Phycologia Volume 55 (2), 165–177 Published 12 February 2016

Wynneophycus geminatus gen. & comb. nov. (, Rhodophyta), based on 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, ) 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 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 geminatum Okamura, Icones Japanese algae maximum likelihood (ML) analysis were conducted with I, p. 156, pl. XXXII, figs 7–12 (1908). 1000 bootstrap replications in MEGA v5 using the GTRþCþI model. A Bayesian inference was performed LECTOTYPE: SAP herbarium (Yoshida 1998). using MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003). The Markov chain Monte LECTOTYPE LOCALITY: Misaki, Kanagawa Prefecture, Japan; fide Carlo runs were carried out for 2 million generations each Yoshida & Mikami, Japanese Journal of Phycology 34: 183 (1986). with one cold chain and three heated chains employing the PREVIOUS TAXONOMIC TREATMENT: Known originally from the GTRþCþI evolutionary model, sampling and printing every collections from syntype localities of Enoshima and Misaki both in 1000 generations. Summary trees were generated using a Sagami Province (now Kanagawa Prefecture), Japan, as H. gemina- burn-in value of 800. tum Okamura 1908, vol. I, pp. 156–159, pl. XXXII, figs 7–12.

 Fig. 6. Opposite branching pattern. Scale bar ¼ 250 lm. Fig. 7. New blades developing from midrib. Scale bar ¼ 200 lm. Fig. 8. Transverse section view of ecorticated midrib in upper part. Scale bar ¼ 25 lm. Fig. 9. Transverse section view of production of new blades (arrows) from a central cell. Scale bar ¼ 25 lm. Fig. 10. Transverse section view of corticated (arrows) midrib in basal part. Scale bar ¼ 50 lm. Fig. 11. Longitudinal section view of blade and cortical cells (arrow). Scale bar ¼ 25 lm. Fig. 12. Marginal multicellular rhizoids. Scale bar ¼ 50 lm. Fig. 13. Cross-section view of short stipe. Scale bar ¼ 50 lm. Fig. 14. Basal attaching disc. Scale bar ¼ 100 lm. 168 Phycologia, Vol. 55 (2)

Figs 15–29. Reproductive structures of Wynneophycus geminatus gen. & comb. nov. (CUK 9904, 12478). Fig. 15. Female plant (CUK12478). Scale bar ¼ 0.1 cm. Figs 16–18. Surface view showing a young prefertilization procarp with supporting cell (sc) bearing a four-celled carpogonial branch (cb) including a carpogonium (cp) with two sterile cells (st1, st2) and a trichogyne (tr). Scale bar ¼ 5 lm. Fig. 19. Two procarps arranged in opposite direction. Scale bar ¼ 5lm. Fig. 20. Hemispherical shape of young cystocarp (arrows) on blade midrib. Scale bar ¼ 200 lm. Fig. 21. Urceolate shape of mature cystocarp (arrow) on blade midrib. Scale bar ¼ 150 lm. Fig. 22. Cross section of young cystocarp showing fusion cell (fc), developing gonimoblasts (g) and remnant sterile groups (st1, st2). Scale bar ¼ 25 lm. Fig. 23. Gonimoblasts with radiating carposporangia in branched chains. Scale bar ¼ 50 lm. Fig. 24. Cross section through a fully developed cystocarp showing carposporangia (arrow) and ostiole (arrowhead). Scale bar ¼ 50 lm. Fig. 25. Male plant (CUK9904). Scale bar ¼ 500 lm. Fig. 26. Male plant bearing mature spermatangia in continuously paired sori (arrows) on both sides of the blade midrib. Scale bar ¼ 200 lm. Fig. 27. Young spermatangial sori in surface view. Scale bar ¼ 5 lm. Jeong et al.: Wynneophycus geminatus gen. & comb. nov. 169

Figs 30–40. Tetrasporophyte of Wynneophycus geminatus gen. & comb. nov. (CUK 6033, 13064). Fig. 30. Tetrasporophyte (CUK6033). Scale bar ¼ 0.2 cm. Fig. 31. Tetrasporangial sori developing from second-order blades. Scale bar ¼ 500 lm. Fig. 32. Tetrasporangia forming from pericentral and second-order cells. Scale bar ¼ 50 lm. Fig. 33. Tetrasporangia in serial arrangement on second-order cell rows (2). Scale bar ¼ 20 lm. Fig. 34. Tetrasporangia protected by cover cells. Scale bar ¼ 200 lm. Fig. 35. Tetrasporangia developing from lateral pericentral cells (lp) and from transverse pericentral cell (tp). Scale bar ¼ 50 lm. Fig. 36. Second-order cells dividing into two periclinal cover cell initials (ci) on both sides of the surface. Scale bar ¼ 10lm. Figs 37, 38. Tetrasporangium (t) developing from stalk cell (st) and two cover initial cells have divided to form four to five small cover cells (c). Scale bar ¼ 10 lm. Figs 39, 40. Cross-section view of tetrasporangial sori situated in a cell layer that is not a midrib. Scale bar ¼ 50 lm.

 Fig. 28. Cross-section view of young spermatangial sori with spermatangial parent cells (arrow). Scale bar ¼ 25 lm. Fig. 29. Spermatangia (arrowhead) formed terminally from their parent cells (arrow). Scale bar ¼ 25 lm. 170 Phycologia, Vol. 55 (2)

DISTRIBUTION: Known from China, Fiji, Japan, Korea and filaments of marginal cells near the apices (Fig. 12) and by a Norfolk Island (Yoshida et al. 1990; Yoshida 1998; Millar 1999a, disc-shaped holdfast at the base (Figs 13, 14). 1999b; Lee & Kang 2001; Zheng et al. 2001; Oak et al. 2002; South & Skelton 2003; Lee 2008; Liu 2008; Nam & Kang 2012; Wynne 2014; Guiry & Guiry 2015). Reproductive Morphology

SPECIMENS EXAMINED: CUK6033, NIBRAL0000148972, NIBRAL0000148973, male and tetrasporic plants, 3 July 2008, The gametophytes were dioecious. Female gameto- Jeongdo-ri beach, Wan-do, Jeollanam-do; CUK9904, male and phytes were 4–6 mm in height and 0.7–2.0 mm in width tetrasporic plants, 24 June 2014, and CUK12478, female, 27 June and had a slightly undulate margin (Fig. 15). A single 2014, Daeseo-ri, Chuja-do, Jeju; CUK13064, tetrasporic plant, 30 procarp was formed from a transverse pericentral cell. October 2014, Gyeongjeong 1-ri, Youngdeok, Gyeongsangbuk-do. Procarps each consisted of a supporting cell, which was a transverse pericentral cell, a single four-celled carpogonial Vegetative Morphology branch and two sterile groups (Figs 16–19). The second cell of the carpogonial branch was the largest cell of the branch. The carpogonium on the carpogonial branch had Plants were epiphytic on Corallina pilulifera Postels & the thin trichogyne ending in a bulbous tip (Figs 18, 19). Ruprecht in the subintertidal zone on rocky shores. Thalli Four to five procarps developed singly in an opposite were greenish brown, membranous, 1–1.5 cm in height, 2– direction and were arranged along the midrib on one 2.5cminwidth,composedofonetotwo(tothree)main surface of a fertile second blade (Fig. 19). One cystocarp blades, and with a small holdfast (Fig. 1). They had terete usually developed on the midrib of the second-order axes with eroded wings in the lower parts (Figs 2, 3). blades (Figs 20, 21). Young cystocarps were hemispherical Branching was entirely monopodial. Blades were generally (Fig. 20). Mature cystocarps were urceolate, 497–655 lm linear to elliptical with pointed apices (Fig. 4), 1–2 mm in diameter, with a wide, open beaked ostiole. Cystocarps broad, 300–360 lm across, with a single distinct corticated were slightly inclined towards the blade apex (Fig. 21). midrib (Fig. 3). Blade margins were entire. The apical After fertilization, the supporting cell cut off an auxiliary organization was easily examined at the tip of a young cell. At a later stage, a fusion cell consisted of the central frond (Fig. 4) in which growth was initiated by a cell and supporting cell, with branched gonimoblast cells transversely dividing apical cell that produced a primary bearing chains of carposporangia (Figs 22, 23). Carpo- sporangia were elongate-ovoid, 10–20 lmindiameter,and cell row or central axis. The main central cell soon formed terminally on branched gonimoblast filaments elongated considerably and formed two lateral pericentral (Figs 23, 24). The pericarp was 2–4 layered, formed from cells to be cut off first, followed by two transverse the extension of cortical cells and central floor cells in the pericentral cells. The lateral pericentral cells became initials cystocarp (Fig. 24). of second-order cell rows and developed the wings, or Male gametophytes (Fig. 25) resembled female gameto- ‘alae’. The cells of these second-order cell rows in turn cut phyte in stature and branching habit. Spermatangia devel- off initials of third-order cell rows abaxially, and third- oped in parallel ovate to long elliptical sori on both surfaces order cells sometimes cut off fourth-order cell rows (Fig. 4). between the midrib and margin of primary and second-order Every cell row reached the thallus margins (Figs 4, 5). At blades (Fig. 26). The sori were separated by a sterile midrib the blade margin, second-order cells remained undivided on each blade. Paired spermatangial sori were 25–180 lm (Fig. 5). Intercalary division was absent in every cell row. broad, or approximately two-thirds the blade width. Cells in New blades were ovate to oblong, arose from a midrib of the sori were produced by the periclinal divisions of cells of older blades on the dorsal face and were arranged in the second and third orders (Fig. 27). These cortical cells opposite pairs from the axial row (Figs 6, 7). Two blade produced three to five spermatangial parent cells on both initials were produced from a central cell in the midrib and surfaces of the blade (Fig. 28). Each spermatangial parent developed into two young blades between pericentral cells cell divided to produce two subterminal or terminal (Figs 7, 9) that were monostromatic and entire, except in spermatangia. Spermatangia were oval, 5 lm wide and 4–5 the midrib region, where the formation of transverse lm long (Fig. 29). pericentral cells resulted in three cell layers (Fig. 8). Tetrasporophytes had a similar stature and branching habit as the gametophytes (Fig. 30). Tetrasporangia Microscopic veins were absent. The midrib was composed developed in circular to elliptical sori on second- and of elongated axial cells and four pericentral cells. It was third-order blades (Fig. 31). Tetrasporangia were first cut initially three-cell layered and slightly corticated in the off from lateral pericentral cells, adaxially from second- middle part of the thallus (Fig. 10). Cortical cells were order cell rows and then from transverse pericentral cells spherical, 2–3 lm long and 2–4 lm broad, and developed (Figs 32, 33, 35). Their originating cells functioned as from pericentral, second-order and third-order cells near tetrasporangial stalk cells. They were arranged in a single the midrib (Figs 10, 11). The midrib of the main blades was row per segment and parallel to second-order cell rows heavily corticated and developed into a stipe-like midrib in (Fig. 33). Each tetrasporangium became covered by the lower thallus (Figs 13, 14). Thalli were attached by protective cover cells (Fig. 34). The cover cell initial was several peg-like haptera formed by outgrowing rhizoidal periclinally cut off from both sides of a second-order cell Jeong et al.: Wynneophycus geminatus gen. & comb. nov. 171

Table 1. List of species, their collection information and the rbcL (denoted by *) and LSU rDNA (denoted by **) accession numbers in GenBank.

GenBank Delesseriaceae Collection information accession number Cumathamnion serrulatum (Harvey) M.J. Wynne & Dongsan-ri, Yangyang-gun, Gangwon-do, Korea, KR604850*, KR604862** G.W. Saunders coll. T.O. Cho, 1 Feb. 2013 (CUK9271) C. serrulatum (Harvey) M.J. Wynne & G.W. Yeolgi-ri, Yangyang-gun, Gangwon-do, Korea, KR604851*, KR604863** Saunders coll. T.O. Cho, 3 Oct. 2013 (CUK10231) sanguinea (Hudson) J.V. Lamouroux Forty Foot Beach, Dublin, Ireland, coll. T.O. Cho, KR604852*, KR604864** 7 May 2014 (CUK11739) Hemineura frondosa (J.D. Hooker & Harvey) Lagoon Beach, Tasmania, Australia, coll. T.O. KR604853*, KR604866** Harvey Cho, 23 Mar. 2014 (CUK10927) Hypoglossum hypoglossoides (Stackhouse) F.S. Portsmouth, Southsea, England, coll. T.O. Cho, 3 KR604846*, KR604858** Collins & Hervey May 2014 (CUK11316) alata (Hudson) Stackhouse Portsmouth, Southsea, England, coll. T.O. Cho, 3 KR604847*, KR604861** May 2014 (CUK11328) M. robbeniensis Tokida Bangpo beach, Taean-gun, Korea, coll. T.O. Cho, KR604848*, KR604859** 5 May 2012 (CUK7845) M. robbeniensis Tokida Bangpo beach, Taean-gun, Korea, coll. T.O. Cho, KR604849*, KR604860** 31 Mar. 2014 (CUK8027) Neoholmesia japonica (Okamura) Mikami Dongsan-ri, Yangyang-gun, Gangwon-do, Korea, KR604854*, KR604865** coll. T.O. Cho, 1 Feb. 2013 (CUK9270) Wynneophycus geminatus S.Y. Jeong, B.Y. Won, S. Daeseo-ri, Chuja-do, Jeju-si, Korea, coll. T.O. Cho, KR604855*, KR604868** Fredericq & T.O. Cho 24 Jun. 2013 (CUK9904) W. geminatus S.Y. Jeong, B.Y. Won, S. Fredericq Daeseo-ri, Chuja-do, Jeju-si, Korea, coll. T.O. Cho, KR604856*, KR604867** & T.O. Cho 27 Jun. 2014 (CUK12478) W. geminatus S.Y. Jeong, B.Y. Won, S. Fredericq Gyengjeong1-ri, Youngdeok-gun, Korea, coll. T.O. KR604857*, KR604869** & T.O. Cho Cho, 30 Oct. 2014 (CUK13064) before the production of the tetrasporangia (Fig. 36). third-order rows (Fig. 42). Every cell row reached the thallus Initially, tetrasporic thalli were composed of three layers of margins. At the blade margin, second-order cells divided (Fig. 43). New blade initials were produced endogenously from a central cell in cells, namely, the row of second-order cells flanked by two the midrib and developed into young blades between pericentral cells cover cell initials (Fig. 36). The cover initial cells never (Fig. 44) that were monostromatic, except in the midrib region, produced tetrasporangia. Four to five cover cells were where transverse pericentral cells and cortical cells were produced. produced per cover initial cell on the dorsal side and two Tetrasporangial sori developed on either side of the midrib and were cover cells on the ventral side (Figs 37, 38). These cover continuous along both sides of the blade (Fig. 45). Tetrasporangia were cut off adaxially by second-order cell rows and third-order cell cells did not completely cover the enlarging tetrasporangia. rows (Fig. 46). They were multilayered (Figs 47, 48) when mature Tetrasporangia were tetrahedrally divided and 15–25 lm 3 and covered by sterile cortical cells (Fig. 49). Cystocarps were 25–40 lm in size (Fig. 40). Mature tetrasporangial sori hemispherical, typically developed singly on the blade midrib (Fig. were composed of a single tetrasporangial layer except for 50). the midrib (Figs 39, 40). Tetrasporangial sori were continuous or discontinuous in a paired arrangement Molecular phylogenetic analyses alongside the midrib, 0.5–1.05 mm in length and 0.2–0.3 A 1197-base-pair (bp) portion of rbcL (81.59% nucleotides mm in width. sequenced) was sequenced for W. geminatus gen. & comb. RbcL sequences were also newly generated for Cumathamn- Hypoglossum hypoglossoides (Stackhouse) F.S. Collins & ion serrulatum (Harvey) M.J. Wynne & G.W. Saunders, Hervey 1917 (Hudson) J.V. Lamouroux, Hemineura Figs 41–50 frondosa (J.D. Hooker & Harvey) Harvey, Hypoglossum hypoglossoides, (Hudson) Stackhouse, LECTOTYPE LOCALITY: Polridmouth Cove, Cornwall, United King- dom. M. robbeniensis Tokida and Neoholmesia japonica (Oka- mura) Mikami. The LSU rDNA data set was analyzed based SPECIMENS EXAMINED: CUK11315, CUK11316, on 900-bp (27.27% nucleotides sequenced) portion including tetrasporic plant, 3 May 2014, Portsmouth, Southsea, sequences from three samples of W. geminatus.TheLSU United Kingdom; CUK11327, CUK11339, female, 3 May rDNA was sequenced from C. serrulatum, D. sanguinea, H. 2014, Portsmouth, Southsea, United Kingdom. CUK11717, frondosa, H. hypoglossoides, M. alata, M. robbeniensis and tetrasporic plant, 7 May 2014, Forty Foot beach, Dun N. japonica. Species and GenBank accession numbers of Laoghaire, Dublin, Ireland. sequences are given in Table 1. MrBayes and ML phylogenetic trees were obtained from rbcLandLSU MORPHOLOGY: Thalli were reddish, membranous, 5–10 cm in height, sequences using Nitophyllum punctatum (Stackhouse) Gre- composed of single or several blades and attached by holdfast ville and Augophyllum delicatum (A.J.K. Millar) S.-M. Lin, consisting of small solid disc (Fig. 41). They had terete axes with eroded wings in the lower parts (Fig. 41). Growth in length occurred S. Fredericq & M. Hommersand as out-groups. Phylogenies by a transversely dividing apical cell in the terminal portion of each inferred from rbcL and LSU demonstrated that Wynneo- branch. All cells of the second-order rows gave rise abaxially to phycus was a distinct clade with robust support values (1 for 172 Phycologia, Vol. 55 (2)

Figs 41–50. Vegetative and reproductive structures of Hypoglossum hypoglossoides (CUK 11315, 11316, 11327, 11339, 11717). Fig. 41. Plant collected in United Kingdom (CUK 11339). Scale bar ¼ 1 cm. Fig. 42. Blade with apex and detail of the apical organization. Scale bar ¼ 20 lm. Fig. 43. Divided marginal cell. Scale bar ¼ 20 lm. Fig. 44. Cross-section view of production of new initial blades (i) from central cell (ce). Scale bar ¼ 50 lm. Fig. 45. Mature tetrasporangial sorus. Scale bar ¼ 100 lm. Fig. 46. Tetrasporangia developing from second- and third-order cell rows. Scale bar ¼ 50 lm. Fig. 47. Tetrasporangia not developing from midrib (arrow) and each tetrasporangia developing from inner cortical cells (arrowhead). Scale bar ¼ 50 lm. Fig. 48. Tetrasporangia situated in multilayer. Scale bar ¼ 50 lm. Fig. 49. Tetrasporangia wholly protected by cover cells. Scale bar ¼ 50 lm. Fig. 50. Mature cystocarp shaped in hemispherical. Scale bar ¼ 100 lm. Jeong et al.: Wynneophycus geminatus gen. & comb. nov. 173

Fig. 51. Phylogenetic tree based on Bayesian analysis of rbcL sequences. Values above branches denote Bayesian posterior probabilities (BPP) . 0.75/maximum likelihood bootstrap values (BS) in % . 50. BPP values of , 0.75 and BS values of , 50% are indicated by hyphens (-). BPP values of 1.00 and BS values of 100% are indicated by asterisks (*) (AQ, Antarctica; AU, Australia; FK, Falkland Islands; IE, Ireland; JP, Japan; KR, South Korea; MX, Mexico; NL, Netherlands; NZ, New Zealand; PA, Panama; PH, Philippines; PT, Portugal; UK, United Kingdom; US, United States; ZA, South Africa).

MrBayes and 100 for ML) (Figs 51, 52). RbcLpairwise disc-shaped holdfasts, urceolate cystocarps, shape of sper- comparison of Wynneophycus with and Para- matangial sori and tetrasporangial development. Okamura glossum was 9.1–10.4% and 8.0%–10.1%, respectively. LSU (1908) originally described H. geminatum with the following rDNA pairwise comparison of Wynneophycus had a 10.8%– features: minute fronds, linear-lanceolate branches, new 14.5% gene sequence divergence with Apoglossum and 8.8%– branches from both sides of the midrib on the upper surface, 10.3% with Paraglossum. These molecular analyses demon- more or less corticated midribs, veinless fronds, entire strated that Wynneophycus wasadistinctgenusinthe blades, disc-shaped attachments from the midrib and subfamily Delesserioideae. urceolate cystocarp on the midrib from central Japan. Yoshida & Mikami (1986) observed the detailed morphology of male and tetrasporic thalli of H. geminatum from DISCUSSION Wakayama Prefecture, Japan, and added the features of ovate to elliptical spermatangial sori produced from both Our specimens from Korea correspond to H. geminatum sides of midrib and tetrasporangia cut off adaxially from previously reported from Japan and Korea based on the lateral pericentral and second-order cells. Oak et al. (2002) following characters: linear-lanceolate branches, corticated also reported the detailed morphology for all stages of H. midribs, origin of branches, veinless thalli, entire margins, geminatum collected along the south and east coasts of 174 Phycologia, Vol. 55 (2)

Fig. 52. Phylogenetic tree based on Bayesian analysis of LSU sequences. Values above branches denote Bayesian posterior probabilities (BPP) . 0.75/maximum likelihood bootstrap values (BS) in % . 50. BPP values of , 0.75 and BS values of , 50% are indicated by hyphens (-). BPP values of 1.00 and BS values of 100% are indicated by asterisks (*) (AQ, Antarctica; AU, Australia; FK, Falkland Islands; IE, Ireland; JP, Japan; KR, South Korea; MX, Mexico; NL, Netherlands; NZ, New Zealand; PH, Philippines; PT, Portugal; UK, United Kingdom; US, United States; ZA, South Africa).

Korea. Although we were not allowed to access the type ment of tetrasporangia in a single row per segment and material of H. geminatum, our samples collected from the parallel to second order cell rows, (3) cover cells partially south and east coasts of Korea appear identical to H. covering the tetrasporangia, (4) inner cover cells never geminatum based on previous detailed accounts of both forming tetrasporangia, (5) tetrasporangial sori composed of vegetative and reproductive morphology. a single tetrasporangial layer except on the midrib, (6) Superficial morphological similarities in members of the spermatangial sori separated by a sterile midrib and margin Delesseriaceae may mask significant developmental differ- on primary and second blades, (7) procarps borne on the ences that result in taxonomic confusion (Lin et al. 2001). midrib of second blades, (8) absence of microscopic veins Molecular-based phylogenies of Delesseriaceae have chal- and (9) lack of intercalary cell divisions in any cell row. lenged the traditional systems of classification based on The origin of tetrasporangia in the Delesseriaceae was not morphology (Lin et al. 2001; Krayesky et al. 2011; Wynne & considered an important generic character because it did not Saunders 2012). Our rbcL and LSU phylogenies propose the appear to resolve current higher-level taxa, and it was recognition of W. geminatus gen. & comb. nov. for the taxon considered variable based on the plant age. However, previously known as H. geminatum Okamura occurring in although the origin of tetrasporangia has been used to China, Fiji, Japan, Australia and Korea. Wynneophycus recognize species, it may be a phylogenetically important geminatus is in a strongly supported clade recognized by the character at the genus level to distinguish Wynneophycus. suite of vegetative and reproductive characters that distin- Wynneophycus geminatus is characterized by tetrasporangia guish it from other genera. The new genus, Wynneophycus,is arranged in a single row per segment and parallel to second- characterized as follows: (1) tetrasporangia adaxially pro- order cell rows. It is distinguished from species of other duced from pericentral and second-order cells, (2) arrange- genera by having tetrasporangia developed only from Jeong et al.: Wynneophycus geminatus gen. & comb. nov. 175 second-order cells. Hypoglossum is the most closely related produced mainly alternately and by a height of 8–20 cm genus to Wynneophycus in tetrasporangial development. The (Womersley & Shepley 1982; Wynne & De Clerk 2000). species of Hypoglossum show variation in cell origin of Hypoglossum dendroides has different apical organization, tetrasporangia. In most species of Hypoglossum, tetraspor- and not all cells of second-order rows bear third-order rows angia are produced by a primary layer of cells and by cortical (Womersley & Shepley 1982). Hypoglossum sagamianum is cells, depending on the species and the age of the sorus distinguished by having intercalary cell divisions in second- (Womersley & Shepley 1982). Wynne & De Clerck (2000) order cell rows (Mikami 1987). Hypoglossum subsimplex provided a list of 16 species developing tetrasporangia from differs by the absence of cortication on the midrib and by a primary layer of cells and from cortical cells. Whereas W. spermatangia produced in discrete sori (Wynne 1994; geminatus develops tetrasporangia only from the primary Ballantine et al. 2002). Hypoglossum minimum can be layer of cells comprising a single tetrasporangial layer in separated from W. geminatus by its simple, rarely branching mature tetrasporangial sori, species in typical Delesserioi- blades and by spermatangial sori arranged in small linear deae have three-layered tetrasporangial sori from both of the patches (Yoshida & Mikami 1986). Although H. caloglos- primary layer of cells and cortical cells (Kylin 1956). In a soides is similar in size and habit to W. geminatus, the blades limited number of species of Hypoglossum, tetrasporangia of the latter do not undergo the regular pattern of are produced only by the primary layer of cells, that is, not constriction (Wynne & Kraft 1985). Like W. geminatus, by cortical cells (Wynne 1988), as in H. annae M.J. Wynne & tetrasporangia of H. revolutum (Harvey) J. Agardh are cut O. DeClerck, H. dendroides (Harvey) J. Agardh, H. minimum off from lateral, transverse pericentral cells and from second- Yamada, H. protendens (J. Agardh) J. Agardh and H. order cells, but the branching is sympodial, and the sagamianum Yamada as well as in H. subsimplex M.J. tetrasporangia are cut off from cortical cells, resulting in Wynne (Wynne 1994; Wynne & De Clerck 2000). Of these many tetrasporangial layers (Womersley & Shepley 1982). species of Hypoglossum, H. annae may be recognized as Our rbcL and LSU sequences revealed sufficient diver- belonging to the Wynneophycus group by having single- gences to warrant recognition of Wynneophycus as a new layered tetrasporangal sori and tetrasporangia produced genus. Intergeneric rbcL and LSU sequences divergence only by second-order cells. between Apoglossum and Paraglossum in the tribe Apoglos- ‘Cortical cells’ and ‘cover cells’ often have been used seae is 9.7%–10.2% and 8.8%–9.3%, respectively. The extent almost synonymously for cells covering the tetrasporangial of rbcL and LSU 8.0%–10.4% and 8.8%–14.5 % gene sori in Delesseriaceae (Womersley & Shepley 1982). Howev- sequence divergence of Wynneophycus compared to Apo- er, Bold & Wynne (1978) defined cover cells as ‘cells that are glossum and Paraglossum indicates that Wynneophycus is a cut off in association with tetrasporangia, serving as new genus in the subfamily Delesserioideae, and phyloge- superficial, protective covers’. They stated that the family netic analysis on the basis of comparative rbcL and LSU Delesseriaceae has tetrasporangial initials cut off prior to the data sets shows separately that Wynneophycus is a sister ‘cover cells’. Wynneophycus geminatus develops two (dorsal clade to the tribe Apoglosseae. and ventral) cover cell initials from the primary cell layer In conclusion, we recognize W. geminatus gen. & comb. (especially from second-order cells) before the tetrasporan- nov. based on morphological and molecular evidence. gial development. These cover cell initials produce cover cell Although this taxon superficially resembles the genus clusters composed of four to five cells in the dorsal side and Hypoglossum, it is distinguished by the tetrasporangial two cells in the ventral side, and these function as a development, the internal structure of tetrasporangial sori protective cover over each tetrasporangium. After develop- and the association of cover cells in the tetrasporangia. The ment of the cover cell initial, the second-order cell develops a comparisons between Wynneophycus and other genera and tetrasporangial initial, and it functions as a tetrasporangial tribes in the subfamily Delesserioideae suggest that the new stalk cell that bears a tetrasporangium and a cover cell genus cannot be placed in any existing group (Table 2). The cluster. However, in the type species of Hypoglossum, H. combination of endogenous development of indeterminate hypoglossoides, the cutting of cover cells from stalk cells after axes, lack of intercalary cell divisions and tetrasporangial initiation of the tetrasporangia has not been observed, and development is considered to be the primary defining feature the cortical layer gives adequate protection to the sporangia of the Wynneophycus group. In this instance, establishing an (Womersley & Shepley 1982). Most authors (e.g. Papenfuss entire tribe for a single species is not without precedent. 1937; Wagner 1954) have referred to the cells covering the Kraft & Huisman (1981) pointed out that some 20 of the 70 tetrasporangial sori as cortical cells, but Papenfuss (1944) red algal families are monogeneric and that four families are referred to them as ‘cover cells’. However, cortical cell and actually monospecific and known only from their type cover cell may be distinguished according to their role in the localities. To this end, we have chosen to formally recognize formation of tetrasporangia. Cover cells are formed after the the Wynneophycus group as a tribe of the subfamily sporangium is initiated in the subfamily Delesserioideae Delesserioideae. (Papenfuss 1944). The order of tetrasporangium and cortical or cover cell initiation has not been satisfactorily document- ed in most Delesseriaceae (Womersley & Shepley 1982). Wynneophycuseae S.Y. Jeong, B.Y. Won, S. Fredericq & Wynneopycus geminatus resembles Hypoglossum annae, H. T.O. Cho tribus nov. protendens, H. dendroides, H. minimum, H. sagamianum and H. subsimplex in habit, tetrasporangial sori and branching pattern. However, H. annae and H. protendens are distin- Thalli with indeterminate branches arising by endogenous guished from W. geminatus by having secondary blades division of the central cell of midribs; growth by means of single 176 Phycologia, Vol. 55 (2)

transversely dividing apical cells; intercalary divisions lacking; all second- and third-order initials reaching the thallus margin; procarps restricted to primary cell rows, each with two groups of . (2012)

— sterile cells and a four-celled carpogonial branch per supporting cell;

et al tetrasporangia developing in a layer. surface cells Lin TYPE GENUS: Wynneophycus.

ACKNOWLEDGEMENTS

We thank Prof. Michael J. Wynne for many constructive suggestions to improve the manuscript. This study was supported by a grant from the research fund of Chosun University 2015 to T.O. Cho. and cortical cells two species) Wynne (1988); Maggs & Hommersand (1993) pericentral, primary

REFERENCES

BALLANTINE D.L. RUIZ H. & WYNNE M.J. 2002. Notes on the marine algae of Puerto Rico VII. Seven new records of benthic . Rhodophyta. Caribbean Journal of Science 38: 252–256. — endogenously — BOLD H.C. & WYNNE M.J. 1978. Introduction to the algae: structure and reproduction. Prentice Hall, Englewood Cliffs, New Jersey. 720 pp.

cells BUSTAMANTE D.E., WON B.Y. & CHO T.O. 2013. Neosiphonia primary and cortical Wynne & Daniels (1966) Yoshida & Mikami (1986); ramirezii sp. nov. (Rhodomelaceae, Rhodophyta) from Peru. Algae 28: 73–82. CHO T.O., FREDERICQ S. & BOO S.M. 2003. Ceramium inkyuii sp. nov. (Ceramiaceae, Rhodophyta) from Korea: a new species based on morphological and molecular evidence. Journal of Phycology 39: 236–247. FRESHWATER D.W. & RUENESS J. 1994. Phylogenetic relationships of

Wynneophycus gen. & comb. nov some European Gelidium (Gelidiales, Rhodophyta) species, based on rbcL nucleotide sequence analysis. Phycologia 33: 187–194. FRESHWATER D.W., FREDERICQ S. & BAILEY J.C. 1999. Character- cells exogenously Womersley (2003) istics and utility of nuclear-encoded large-subunit ribosomal gene Papenfuss (1961); sequences in phylogenetic studies of . Phycological Research 47: 33–38. GUIRY M.D. & GUIRY G.M. 2015. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://algaebase.org; searched on 23 June 2015. HUELSENBECK J.P. & RONQUIST F. 2001. MrBayes: Bayesian . (2012) inference of phylogenetic trees. Bioinformatics 17: 754–755. KRAFT G.T. & HUISMAN J.M. 1981. New record of the marine red et al

Apoglossum Caloglossa Cumathamnion Hypoglossum Paraglossum alga Cubiculosporum (Gigartinales) from the southern Great

Lin Barrier Reef. Journal of Phycology 17: 278–280.

inner cortical cells pericentral, primary KRAYESKY D.M., NORRIS J.N. & FREDERICQ S. 2011. The Caloglossa leprieurii complex (Delesseriaceae, Rhodophyta) in the Americas: the elucidation of overlooked species based on molecular and

. morphological evidence. Cryptogamie, Algologie 32: 37–62. KU¨ TZING F.T. 1843. Phycologia generalis. . . . Leipzig. 458 pp., 80 pls. KYLIN H. 1924. Studien u¨ber die Delesseriaceen. Lunds Universitets A˚ rsskrift, NyFo¨ljd, Andra Afdelningen, vol. 20(6). Lund Univer-

& comb. nov sity, Lund. 111 pp. second-order cells YLIN type 1allpericentral, type 2 some type 2 all type 2 some type 1/type 2 all/some type 2 some Wynneophycus gen. 1K 2H. 1956. Die Gattungen 1 der Rhodophyceen. CWK multilayers Gleerup, multilayers (except Lund. 673 pp. LEE Y. 2008. Marine algae of Jeju. Academy Publication, Seoul. 477 pp.

1 LEE Y. & KANG S. 2001. A catalogue of the seaweeds in Korea. Cheju National University Press, Jeju, Korea. 662 pp. LIN S.-M., FREDERICQ S. & HOMMERSAND M.H. 2001. Systematics of the Delesseriaceae (Ceramiales, Rhodophyta) based on large subunit rDNA and rbcL sequences, including the Phycoryoideae, Comparisons among genera belonging to some genera of Delesserioideae and subfam. nov. Journal of Phycology 37: 881–899. LIN S.-M., FREDERICQ S. & HOMMERSAND M.H. 2012. Molecular Type 1: all cells of second-order rows bear third-order rows; type 2: only some cells of second-order rows bear third-order rows. blade margin tetrasporangia layers phylogeny and developmental studies of Apoglossum and Para- 1 Third-order cells reaching Male soriTetrasporangial soriOrigin cells of continuous sori paired paired/continuous sori sori paired/continuous sori paired sori striae/continuous sori paired sori paired/continuous sori continuous sori paired/striae sori striae/paired sori continuous sori Origin of new blades endogenously endogenously endogenously and Table 2. Tetrasporangial sori: References this study Wynne (1984b); Branching patternCorticationLateral veinsMarginal rhizoidsIntercalary cell divisionApical opposite organization absent abundant present absent simple absent present present present alternate present absentglossum absent absent(Delesseriaceae, alternate present absent present present Rhodophyta) alternate/opposite absent present/absentwith present opposite absent a description absent of present present present Jeong et al.: Wynneophycus geminatus gen. & comb. nov. 177

Apoglosseae trib. nov. European Journal of Phycology 47: 366– WOMERSLEY H.B.S. & SHEPLEY E.A. 1982. Southern Australian 383. species of Hypoglossum (Delesseriaceae, Rhodophyta). Australian LIU R. 2008. Checklist of biota of Chinese seas. Science Press, Journal of Botany 30: 321–346. Academia Sinica, Beijing. 1267 pp. WYNNE M.J. 1983. The current status of genera in the Delesser- MAGGS C.A. & HOMMERSAND M.H. 1993. Seaweeds of the British iaceae (Rhodophyta). Botanica Marina 26: 437–450. Isles. Vol. 1 Rhodophyta. Part 3A Ceramiales. The Natural WYNNE M.J. 1984a. The correct name for the type of Hypoglossum History Museum. HMSO Publications, London. 444 pp., 1 map. Ku¨tzing (Delesseriaceae, Rhodophyta). Taxon 33: 85–87. MIKAMI H. 1987. On Erythroglossum pulchrum Yamada and WYNNE M.J. 1984b. The occurrence of Apoglossum and Delesseria Hypoglossum sagamianum Yamada (Rhodophyta, Delesseria- (Ceramiales, Roodphyta) in South Africa. South African Journal ceae). Japanese Journal of Phycology 35: 124–129. of Botany 3: 136–145. MILLAR A.J.K. 1999a. Marine benthic algae of North East Herald WYNNE M.J. 1988. A reassessment of the Hypoglossum group Cay, Coral Sea, South Pacific. Proceedings of the International (Delesseriaceae, Rhodophyta), with a critique of its genera. Seaweed Symposium 16: 65–74. Helgola¨nder Meeresuntersuchungen 42: 511–534. MILLAR A.J.K. 1999b. Marine benthic algae of Norfolk Island, WYNNE M.J. 1994. The description of Hypoglossum subsimplex sp. South Pacific. Australian Systematic Botany 12: 479–547. nov. (Delesseriaceae, Rhodophyta) from the Florida Keys, Gulf NAM K.W. & KANG P.J. 2012. Algal flora of Korea. Volume 4, of Mexico. Cryptogamie, Algologie 15: 253–262. Number 7 Rhodophyta: Florideophyceae: Ceramiales: Delesseria- WYNNE M. J. 1996. A revised key to genera of the red algal family ceae: 22 genera including . National Institute of Delesseriaceae. Beihefte zur Nova Hedwigia 112: 171–190. Biological Resources, Incheon, Korea. 129 pp. WYNNE M.J. 2014 [‘2013’]. The red algal families Delesseriaceae and OAK J.H., PARK M.R. & LEE I.K. 2002. Taxonomy of Hypoglossum Sarcomeniaceae. Koeltz Scientific Books, Ko¨nigstein, Germany. (Delesseriaceae, Rhodophyta) from Korea. Algae 17: 21–31. 326 pp. OKAMURA K. 1908. Icones of Japanese algae, vol. 1(1). Kazama- WYNNE M.J. & BALLANTINE D.L. 1986. The genus Hypoglossum shobo, Tokyo. 258 pp. Ku¨tzing (Delesseriaceae, Rhodophyta) in the tropical western PAPENFUSS G.F. 1937. The structure and reproduction of Atlantic, including H. anomalum sp. nov. Journal of Phycology 22: multifida, Vanvoorstia spectabilis and Vanvoorstia coccinea. 185–193. Symbolae Botanicae Upsalienses 2(4): 1–66. WYNNE M.J. & DANIELS K. 1966. Cumathamnion, a new genus of the PAPENFUSS G.F. 1944. Structure and taxonomy of Taenioma, Delesseriaceae (Rhodophyta). Phycologia 6: 13–28. including a discussion of the phylogeny of the Ceramiales. WYNNE M.J. & DE CLERCK O. 2000. Taxonomic observations of Madron˜o 7: 193–214. Hypoglossum (Delesseriaceae, Rhodophyta) in the Indian Ocean PAPENFUSS G.F. 1961. The structure and reproduction of Caloglossa and Malayan region, including the description of two new species. leprieurii. Phycologia 1: 8–31. Cryptogamie, Algologie 21: 111–131. RONQUIST F. & HUELSENBECK J.P. 2003. MrBayes 3: Bayesian WYNNE M.J. & KRAFT G.T. 1985. Hypoglossum caloglossoides sp. phylogenetic inference under mixed models. Bioinformatics 19: nov. (Delesseriaceae, Rhodophyta) from Lord Howe Island, 1572–1574. South Pacific. British Phycological Journal 20: 9–19. SOUTH G.R. & SKELTON P.A. 2003. Catalogue of the marine benthic WYNNE M.J. & SAUNDERS G.W. 2012. Taxonomic assessment of macroalgae of the Fiji Islands, South Pacific. Australian North American species of the genera Cumathamnion, Delesseria, Systematic Botany 16: 699–758. Membranoptera and Pantoneura (Delesseriaceae, Rhodophyta) STEGENGA H., ANDERSON R.J. & BOLTON J.J. 2001. Hypoglossum using molecular data. Algae 27: 155–173. imperfectum nov. spec. (Rhodophyta, Delesseriaceae), from the YOSHIDA T. 1998. Marine algae of Japan. Uchida Rokakuho South African south coast. Botanica Marina 44: 157–162. Publishing Co., Ltd, Tokyo. 1222 pp. (in Japanese) TAMURA K., PETERSON D., PETERSON N., STECHER G., NEI M. & YOSHIDA T. & MIKAMI H. 1986. Observations on morphology of KUMAR S. 2011. MEGA5: molecular evolutionary genetics Hypoglossum minimum Yamada and H. geminatum Okamura analysis using maximum likelihood, evolutionary distance, and (Delesseriaceae, Rhodophyta). Japanese Journal of Phycology 34: maximum parsimony methods. Molecular Biology and Evolution 177–184. 28: 2731–2739. YOSHIDA T., NAKAJIMA Y. & NAKATA Y. 1990. Check-list of marine THOMPSON J.D., HIGGINS D.G. & GIBSON T.J. 1994. Clustal W: algae of Japan (revised in 1990). Japanese Journal of Phycology improving the sensitivity of progressive multiple sequence 38: 269–320. alignment through sequence weighting, position-specific gap ZHENG B., LIU Z. & CHEN Z. 2001. Flora algarum marinarum penalties and weight matrix choice. Nucleic Acids Research 22: sinicarum Tomus II Rhodophyta No. VI Ceramiales (I). Science 4673–4680. Press, Beijing. 159 pp. WAGNER F.S. 1954. Contributions to the morphology of the ZHENG Y. 1998. Hypoglossum fujianensis sp. nov. (Delesseriaceae, Delesseriaceae. University of California Publications in Botany Rhodophyta) from Fujian coast, China. Chinese Journal of 27: 279–346. Oceanology and Limnology 16: 369–372. WOMERSLEY H.B.S. 2003. The marine benthic flora of southern Australia. Rhodophyta – part IIID. Ceramiales-Delesseriaceae, Sarcomeniaceae, Rhodomelaceae. Australian Biological Resources Study and the State Herbarium of South Australia, Canberra. 533 Received 31 August 2015; accepted 22 November 2015 pp. Associate Editor: Mariana Cabral Oliveira