C., Mexico (PTM9167 This study FCME, 09/30/2009) Neoralfsia expansa AB250077.1 Okinawa, Ishigaki Island, Hamasaki, Japan Lim et al. 2007 AB250078.1 Johor, Desaru, Malaysia Lim et al. 2007 AB250079.1 Hyogo, Igumi, Japan Lim et al. 2007 R. verrucosa AB250072.1 Roscoff, Brittany, France Lim et al. 2007 R. fungiformis AB250071 Hokkaido, Akkeshi, Japan Lim et al. 2007 EU579936.1 Cap du Bon Désir, Quebec, Canada Bittner et al. 2008 Ralfsia sp. A AB250073.1 Hyogo, Ako, Japan Lim et al. 2007 Ralfsia sp. B AB250074.1 Bergen, Norway Lim et al. 2007 Ralfsia sp. C AB250075.1 Katiki Beach, New Zealand Lim et al. 2007 Ralfsia sp. D AB250076.1 Durban, South Africa Lim et al. 2007 Ralfsia sp. H AB250080.1 Shimoda, Nabeta, Japan Unpublished Ralfsia sp. I AB250081.1 Shimoda, Nabeta, Japan Unpublished Ralfsia sp. J AB250082.1 Hyogo, Awaji Island, Maruyama, Japan Unpublished Ralfsia sp. K AB250083.1 Hyogo, Takeno, Oura, Japan Unpublished Mesospora JQ620004.1 Lombok island, Gili Genting, Indonesia Poong et al. 2013 elongata M. elongata B JQ620005.1 Lombok Island, Nipah, Indonesia Poong et al. 2013 M. elongata C JQ620003.1 Okinawa Prefecture, Ishigaki Island, Japan Poong et al. 2013 M. schmidtii JQ620000 Port Dickson, Malaysia Poong et al. 2013 Mesospora sp. A AB250063.1 Okinawa, Ishigaki Island, Fusaki, Japan Unpublished Mesospora sp. B AB250064.1 Johor, Desaru, Malaysia Unpublished Mesospora sp. C AB250065.1 Johor: Tg. Gemoh, Malaysia Lim et al. 2007 Mesospora sp. G AB250069.1 Shizuoka, Shimoda, Japan Lim et al. 2007 Heteroralfsia AB250070.1 Ohma, Aomori Pref., Japan Lim et al. 2007 saxicola Endoplura aurea AB264039 Inubouzaki, Chiba Pref., Japan Lim et al. 2007 Tilopteris mertensii AB045260 Helgoland, Germany Sasaki et al. 2001 Sargassum AJ287854 Zeeland, Oosterschelde, Zeelandbrug, Netherlands Draisma et al. 2001 muticum

basal cells, l/d l/d cells, ∅ basal Present, 100–110 μ m, ∅ 1.6–5.2, 11–13 cells, 1.6–2.1 l/d Present, 95–107 μ m, 25 basal ∅ 2–6.5, l/d cells, 2–2.4 cells Present Present Present Present Present, 100–150– (175) ∅ μ m, 10–13 (18) cells, 2.2–3.7 3.1–7.3, l/d Paraphyses: length, no. Paraphyses: cells, of Present, 100–190 (220) ∅ μ m, 12–16 cells, 4–7 2.5–3, l/d basal cells

Terminal on 3–6- Terminal stalks celled Terminal on 3-celled on 3-celled Terminal stalks – on 3-celled Terminal stalks on 3-celled Terminal stalks on 6-celled Terminal stalks Terminal on 4–5- Terminal stalks celled Unangia Terminal on 3–6- Terminal stalks celled

Not seen Not Not seen Not – with Subterminal, cell one sterile with Subterminal, cell one sterile with Subterminal, cell one sterile Not seen Not Plurangia Subterminal, with with Subterminal, cell one sterile

, diameter. –, not available. –, not 1980: 231–232, fig. 1B. ∅ , diameter. Chihara and Tanaka c Yes Yes Yes Yes Yes Yes Yes Delimitation Delimitation between medulla cortex and Yes

Lim et al. (2007). Lim et al. b Mainly unilateral; curving curving unilateral; Mainly a from downwardly and upwardly layer central Mainly unilateral; curving curving unilateral; Mainly a from downwardly and upwardly layer central Bilateral; tightly adhered; curving adhered; curving tightly Bilateral; a from downwardly and upwardly layer central and upwardly curving Bilateral; layer a central from downwardly and upwardly curving Bilateral; layer a central from downwardly and upwardly curving Bilateral; layer a central from downwardly Mainly unilateral, also curving curving also unilateral, Mainly in part downwardly Symmetry of vegetative filaments vegetative of Symmetry Bilateral; curving upwardly and and upwardly curving Bilateral; layer a central from downwardly

μ m) 160–300 130–210 457–700 138–390 110–280 141–160 Thickness ( Thickness 108–306 (vegetative), 108–306 (vegetative), 198–360 (reproductive) 200–800 (1100)

sp. D sp. Ralfsia Neoralfsia N. expansa N. expansa R. hancockii (2003). González-González and in UC, León-Álvarez HAHF9 in LAM500460, now (Japan)N. expansa (PTM (PTM R. hancockii 9167) hancockii Ralfsia 9165) (PTM (Okinawa) expansa (Malaysia) (Hyogo) and related species. related and R . hancockii of specimens of comparison 2 Morphological Table Taxa a b b b b c a (holotype)

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM 142 D. León-Álvarez et al.: Characterization of Neoralfsia hancockii comb. nov. from topotype

Molecular analysis sequences were analyzed with maximum parsimony (MP) using PAUP* 4.0b 10 (Swofford 2002). The MP tree DNA was extracted from 20 to 40 mg of tissue lyophilizate, was constructed using the heuristic search option, 100 using a Qiagen DNeasy Plant Mini Kit (Quiagen, México) random sequence additions, tree bisection reconnection according to the manufacturer’s protocols, and subse- branch swapping, and unordered and equally weighted quently purified with Qiagen DNeasy Blood and Tissue kit characters. Bootstrap support was computed with 10,000 and stored at -70°C. replications. The uncorrected distances (“p”) for partial The rbcL region was amplified by PCR using the sequences of rbcL were calculated in PAUP* 4.0b 10. primers rbc-F0, rbc-F3, and rbc-R2 (Kawai and Sasaki To find the model of nucleotide substitution that best 2004) and Ral-R952 (Lim et al. 2007) in a Flexigene Techne fits the data set, the ModelTest v.3.7 program (Posada and thermocycler, with Taq PCR Core Kit (QIAGEN Quality, Crandall 1998) was used with the Akaike information cri- Quiagen, México). The final volume of the PCR reaction terion. The model selected for the present study data set was 25 μl: 1 μl DNA, 2.5 μl 10 × buffer, 1 μl BSA, 1 μl MgCl2, was general-time-reversible (GTR)+proportion of invari- 0.5 μl dNTP, 0.125 μl Taq DNA polymerase, 1 μl primer F, 1 able sites (I)+variable sites (G). The Bayesian analysis was

μl primer R, and 16.875 μl H2O. The parameters of the PCR performed using the program MrBayes 3.1.1 (Huelsenbeck amplification were those described by Lim et al. (2007). and Ronquist 2001), using a GTR model with a gamma dis- The sequences were edited and aligned in the tribution. Five chains of Markov chain Monte Carlo were program Bioedit (Hall 1999) together with the 26 GenBank used, starting with a random tree and sampling the data sequences [24 Ralfsiales and Tilopteris mertensii (Turner) every 100 generations for 5 million generations. The likeli- Kützing and Sargassum muticum (Yendo) Fensholt des- hood scores stabilized after 250,000 generations, but we ignated as outgroup species (Table 1)]. The aligned used a “burn-in” of 25% (1,250,000 generations).

Figures 1–6 Neoralfsia hancockii. (1) Irregular crustose habit. Scale bar = 1 cm. (2) Radial longitudinal section of thallus, showing medullary filaments branching from a basal to central core and mostly unilateral symmetry. Scale bar = 70 μm. (3) Detail of radial longitudinal section, showing medullary branched filaments and cortex. Scale bar = 10 μm. (4) Radial longitudinal section, showing a part of the sorus with unangia surrounded by numerous paraphyses. Scale bar = 30 μm. (5) Three-cell stalked unangium accompanied by a paraphysis. Scale bar = 18 μm. (6) Detail of paraphyses. Scale bar = 20 μm.

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM D. León-Álvarez et al.: Characterization of Neoralfsia hancockii comb. nov. from topotype 143

Results consisted of 1347 bp. The bootstrap values resulting from MP analysis were incorporated in the Bayesian tree of Figure 7. MP analysis resulted in one strict consensus Morphological observations derived from six most parsimonious trees of 1436 steps. The overall topologies and branching order of the main clades The diagnostic morphological characters for specimens were similar in MP and Bayesian trees, with only minor dif- of Ralfsia hancockii and related species are summarized ferences. MP analysis recovered the species Ralfsia sp. B in Table 2. Data for Ralfsia sp. A, B, C, D, H, I, J, K speci- and Ralfsia sp. C in a clade that is, in turn, a sister subclade mens are not available. The following descriptions and of the species of the families Ralfsiaceae and Neoralfsi- Figures 1–6 show the main characteristics of the specimens aceae, all inside the large clade of the species of the Ralf- of R. hancockii from the type locality: siales order, while in the Bayesian analysis, the species –– Thallus crustose, irregular or orbicular, with lobulate Ralfsia sp. B and Ralfsia sp. C were grouped outside, as the or irregular margins, 1–5(–8) cm diameter, dark basal clade of the remaining species of the order Ralfsiales, brown to green when wet and dark brown, almost as shown in Figure 7. The rest of the clades and the location black when dry, epilithic, adherent to the substrate by of the R. hancockii clade were identical in both analyses. rhizoids, wrinkled, coriaceous, growing radially with The resulting topology of the MP and BPP trees conspicuous growth lines, not disintegrating when (Figure 7) showed a clade corresponding to the order Ralf- rubbed between fingers, smooth surface, markedly siales, rooted by Sargassum muticum and Tilopteris merten- lobed margins, growing over each other (Figure 1). sii. Two major well-supported groups were recognized –– In radial longitudinal section, 130–210 μm thick. (BPP/MP = 1.00/98), both of which contained representative Vegetative filaments with primarily unilateral species of the order Ralfsiales Nakamura ex Lim et Kawai. symmetry (Figure 2), differentiated in medulla and The first group includes Ralfsia sp. B and Ralfsia sp. C and cortex by their diameter (Figures 2 and 3). Medullary is also well supported (1.00/100). However, the clade I and filaments branching (Figure 3) from a basal to central clade II second group is not well supported in BPP (0.56). core, curved mostly upward (Figure 2), although in Clade I includes species of the family Ralfsiaceae and it is some thick parts of the thallus the filaments also not well supported in MP (58). Clade II includes Mesospora diverge downward; the terminal cells frequently giving sp. G appearing as one terminal branch in an unknown rise to rhizoids. Medullary filaments with cylindrical taxonomic position and two sister clades, one of them not basal cells 32–36 μm in length × 12–14 μm in diameter. well supported (subclade IIC, MP/58) with species of Meso Subcortical cells cylindrical or irregular, 22–25 μm sporaceae J. Tanaka et Chihara and the other well sup- in length × 10–12 μm in diameter. Cortical layer one- ported (1.00/95) with species of the family Neoralfsiaceae. half to one-third of the diameter of the medullary In turn, the clade of the former family was separated into filaments with five to eight cylindrical cells 6–9 μm in two sister subclades: subclades IIA and IIB. Subclade IIA length × 5–7 μm in diameter. One chloroplast per cell, is well supported (1.00/100) and has the topotype speci- without pyrenoid. men of R. hancockii. However, subclade IIB is not well sup- –– Unangial sori (Figure 4) scattered, inconspicuously ported in MP (52) and includes the specimens of Neoralfsia differentiated (as discolored areas) on the surface of expansa and Ralfsia (Ralfsia sp. D, H, I, J, K). the thallus. Unangia clavate, 70–86 μm in length × 28– Among the Ralfsiaceae, the values of uncorrected dis- 36 μm in diameter, one per filament, with three- to tance (“p”) ranged from 0.6% when intraspecific (speci- six-celled stalks (Figure 5), surrounded by paraphyses mens of R. fungiformis) to 4.9% between species of the that are differentiated morphologically from the genus Ralfsia (e.g., R. fungiformis and R. verrucosa). In reproductive filaments (Figure 6). Paraphyses clavate, turn, these diverged by 8.2–10.9% from other genera in 95–110 μm in length, with 11–13 (–25) cells, 1.6–6.5 μm the family Ralfsiaceae (e.g., from Heteroralfsia saxicola). in diameter in basal parts, and a length/diameter Among the specimens of Neoralfsia expansa located in sub- ratio of basal cells of 1.6–2.4. Plurangia unknown. clade IIB (Figure 7), divergence values ranged from 2.3% to –– rbcL gene sequence distinct. 4.6%. In turn, these specimens diverged from R. hancockii specimens by 6.5–9.7%. The closest sampled relative to Molecular analysis R. hancockii in the tree of Figure 7 is Ralfsia sp. D, diverging by 10.10%. The R. hancockii specimens located in subclade Molecular sequences were obtained for the rbcL gene of IIA did not diverge from each other. However, both speci- the topotype of Ralfsia hancockii. The aligned sequences mens diverged from R. fungiformis by 11.5–12.8%.

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM 144 D. León-Álvarez et al.: Characterization of Neoralfsia hancockii comb. nov. from topotype

Figure 7 Bayesian phylogenetic tree of rbcL gene with MP bootstrap values added. Support values are shown at the nodes of the branches (BPP/MP bootstrap). Consistency index (CI) = 0.4659 and retention index (RI) = 0.6461 for MP analysis.

Discussion species thus far recognized in the genus Ralfsia sensu Euralfsia (Batters 1889, Batters 1902, De Toni 1895) and Our specimens of Ralfsia hancockii from the type locality Neoralfsia (Lim et al. 2007), which also have a multilayered have vegetative filaments with a monomerous arrange- cortex, such as R. fungiformis (fide Tanaka and Chihara ment. This feature is shared with crustose brown algal 1980: p. 233, fig. 3) and Neoralfsia expansa (as R. expansa

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM D. León-Álvarez et al.: Characterization of Neoralfsia hancockii comb. nov. from topotype 145 fide: Børgesen 1912, fig. 1b; Schnetter 1976: p. 45, table IIIF; The topotype specimens of R. hancockii formed a well- Tanaka and Chihara 1980: pp. 231–232, fig. 1B, León-Álva- supported clade (subclade IIA, 1.00/100) distinct from the rez and González-González 2003) and distinguishes these nearest clade (subclade II) with their relatives: N. expansa from the species without a cortex of multiple cell layers, (the type species of the genus) and five closely related such as R. verrucosa (Areschoug) Areschoug in Fries (Fries specimens. These specimens were identified as Ralfsia 1845, vide Reinke 1889) and R. integra Hollenberg (holo- spp. in GenBank but are likely to be unidentified species of type US61155), or those of Ralfsia sensu Stragularia (Ström- Neoralfsia given the difficulty in separating these genera felt 1886, 1888, Batters 1889) with dimerous arrangement. by morphology. (e.g., Ralfsia confusa Hollenberg 1969). Also, our speci- Ralfsia hancockii (subclade IIA) was also clearly sepa- mens from the type locality have a cortical layer with veg- rated from the specimens of N. expansa (subclade IIB) by etative filaments one-half to one-third of the diameter of nucleotide divergence values of 6.5–9.7%, covering the the medullary filaments and paraphyses with broad and range of those found among species of different genera short basal cells (length-diameter ratio of basal cells 1.6– in the Ralfsiaceae (e.g., 8.2–10.9% between specimens of 2.4, holotype 2.2–3.7; Table 2). Plurangia have never been R. fungiformis and Heteroralfsia saxicola), but also among found in several years of observations of this species along species of a genus of Mesosporaceae (e.g., specimens of the Mexican Pacific coast (León-Álvarez and González- Mesospora elongata and M. schmidtii range 7.7–8.5%), González 1993, 1995, 2003), and this, together with the which suggests caution in the interpretation of the taxo- presence of unangia with (2–) 3–6 stalk cells and mostly nomic level to which R. hancockii ought be separated in unilateral symmetry with partial bilateral development, the Neoralfsiaceae. support the view of León-Álvarez and González-González On the basis of the joint analysis of morphologi- (2003) that these are traits characteristic of R. hancockii, cal and molecular evidence generated in this study, while the presence of sessile unangia or with stalks of only the remoteness of R. hancockii from the type species of one cell and mostly bilateral symmetry (Børgesen 1912, the genus (R. fungiformis; a nucleotide divergence 11.5– León-Álvarez and González-González 2003, León-Álvarez 12.8%), and its distance from other presumed species of 2005) are typical characters of N. expansa (as R. expansa Neoralfsia (e.g., Ralfsia D, 10.10%), we propose Neoralfsia sensu Borgesen non sensu Tanaka and Chihara 1980). hancockii comb. nov., including it in the Neoralfsiaceae However, R. hancockii and N. expansa (as R. expansa sensu pending more studies with N. expansa specimens from Tanaka and Chihara non sensu Børgesen 1912) are almost Mexico. morphologically identical, except only that N. expansa has paraphyses with thin and long basal cells (length- diameter ratio of basal cells 4–7, Table 2; Tanaka and Chihara 1980: p. 232) and specimens of N. expansa from Taxonomic proposition Japan sometimes have unangia and plurangia in the same thallus (Tanaka and Chihara 1980: p. 231, Lim et al. 2007). Neoralfsia hancockii (E.Y. Dawson) D. León- Preliminary unpublished data (a short c. 900-bp frag- Álvarez et M.L. Núñez-Reséndiz comb. nov. ment of rbcL obtained by the authors) from specimens of N. expansa from Mexico appear to show that these speci- Basionym Ralfsia hancockii E.Y. Dawson (The Univer- mens are genetically distinct from R. hancockii, and also sity of Southern California Publications, Allan Hancock from Japanese and Malaysian specimens. Confirmation of Pacific Expeditions, 1944, vol. 3, number 10: p. 223, plate these results with complete sequences could indicate that 31, figs. 6,7; plate 54, fig. 2). different species are found on the Atlantic coast of Mexico This species corresponds with the detailed descrip- and in the Asian Pacific. tion of the holotype of Ralfsia hancockii Dawson (D650 in The resulting topology from MP and BPP analyses LAM500460, after HAHF9, now in UC) and of the isotype (Figure 7) showed all the species are corresponding to (UC700549), provided by León-Álvarez and González- the order Ralfsiales, and these are representative species González (2003), except that the isotype lacks rhizoids. In of the families Ralfsiaceae (clade I), Neoralfsiaceae, and our specimens from the tropical Pacific coast of Mexico, Mesosporaceae (clade II, Figure 7). The group of two we have also observed unangial stalks with three to unidentified Ralfsia specimens (sp. B and sp. C) included seven stalk cells (in rare cases, when two stalk cells are in the Ralfsiales clade (Figure 7) corresponds to clade Id present, the same sorus has up to six). Also, this species from Lim et al. (2007); as?Ralfsia? sic.), which remains in corresponds with Morph A of N. hancockii described by an unknown position. León-Álvarez and González-González (1995), as Ralfsia

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM 146 D. León-Álvarez et al.: Characterization of Neoralfsia hancockii comb. nov. from topotype hancockii) and its distribution in the same environmental support was partially supplied by DGAPA, UNAM projects conditions along the Pacific coast of Mexico. PAPIIT IN215108 and IN229711.

Acknowledgments: The authors are indebted to Drs. M. Wynne and J. Buchanan for their valuable comments. Received 13 September, 2013; accepted 26 February, 2014; online Also, we thank one anonymous reviewer. Financial first 22 March, 2014

References

Agardh, J.G. 1847. Nya alger från Mexico. Öfversigt af Kongliga. eds.) Biodiversidad Marina y Costera de México. Comisión Veternskaps-Akademiens Förhandligar 4: 5–17. Nacional para la Biodiversidad y CIQRO, México. pp. 456–474. Batters, E.A.L. 1889. A list of the marine algae of Berwick-on-Tweed. León-Álvarez, D. and J. González-González. 1995. Characterization of Hist. Berwickshire Nat. Club 12: 221–392. the environmental distribution and morphs of Ralfsia hancockii Batters, E.A.L. 1902. A catalogue of the British marine algae being Dawson (Phaeophyta) in the Mexican tropical Pacific. Bot. Mar. a list of all the species of seaweeds known to occur on the 38: 359–367. shores of the British Islands, with the localities where they are León-Álvarez, D. and J. González-González. 2003. The morphological found. J. Bot. Lond. 40(suppl.): 1–107. distinction of Ralfsia expansa and R. hancockii (Ralfsiaceae, Bittner, L., C.E. Payri, A. Couloux, C. Cruaud, B. de Reviers and Phaeophyta) from Mexico. Phycologia 42: 613–621. F. Rousseau. 2008. Molecular phylogeny of the Dictyotales León-Álvarez, D. and J.N. Norris. 2005. Terminology and position of and their position within the Phaeophyceae, based on reproductive structures in crustose brown algae: misapplication, nuclear, plastid and mitochondrial DNA sequence data. Mol. confusion and clarification. Cryptogamie Algol. 26: 91–102. Phylogenet. Evol. 49: 211–226. León-Álvarez, D. and J.N. Norris. 2010. Ralfsiales. p. 155–161. In: Børgesen, F. 1912. Two crustaceous brown algae from the Danish (J.N. Norris, ed.) Marine algae of the northern Gulf of California: West Indies. Nuova Notarisia [Padova] Ser. XXIII: 1–7. Chlorophyceae and Phaeophyceae, Smithsonian Contributions Dawson, E.Y. 1944. The marine algae of the Gulf of California. Allan to Botany Number 94. Smithsonian Institution Scholarly Press, Hancock Pac. Exped. 3: i–v+189–432, pls 31–77. Washington D.C. USA. pp. 277. Dawson, E.Y. 1953. Preliminary results of a marine algal Lim, P.-E., M. Sakaguchi, T. Hanyuda, K. Kogame, S.-M. Phang and reconnaissance of the Pacific Mexican coast. Proc. Sev. Pac. Sc. H. Kawai. 2007. Molecular phylogeny of crustose brown algae Congr. (Auckland, 1949) 5: 43–46. (Ralfsiales, Phaeophyceae) inferred from rbcL sequences Dawson, E.Y. 1954. The marine flora of San Benedicto Island, Mexico resulting in the proposal for Neoralfsiaceae fam. nov. following the volcanic eruption of 1952–1953. VIII Congr. Int. Phycologia 46: 456–466. Bot. (Paris, 1954) 17: 157–158. Poong, S.W., P.E. Lim, S.M. Phang, H. Sunarpi, J.A. West and De Toni, J.B. 1895. Sylloge Algarum, vol. 3. Fucoideae, Padua. H. Kawai. 2013. A molecular-assisted floristic survey of pp. 638. crustose brown algae (Phaeophyceae) from Malaysia and Draisma, S.G.A., W.F. Prud’homme van Reine, W.T. Stam and J.L. Lombok Island, Indonesia based on rbcL and partial cox 1 Olsen. 2001. A reassessment of phylogenetic relationships genes. J. Appl. Phycol. DOI 10.1007/s10811-013-0081-9. within the Phaeophyceae based on RUBISCO large subunit and Posada, D. and K.A. Crandall. 1998. MODELTEST: testing the model ribosomal DNA sequences. J. Phycol. 37: 586–605. of DNA substitution. Bioinformatics 14: 817–818. Fries, E.M. 1845. Summa Vegetabilium Scandinaviae. Holmiae y Reinke, J. 1889. Atlas deutscher Meeresalgen. part 1, pls 1–25. Paul Lipsiae. pp. 123–134. Parey, Berlin. 34 pp. Hall, T.A. 1999. BioEdit a user-friendly biological sequence Sasaki, H., A. Flores-Moya, E.C. Henry, D.G. Muller, and H. Kawai. alignment editor and analysis program for Windows 95/98/NT. 2001. Molecular phylogeny of Phyllariaceae, Halosiphonaceae Nucleic Acids Symp. Ser. 41: 95–98. and Tilopteridales (Phaeophyceae). Phycologia 40: 123–134. Hollenberg, G.J. 1969. An account of the Ralfsiaceae (Phaeophyta) of Schnetter, R. 1976. Marine algen der Karibische Küsten von California. J. Phycol. 5: 290–301. Kolumbien I. Phaeophyceae, Bibl. Phycol. 24: 125. Huelsenbeck, J.P. and F. Ronquist. 2001. MRBAYES: Bayesian Strömfelt, H.F.G. 1886. Einige fur die Wissenschaft neue inference of phylogenetic trees. Department of Biology, Meeresalgen aus Island. Bot. Zentralblatt 26: 172–173. University of Rochester, Rochester, NY. Bioinformatics 17: Strömfelt, H.F.G. 1888. Algae novae quas ad litora Scandinavie 754–75. indaganit. Notarisia 3: 381–384. Kawai, H. and H. Sasaki. 2004. Morphology, life history, and Swofford, D.L. 2002. PAUP*. Phylogenetic analysis using parsimony molecular phylogeny of Stschapovia flagellaris (Tilopteridales, (*and other methods). Version 4. Sinauer Associates, Phaeophyceae) and the erection of the Stschapoviaceae. fam. Sunderland, MA. nov. J. Phycol. 40: 1156–1169. Tanaka, J. and M. Chihara. 1980. Taxonomic study of the Japanese León-Álvarez, D. 2005. The morphology of Ralfsia expansa crustose brown algae (2). Ralfsia (Ralfsiaceae, Ralfsiales), part (J. Agardh) J. Agardh (Ralfsiaceae, Phaeophyta) from Veracruz, 1. J. Jpn. Bot. 55: 225–236. México. Cryptogamie Algol. 26: 343–354. Woelkerling, W.J. 1988. The coralline red algae: an analysis of the genera León-Álvarez, D. and J. González-González. 1993. Algas costrosas and subfamilies of nongeniculate Corallinaceae. British Museum del Pacífico Tropical. In: (S.I. Salazar-Vallejo y N.E. González, (Natural History). Oxford University Press, London. pp. 267.

Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM