Botanica Marina 2014; aop Daniel Le ó n- Á lvarez * , Maria L. N ú ñ ez-Resendiz and Michael J. Wynne Morphological and molecular studies on topotype material of Neoralfsia expansa (Phaeophyceae) reveal that Asian specimens assigned to this taxon are genetically distinct Abstract: The genus Neoralfsia was newly described by specimens, collected by Liebmann from an unspecified P.-E. Lim and Kawai on the basis of rbc L sequence data area on the coast of Veracruz, Mexico, in February 1841 from specimens from Japan and Malaysia identified ( God í nez 2008 ). B ø rgesen (1912) reported on specimens as Neoralfsia expansa , and N. expansa [ Myrionema ( ? ) from the Danish West Indies (now the U.S. Virgin Islands) expansum ] from Mexico was designated as the type spe- that were very similar to the type of N . expansa (as Ralfsia cies. Our maximum likelihood and Bayesian analyses expansa ), and he described his material in detail includ- show that specimens of N. expansa from the type locality ing reproductive characteristics. His study was directly are grouped into a new, not previously described, clade or indirectly the basis for the recognition of the species in Neoralfsiaceae, distant from that of the putative speci- by other authors ( Weber-van Bosse 1913 , B ø rgesen 1914 , mens attributed to N . expansa from Japan and Malaysia. Taylor 1960 , Joly 1965 , Earle 1969 , Schnetter 1976 , Lawson We show that genuine “ expansa ” does not include Asian and John 1982 ). However, Tanaka and Chihara (1980a,b,c, material. The distribution of the species is not as wide as 1981a,b) , in their review of the order Ralfsiales Nakamura has been previously recorded. ex Lim et Kawai, described the reproductive characteris- tics of specimens of what they called “R. expansa ” from Keywords: brown crustose algae; distribution; morphology; various Japanese islands and generated a different inter- Neoralfsia ; Ralfsia ; rbc L. pretation of the species than that of B ø rgesen. Based on a morphological study of specimens from various localities in Veracruz and the Mexican Tropical Pacific, Le ó n- Á lva- DOI 10.1515/bot-2014-0029 Received May 15 , 2014 ; accepted July 24 , 2014 rez and Gonz á lez-Gonz á lez (2003) were of the opinion that specimens with mainly unilateral symmetry, unangial specimens with sessile unangia, or with unangia with a single stalk cell correspond to N. expansa sensu B ø rgesen Introduction (as R. expansa sensu B ø rgesen), whereas specimens with unilateral to bilateral symmetry and unangia with multi- Neoralfsia expansa (J. Agardh) P.-E. Lim et H. Kawai ex cellular stalks correspond to N. expansa sensu Tanaka and Cormaci et G. Furnari in Cormaci et al. (2012) is a commonly Chihara (as R. expansa sensu Tanaka and Chihara). Lim recorded species [as Ralfsia expansa (J. Agardh) J. Agardh] et al. (2007) , however, considered the circumscription of around the world. Poong et al. (2013) described its distri- N. expansa (as R. expansa ) based on these characteristics bution as “cosmopolitan”. It was originally described by J. to be insufficient, and they used molecular data from the Agardh (1847) as Myrionema ( ? ) expansum and transferred rbc L region of chloroplast DNA to show that specimens to Ralfsia the following year by J. Agardh (1848) . The origi- identified as R. expansa from Japan and Malaysia were nal description was very brief and based on vegetative located in a distinct clade from other Ralfsia species and described the new genus Neoralfsia in the new family *Corresponding author: Daniel Le ó n- Á lvarez, Laboratorio de Neoralfsiaceae of the order Ralfsiales. Their proposed new Ficolog í a and Secci ó n de Algas del Herbario de la Facultad de combination N. expansa as the type species of the genus, Ciencias, Universidad Nacional Aut ó noma de M é xico, UNAM, with Myrionema ( ? ) expansum J. Agardh as the basionym, Mexico 04510 D.F., e-mail: [email protected] was later validated by Cormaci and Furnari (in Cormaci Maria L. N ú ñ ez-Resendiz: Laboratorio de Ficolog ía and Secció n de Algas del Herbario de la Facultad de Ciencias, UNAM, Mexico et al. 2012 ). Recent molecular studies on rbc L showed Michael J. Wynne: University of Michigan Herbarium, 3600 Varsity Neoralfsia hancockii (E.Y. Dawson) D. Le ó n- Á lvarez and Drive, Ann Arbor, Michigan 48108, USA M.L. N ú ñ ez-Res é ndiz, another common species in the Brought to you by | UNAM Authenticated | [email protected] author's copy Download Date | 8/15/14 11:49 AM 2 D. Le ó n- Á lvarez et al.: Topotype material of Neoralfsia expansa eastern Tropical Pacific ( Le ó n- Á lvarez et al. 2014 ), to be Faculty of Sciences, National Autonomous University of distinct from N. expansa (as Ralfsia expansa sensu Tanaka Mexico, FCME (GM649, GM650, GM651) ( Table 1 ). These and Chihara 1980b , as Neoralfsia expansa sensu Lim et al. collections were also compared with three specimens of 2007 ). Given the geographic distance between Japan N. expansa (FCME: GM289 and GM635), from the Morro de or Malaysia and Veracruz, Mexico, and the differences la Mancha, Veracruz, one of them in unangial reproduc- in morphological characteristics, we have conducted tive condition, and other one identified as Ralfsia expansa molecular studies on topotype material to determine the in plurangial reproductive condition from Punta Lim ó n, relationship between the Mexican (topotype) and Asian Veracruz, from the Herbarium of the National School specimens. of Biological Sciences, National Polytechnic Institute, ENCB4257. Materials and methods Morphology For this study, we collected samples of Neoralfsia expa nsa from Veracruz, Mexico, on multiple occasions ( Table 1 ). Under an optical microscope, preparations were observed Specimens were collected using a chisel and hammer and starting with vertical-radial sections of tissue made using a knife to remove scrapings from rock substratum. For the a manual rotation microtome, which were stained with molecular studies, samples collected on the dates Decem- safranin and fast green (slides in FCME, Table 1 ). Obser- ber 4, 2009, May 18, 2010, and April 29, 2010 were used, vations were made on diagnostic characteristics of this and these samples were each subdivided into three por- species, such as thickness of the thallus, crust medul- tions. One portion of each was preserved with silica gel lary differentiation, chloroplast number, presence, posi- (for molecular analysis), a second portion with a dilute tion and number of cells in the plurangia, presence and formalin solution in seawater, and the third portion on position of unangia and number of stalk cells, presence/ blotting paper (these latter two portions being used for absence of paraphyses, according to the characteristics morphological analysis and herbarium reference). All described by Tanaka and Chihara (1980a,b) , Le ó n- Á lvarez samples were incorporated into the collection of the Gulf and Gonz ález-Gonz ález (1995, 2003) , Le ó n- Á lvarez (2005) , of Mexico of the Algae Section of the Herbarium of the and Lim et al. (2007) for Ralfsia species and with the Table 1 Herbarium references and data collection sites for the Mexican samples of Neoralfsia expansa and Neoralfsia hancockii included in this study. Species Locality Collection date Collector Habitat Catalog no. Slides nos. in FCME in FCME N. expansa Mexico: Morro de la Mancha, 27/09/2010 Mar í a L. Karst rock platform GM635 1174 – 1177 Veracruz. N ú ñ ez and exposed to waves at sea 19 ° 35 ′ 22 ″ N; 96 o 22 ′ 43 ″ W Daniel Le ó n ” 29/04/2010 ” ” GM651 1170 – 1174 ” 18/05/2010 ” Vertical walls of rocky GM649 1153 – 1156 barriers or crags ” 04/12/2009 ” Boulders exposed to light GM650 1165 – 1169 and waves ” 14/02/2003 Daniel Le ó n Karst rock platform GM289 244 exposed to waves at sea a Mexico: Punta Lim ó n, Veracruz 09/11/1976 – Unspecified ENCB4257 1758 N. hancockii Mexico: San Jos é del Cabo (La 30/09/2009 ” Granite boulders directly PTM9165 1139 – 1142 Palmilla), B.C.S. exposed to waves and 23 ° 03 ′ 31.8 ″ N; 109 ° 41 ′ 23.3 ″ W light San Jos é del Cabo (Club 30/09/2009 ” Rocky irregular platform PTM9167 1132 – 1138 Campestre), B.C.S. exposed to light, protected 23 ° 01 ′ 04.0 ″ N; 109 ° 42 ′ 56.9 ″ W from direct waves a As Ralfsia. GM and PTM, Gulf of Mexico and Mexican Tropical Pacific catalog numbers in FCME; ENCB, Escuela Nacional de Ciencias Biol ó gi- cas, Herbarium. Brought to you by | UNAM Authenticated | [email protected] author's copy Download Date | 8/15/14 11:49 AM D. Le ó n- Á lvarez et al.: Topotype material of Neoralfsia expansa 3 terminology proposed by Le ó n- Á lvarez and Norris (2005) and PRB-R3 ( Kogame et al. 1999 ). The PCR product was for crustose brown algae. Because the exact nature of the sequenced at the Molecular Biology Laboratory of the reproductive cells (i.e., gametes or spores) cannot be estab- Institute of Biology, UNAM. lished, we use the morphological terms unangium and plu- rangium instead of unilocular or plurilocular sporangia. Phylogenetic analysis Molecular analysis Thirty-four rbc L sequences of the order Ralfsiales from GenBank (NCBI) and three sequences from this study were DNA extraction and amplification followed the methods included in the molecular analyses, together with Tilop- described in Le ó n- Á lvarez et al. (2014) , with the addition teris mertensii (Turner) K ü tzing and Sargassum muticum of the following primers: rbc-F4 ( Kawai and Sasaki 2004 ) (Yendo) Fensholt as outgroups ( Table 2 ). Table 2 Molecular sequence data (NCBI, 2014 and this study) and herbarium references. Species Accession no.