Phycologia Volume 55 (2), 165–177 Published 12 February 2016 Wynneophycus geminatus gen. & comb. nov. (Delesseriaceae, Rhodophyta), based on Hypoglossum geminatum Okamura 1 1 3 1,2 SO YOUNG JEONG ,BOO YEON WON ,SUZANNE FREDERICQ AND TAE OH CHO * 1Department of Life Science, Chosun University, Gwangju 501-759, Korea 2Marine Bio Research Center, Chosun University, Wando, Jeollanam-do 537-861, Korea 3Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-3602, USA ABSTRACT: Wynneophycus gen. nov. (Delesseriaceae, Ceramiales) is a new monotypic genus based on Hypoglossum geminatum Okamura, a species originally described from Japan. Wynneophycus geminatus (Okamura) comb. nov.is characterized by a discoid holdfast, erect or decumbent monostromatic blades with percurrent midribs, production of new blades from the midrib axial cells and absence of microscopic veins. In addition, it has apical cell division, several orders of lateral cell rows and paired transverse periaxial cells and formation of second-order cell rows from lateral cells with all forming third-order cell rows, with the midrib becoming corticated and forming a subterete stipe below as the blade wings are lost. Distinctive features of the new genus include tetrasporangia initiated from and restricted to single rows of second-order cells arranged in a single layer, cover cells developing prior to the tetrasporangia and an absence of intercalary cell divisions. Phylogenetic analyses of rbcL and large-subunit rDNA sequence data support the separation of Wynneophycus from Hypoglossum. We herein report on W. geminatus gen. & comb. nov. and delineate the new tribe Wynneophycuseae within the subfamily Delesserioideae of the family Delesseriaceae. KEY WORDS: Delesserioideae, LSU rDNA, Morphology, Phylogeny, rbcL, Rhodophyta, Wynneophycus, Wynneophycus geminatus, Wynneophycuseae INTRODUCTION Zheng 1998; Wynne & De Clerck 2000; Stegenga et al. 2001; Wynne 2014). Of these, H. geminatum Okamura (1908) was The Delesseriaceae are a large worldwide family of nearly 100 originally described from Japan. It is distinct by having a genera composed of the three subfamilies: Delesserioideae, small thallus, with the primary frond creeping over other Nitophylloideae and Phycodryoideae (Lin et al.2001).The algae by forming holdfasts at several points, pairs of blades subfamily Delesserioideae (as Delesserieae) was originally arising from the dorsal side of the midrib. It also has ovate to recognized by Kylin (1924) based on the procarps being elongate elliptical shaped spermatangial sori and tetraspor- restricted to primary cell rows, the presence of midribs with angia cut off adaxially from the cells of second-order cell rhizoidal filaments and the absence of intercalary cell divisions rows including lateral pericentral cells (Okamura 1908; in the primary cell rows. The Delesserioideae comprise 12 Yoshida & Mikami 1986). tribes based on molecular and morphological evidence (Kylin We collected several red algal plants that we identified as 1924, 1956; Lin et al. 2001; Wynne 1983, 1996, 2014). Lin et H. geminatum from the subintertidal zone in Wan-do, Chuja- al. (2001) evaluated the phylogenetic relationships at the do and Youngdeok, Korea. In this study, we propose that subfamily, tribal and generic levels based on rbcLandlarge- this species does not conform with the concept of Hypo- subunit (LSU) rDNA data and morphological evidence. glossum but that it instead forms the basis of a new genus, The genus Hypoglossum was established by Ku¨tzing (1843, Wynneophycus gen. nov. The new genus is assigned to the p. 444, pl. 65, I) based on H. woodwardii Ku¨tzing, a Delesserioideae on the basis of comparative morphological taxonomic synonym of H. hypoglossoides (Stackhouse) F.S. evidence and phylogenetic analysis of the chloroplast- Collins & Hervey, with the lectotype locality of Cornwall, encoded rbcL gene and the nuclear LSU rDNA. United Kingdom (Wynne 1984a). Hypoglossum is charac- terized by having branches developed endogenously along the midrib, absence of intercalary cell divisions, formation of MATERIAL AND METHODS tetrasporangia from second- and third-order cell rows, pericentral cells, cortical cells and tetrasporangia usually We collected our samples at Chuja-do (33857054.7300 N, produced in multiple layers (Womersley & Shepley 1982; 126817045.2400 E), Wan-do (34817044.9700 N, 126842004.7300 E) Wynne 2014). Hypoglossum has 31 species and is well and Youngdeok (36829014.0100 N, 129826027.5200 E) in Korea represented in both northern and southern hemispheres, from 2008 to 2014. We also collected the materials of H. primarily in warm temperate to tropical waters throughout hypoglossoides from Portsmouth (50846025.7900 N, 0 00 the world (Wynne & Kraft 1985; Wynne & Ballantine 1986; 1802 29.06 E), Southsea, England, near the lectotype locality to compare with our samples of H. geminatum. The samples were sorted according to reproductive stages using a * Corresponding author ([email protected]). DOI: 10.2216/15-94.1 stereomicroscope and preserved in 4%–5% formalin/seawater Ó 2016 International Phycological Society for morphological examination and in silica gel for molecular 165 166 Phycologia, Vol. 55 (2) Figs 1–14. Vegetative structures of Wynneophycus geminatus gen. & comb. nov. (CUK 6033, 9904, 12478, 13064). Fig. 1. Whole plant (CUK12478). Scale bar ¼ 1 cm. Fig. 2. Irregular branching pattern of blades, short stipe and discoid holdfast. Scale bar ¼ 0.1 cm. Fig. 3. Corticated midrib. Scale bar ¼ 50 lm. Fig. 4. Blade with apex and detail of the apical organization. Scale bar ¼ 10 lm. Fig. 5. Blade margin. Scale bar ¼ 20 lm. Jeong et al.: Wynneophycus geminatus gen. & comb. nov. 167 investigation. Microscopic observations of internal anatomy RESULTS were prepared using a freezing microtome (Shandon Cry- otome FSE, Thermo Fisher Scientific, London, UK) with Wynneophycus S.Y. Jeong, B.Y. Won, S. Fredericq & T.O. materials stained with 1% aqueous aniline blue acidified with Cho gen. nov. 0.1% diluted HCl. Photomicrographs were taken by using an Olympus microscope (BX51TRF, Olympus, Tokyo, Japan) with an Olympus DP71 camera. Voucher specimens are Plants epiphytic, erect, membranous, attached to host by deposited in the herbarium of Chosun University, Gwangju, small bundles of multicellular rhizoids. Blades originating Korea (CUK), and in the National Institute of Biological from an endogenous budding of central axial cells of the Resources (NIBR), Incheon, Korea. midrib, lanceolate to ovate, monostromatic except along the Genomic DNA was extracted from silica gel-dried samples midrib, monopodial, with a distinct corticated midrib. using a NucleoSpin Plant II Kit (Macherey-Nagel, D ren, u¨ Apices blunt and tapering. Apical cells obconical, segment- Germany) following the instructions of the manufacturer. ing to form an axial filament of cells with two lateral Polymerase chain reaction (PCR) was performed with a final pericentral cells followed by two transverse pericentral cells. volume 30 ll using 2.8 ll of genomic DNA, 1 ll of 10 pmol Lateral veins absent. Gametophytes dioecious. Procarps forward and reverse primers and Ready-2x-Go Series borne on transverse pericentral cells on one side of the (NanoHelix Co., Ltd, Daejeon, Korea). The rbcL was blades, each with two sterile groups and a four-celled amplified using the primer combinations F7-R753 and carpogonial branch. Female plants with a basal fusion cell F645-RrbcS start (Lin et al. 2001; Bustamante et al. 2013) and much-branched gonimoblasts. Pericarp formed by one and purified with PCRquick-spin PCR product purification to three layers of outer cortical cells. Male plants with kit (iNtRON Biotechnology, Inc., Seongnam, Korea). Cycle spermatangial sori between midrib and margin on both sides sequencing was performed with the primers F7, F645, F993, R376, R753, R1150 and RrbcStart (Freshwater & Rueness of primary and second-order blades. Tetrasporangial plants 1994; Cho et al. 2003; Bustamante et al. 2013). The partial with tetrasporangial sori on second-order blades. Tetraspor- fragments of LSU rDNA were amplified with 28C-28D and angia cut off from pericentral cells and second-order cells, W-28F (Freshwater et al. 1999) and purified with PCRquick- spherical, tetrahedrally divided, each associated with four to spin PCR product purification kit (iNtRON Biotechnology). five cover cells, arranged in primary cell layer except at Cycle sequencing was performed with the primers 28C-28D midrib. and W-28F (Freshwater et al. 1999). Sequences were ETYMOLOGY:‘Wynneophycus’ – ‘Wynne’ plus ‘phycus’. This generic determined for both forward and reverse strands using an name honours Dr. Michael J. Wynne for his valuable contributions to ABI Prism 3100 Genetic Analyzer (Life Technologies, Seoul, phycology, especially in the taxonomy of the family Delesseriaceae. Korea). New sequences were obtained from Wynneophycus geminatus and have been deposited in EMBL/GenBank TYPE SPECIES: Wynneophycus geminatus (Okamura) S.Y. Jeong, B.Y. under accession numbers KR604855, KR604856 and Won, S. Fredericq & T.O. Cho, comb. nov. KR604857 for rbcL and KR604867, KR604868 and KR604869 for LSU rDNA. Sequences generated in the Wynneophycus geminatus (Okamura) S.Y. Jeong, B.Y. Won, present study and others obtained from GenBank were S. Fredericq & T.O. Cho comb. nov. aligned with Clustal W (Thompson et al. 1994) and corrected manually using the MEGA v5 software (Tamura et al. 2011). Figs 1–40 Phylogenetic and molecular evolutionary analyses for BASIONYM: Hypoglossum