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: holo- type 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 plurilocu- lar 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 Brought to you by | UNAM Authenticated | 132.248.181.237 Download Date | 4/8/14 10:04 PM Table 2 Morphological comparison of specimens of R. hancockii and related species. Taxa Thickness (μm) Symmetry of vegetative filaments Delimitation Plurangia Unangia Paraphyses: length, no. No. of between medulla of cells, ∅ basal cells, l/d chloroplast and cortex basal cells per cell of Characterization et al.: D. León-Álvarez Ralfsia hancockii 130–210 Mainly unilateral; curving Yes Not seen Terminal on 3-celled Present, 95–107 μm, 25 Single (PTM 9165) upwardly and downwardly from a stalks cells, ∅ 2–6.5, l/d basal central layer cells 2–2.4 Download Date |4/8/14 10:04 PM Authenticated |132.248.181.237 R. hancockii (PTM 160–300 Mainly unilateral; curving Yes Not seen Terminal on 3–6- Present, 100–110 μm, Single Brought to youby|UNAM 9167) upwardly and downwardly from a celled stalks 11–13 cells, ∅ 1.6–5.2, central layer l/d 1.6–2.1 aR. hancockii 108–306 (vegetative), Mainly unilateral, also curving Yes Not seen Terminal on 4–5- Present, 100–150– (175) One (holotype) 198–360 (reproductive) downwardly in part celled stalks μm, 10–13 (18) cells, ∅ 3.1–7.3, l/d 2.2–3.7 bRalfsia sp. D 457–700 Bilateral; tightly adhered; curving Yes – – Present Single upwardly and downwardly from a central layer Neoralfsia hancockii Neoralfsia bNeoralfsia 138–390 Bilateral; curving upwardly and Yes Subterminal, with Terminal on 3-celled Present Single expansa (Okinawa) downwardly from a central layer one sterile cell stalks bN. expansa 110–280 Bilateral; curving upwardly and Yes Subterminal, with Terminal on 3-celled Present Single (Malaysia) downwardly from a central layer one sterile cell stalks bN. expansa 141–160 Bilateral; curving upwardly and Yes Subterminal, with Terminal on 6-celled Present Single (Hyogo) downwardly from a central layer one sterile cell stalks cN. expansa (Japan) 200–800 (1100) Bilateral; curving upwardly and Yes Subterminal, with Terminal on 3–6- Present, 100–190 (220) One topotype nov.from comb.