Journal 
INTRODUCTION
The crustose brown algal genus, Ralfsia, was described by Berkely (in Smith and Sowerby 1843). It is characterized by having thalli crustose, flat and expanded, more or less circular in outline, attached to the substratum with or without rhizoids; erect vegetative filaments tightly adherent, straight or curved, branched or simple, arising from a basal layer; unilocular sporangia clavate, ovoid to pyriform, sessile or pedicellate arising from the supporting cells of paraphysis; plurilocular reproductive organs intercalary, arising from a cell located in the submerged part of the erect filament, composed of one to four rows of plurilocular reproductive filaments with one to several terminal sterile cells; chloroplasts mostly plate-shaped, generally one per cell; hairs in tufts, scattering over thallus (Setchell and Gardner 1924;Hamel 1931-1939;Hollenberg 1969;Abbott and Hollenberg 1976;Tanaka and Chihara 1980;Fletcher 1987;Womersley 1987;Parente and Saunders 2019). Currently, 17 Ralfsia species are recognized worldwide (Guiry and Guiry 2020). Of them, four Ralfsia species were reported from Korea: Ralfsia fungiformis (Gunnerus) Setchell and N.L. Gardner, R. verrucosa (Areschoug) Areschoug, R. confusa Hollenberg, and R. integra Hollenberg (Lee and Kang 1986;Lee and Kang 2002;Keum 2010).
Ralfsia longicellularis was described from Peter the Great Bay, Far East Russia by Perestenko in 1980. It is mainly characterized by a thick dark brown thallus, curved cells in the creeping and ascending parts of the filaments forming a unilateral symmetry, basal layer and erect filament cells’ width to length ratio of 1 : 1.5-10, and narrow-shaped sporangia on stalk cells at the base of paraphyses (Perestenko 1980). Since its description, R. longicellularis has only one report worldwide (Guiry and Guiry 2020). In the analysis of the biogeographical structure of flora in Vostok Bay, Sea of Japan, it was reported as part of Asian low-boreal species (A lB) (Kozhenkova 2009). This may allude to the fact that R. longicellularis is endemic to the northern region of Far East Asia.
We collected the unidentified samples that fit the genus Ralfsia’s description, from coasts of Korea. We observed their detailed morphology and analyzed molecular data based on the plastid-encoded rbcL gene for their phylogenetic relationships. In this study, we add Ralfsia longicellularis` to the Korean marine algal inventory.
MATERIALS AND METHODS
1. Morphology
Samples of Ralfsia were collected from shallow intertidal zones of Korea (Taean, Buan, and Wando) and Russia (Vladivostok). Vouchers were air-dried and preserved in silica gel for morphological and molecular studies. For morphology, samples were detached from substrate by use of a single-edged blade, then embedded in a matrix (O.C.T., CellPath, Ltd., Newtown, Wales, UK) and sectioned (8- 10 μm thickness) using freezing microtome (Shandon Cryotome FSE, Thermo Shandon, Ltd., Loughborough, UK), stained in a 1 : 1 mixture of aqueous aniline blue and acetic acid. Photomicrographs were taken using an Olympus DP71 camera mounted on an Olympus microscope (BX51TRF; Olympus, Tokyo, Japan) and a digital camera (Nikon D40; Nikon, Japan). Representative voucher specimens examined in this study are deposited in the herbarium of Chosun University (CUK) and the National Institute of Biological Resources (NIBR), Korea.
2. Molecular study
Genomic DNA was extracted using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). The extracted DNA was stored at -20°C and used to amplify rbcL. The rbcL gene was amplified using the primer combinations NDrbcL2-DRL1R and DRL2F-R3A (Kogame et al. 1999;Hwang et al. 2005) with HelixAmp Ready-2x-Go Series (NanoHelix Co., Ltd., Daejeon, Korea). All PCR amplifications were carried out with a Veriti 96 well Thermal cycler (Applied Biosystem). PCR products were purified using a PCRquick-spinTM PCR product purification kit (iNtRON Biotechnology, Inc, Seongnam, Korea). New rbcL sequences obtained from Ralfsia longicellularis have been deposited in EMBL/GenBank under the accession numbers MT000915 (CUK18752), MT000917 (CUK18820), MT000918 (CUK19216), MT000916 (CUK19269) and MT000914 (CUK20024).
Nineteen rbcL sequences (781 bp) were aligned, including sequences of other species of Ralfsiales previously published in GenBank and two outgroup taxa, Tilopteris mertensii (Turner) Kützing and Sargassum muticum (Yendo) Fensholt, with ClustalW (Thompson et al. 1994). Phylogenetic analyses were conducted using raxmlGUI1.5b2 (Silvestro and Michalak 2012). Maximum likelihood analyses were conducted using the GTR+G+I model, with 1,000 bootstrap replicates. Bayesian inference was performed using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003). Markov chain Monte Carlo runs were conducted for 2 million generations, each with one cold chain and three heated chains using the GTR+Γ+I evolutionary model and sampling and printing every 1,000 generations. Summary trees were generated using a burn-in value of 800.
RESULTS AND DISCUSSION
1. Morphological observations
Class Phaeophyceae F.R. Kjellman, 1891 갈조강
Order Ralfsiales Nakamura ex P.-E. Lim & H. Kawai in Lim et al., 2007 바위딱지목
Family Ralfsiaceae W.G. Farlow, 1881 바위딱지과
Genus Ralfsia M.J. Berkeley, 1843 바위딱지속
Ralfsia longicellularis L. P. Perestenko, 1980 긴세포바위딱지 (신칭) (Fig. 1A-E)
Material examined. NIBROR0000001763 (deposited in the National Institute of Biological Resources), CUK19216 (=MBRB0103TC19216D1), MT000918, Pado-ri, Sowuon- myeon, Taean-gun, Chungcheongnam-do, Korea (36°43′05.65″N, 126°07′33.79″E), November 3, 2018, T.O. Cho and B.Y. Won, at 1 m depth by hand; CUK18752 (=MBRB0103TC18752D1), MT000915, Pado-ri, Sowuon- myeon, Taean-gun, Chungcheongnam-do, Korea (36°43ʹ05.65ʺN, 126°07ʹ33.79ʺE), March 3, 2018, T.O. Cho and B.Y. Won, at 1 m depth by hand; CUK18820 (=MBRB0103TC18820D1), MT000917, Jeongdo-ri, Wando-eup, Wando-gun, Jeollanam-do, Korea (34°17ʹ 50.04ʺN, 126°42ʹ08.14ʺE), April 1, 2018, T.O. Cho and B.Y. Won, at 1 m depth by hand; CUK19269 (=MBRB 0103TC19269D1), MT000916, Chaeseokang, Byeonsanmyeon, Buan-gun, Jeollabuk-do, Korea (35°37ʹ27.04ʺN, 126°27ʹ56.88ʺE), January 26, 2019, T.O. Cho and B.Y. Won, at 1 m depth by hand; CUK20024 (=MBRB0103 TC20024D1), MT000914, Ulitsa Leytenanta Shmidta, Vladivostok, Russia (43°06ʹ21.78ʺN, 131°52ʹ11.07ʺE), October 10, 2019, T.O. Cho and B.Y. Won, at 1 m depth by hand.
Description. Thallus thick, crustose, epilithic, dark brown (almost black in the dry state), with uneven surface, may have overlapping lobes (Fig. 1A, arrows) at margins, several centimeters across, 770-1200 μm thick, undersurface irregularly distributed with rhizoids 8.4-14 μm wide. In the creeping and ascending parts of the filaments (Fig. 1B, bottom), the cells are curved, often with oblique partitions, 8.5-11(14) μm width, with a ratio of width to length 1 : 1.5-7, forming horizontal rows. The lower layer of creeping ascending filaments is 0.3 times the thickness of the thallus. The cells of vertical filaments (Fig. 1B, middle) are cylindrical 5.5-8.5 μm width, with a ratio of width to length 1 : 1.5-10. Cells of the upper third (Fig. 1B, top) of vertical filaments taper to the top with 4-8 μm width and a ratio of width to length 1 : 1-5. Chloroplasts (Fig. 1C, arrows) parietal, single per cell, mostly on the apical part of the cell. Reproductive portions of sori (Fig. 1D, arrowhead), scattered throughout the thallus. Narrow-shaped unilocular sporangia, on 1-2(3) stalk cells, 22.5-31×65- 92 μm. Paraphyses of 10-13 cells, 190-210 μm long. Apical cells of paraphysis 8.5-10 μm wide. Plurilocular reproductive organs 90-265 μm long, uniseriate, occasionally biseriate, capped with one (Fig. 1E, asterisk) to two (Fig. 1E, arrowheads) sterile cells. Sterile cell 5-8 μm width and 1.8-3 times as long as the width. Hair unknown.
Habitat. Epilithic at intertidal zone.
World Distribution. Asia; Russia (Far East) (Guiry and Guiry 2020) and Korea.
Identifier. Antony Otinga Oteng’o and Boo Yeon Won.
2. Phylogenetic analyses
The 781 rbcL bp portion was sequenced for Ralfsia longicellularis. Phylogenetic analyses revealed that both Korean and Russian R. longicellularis sequences were placed within the same clade (Fig. 2). The analyses revealed R. longicellularis differs from its nearest neighbor R. tenebris by 7.7- 7.9% and the generitype, R. fungiformis, by 8.7-8.8% gene sequence divergence. Intra-specific genetic divergence within R. longicellularis was 0.0-1.0%.
Remarks.Ralfsia longicellularis was originally described by Perestenko (1980) from Russia. This species has only been described and reported (Kozhenkova 2009) from Russia. Most characteristics from the protologue of R. longicellularis from Peter the Great Bay, Russia (Perestenko 1980) are an exact match with our Korean samples. However, the protologue had unilocular sporangia, while Korean samples presented plurangial reproductive structures. This shows that both types of reproductive organs are borne on separate thalli. The original description had no molecular analysis. Our study included molecular analyses. The rbcL genetic divergence between Korean samples and those from Vladivostok (within Peter the Great Bay) was 1%. Therefore, Korean samples are the same species as Russian ones. We add this species as Ralfsia longicellularis in list of Korean macroalgal flora.
