INTRODUCTION

The crustose brown algal genus, Petroderma, was described by Kuckuck in 1897. It is characterized by having small dark brown confluent spots, single-layer basal cells giving rise to erect filaments that easily separate by pressure, one plate-shaped chromatophore per cell, unilocular and plurilocular sporangia arising from the transformation of the top vegetative cells, unilocular sporangia on erect filaments without paraphyses, successive production of unilocular sporangia on the same filament results in collar-like remnants of old sporangial walls along the filament, multirow erect filament plurilocular sporangia on a terminal position (Kuckuck 1897;Waern 1949;Boraso de Zaixso 2013). Currently, three Petroderma species are recognized from worldwide (Guiry and Guiry 2020): P. maculiforme (Wollny) Kuckuck from Helgoland Island, German (Kuckuck 1897;Athanasiadis 1996), P. steinitziiRayss et Dor (1963) from Eilat, Israel, and P. vietnamensisPham-Hoàng (1969) from Vietnam. None of these has been reported in Korea even if P. maculiforme has a wide distribution range.

The genus Hincksia was first described by Gray (1864) and characterized by thalli having secundly branched frond, intercalary growth, discoid chloroplast with a pyrenoid, unilocular and plurilocular sporangia produced from the adaxial side of the lateral branches (Gray 1864; Womershttps ley 1987; Guiry and Guiry 2020). Currently, twenty-nine Hincksia species are recognized from worldwide (Guiry and Guiry 2020). Of them, H. granulosa (Smith) P.C. Silva, H. onslowensis (Amsler & Kapraun) P.C. Silva, H. ovata (Kjellman) P.C. Silva (Silva et al. 1996), H. sandriana (Zanardini) P.C. Silva, and H. secunda (Kützing) P.C. Silva have been reported in Korea (Kim 2010;Guiry and Guiry 2020).

We collected some unidentified crustose and filamentous brown alga along the coasts of Korea. We observed their detailed morphology and analyzed the phylogenetic relationship based on the rbcL. We add Petroderma maculiforme and Hincksia sordida to the Korean marine algal inventory.

MATERIALS AND METHODS

1. Morphology

Samples of Petroderma maculiforme were collected from the west (Byeonsan) coast of Korea. They were sorted into voucher specimens, air-dried, and preserved in silica gel for morphological and molecular analyses. 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 a freezing microtome (Shandon Cryotome FSE, Thermo Shandon, Ltd., Loughborough, UK), stained in a 1 to 1 mixture of aqueous aniline blue and acetic acid. Samples of Hincksia sordida were collected from southeastern (Gampo) coast of Korea. They were sorted into voucher herbarium specimens, silica gel samples, and formalin samples. Formalin samples were preserved in 4-5% formalin/seawater. Silica gel samples were used for molecular analyses. Formalin sample was stained with aniline blue and morphological observations carried out. 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). PCR amplification was carried out with a Veriti 96 well Thermal cycler (Applied Biosystems, Foster city, USA). PCR products were purified using a PCRquick-spinTM PCR product purification kit (iNtRON Biotechnology, Inc, Seongnam, Korea). New rbcL sequences obtained from Petroderma maculiforme and Hincksia sordida and been deposited in EMBL/ GenBank under the accession numbers MT023108 (CUK19777) and MT469950 (CUK18158) respectively. For P. maculiforme molecular analysis, eighteen rbcL sequences (1069 bp) including sequences from GenBank and two outgroup taxa were aligned using ClustalW (Thompson et al. 1994). Canistrocarpus cervicornis (Kützing) De Paula and De Clerck and Dictyota dichotoma (Hudson) J.V. Lamouroux have been chosen as outgroup. For H. sordida, twenty-three rbcL sequences (1300 bp) were aligned using ClustalW. Hincksia granulosa and H. sandriana were included in this study for molecular analyses as MT569437 (CUK18909) and MT469949 (CUK18929) respectively. Asterocladon rhodochortonoides and A. interjectum selected as outgroups. 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

Family Petrodermataceae Silberfeld, F. Rousseau et Reviers, 2014 납작패과 (신칭) Genus PetrodermaKuckuck, 1897 납작패속 (신칭)

Petroderma maculiforme (Wollny) Kuckuck, 1897: 382 원반납작패 (신칭) (Figs. 1, 2)

Basionym: Lithoderma maculiformeWollny 1881. Hedwigia. 20: 31.

Heterotypic Synonym: Lithoderma lignicolaKjellman 1883. Vega-expeditionens Vetenskapliga Iakttagelser. 3: 318.

Material examined. CUK19777 (=MBRB0104TC19 777S1) & NIBROR0000001764 (National Institute of Biological Resources); Chaeseokang, Byeongsan-myeon, Buan-gun, Jeollabuk-do, Korea (35°37′38.18″N, 126°28′ 05.52″E); July 26, 2019; T.O. Cho and B.Y. Won.

Morphological observations. Plants are small irregular epilithic crusts (Fig. 1A), light to dark brown color, a few millimeters to 1 cm diam., gelatinous but adherent to the substratum. Short multicellular rhizoids were rarely observed. Thalli are 139-175 μm high, composed of a monostromatic basal layer and erect filaments (Fig. 1B), easily separate with pressure (Fig. 1C). They are mostly composed of up to 11 cells. Each cell contains a single parietal chromatophore (Fig. 1D). Hairs are infrequent, developed from terminal of erect filaments, and not arranged in clusters (Fig. 1E). Unilocular sporangia are on the terminal of erect filaments, spherical to cylindrical, 20-35 μm long, 10-15 μm wide, without associated paraphyses (Fig. 1F). Plurilocular sporangia (Fig. 1G) are on terminal of erect filaments, without associated paraphyses. Some plurilocular sporangia are elongate uniseriate with occasional oblique or longitudinal partitions and not wider than erect filaments while others are short wider than erect filaments and may be branched.

Habitat. Epilithic at the intertidal zone.

World distribution. North America: Alaska, California, Maine, New Hampshire, Oregon, and Washington; Arctic: Svalbard (Spitsbergen). Europe: Britain, Denmark, Germany, Helgoland, Ireland, Norway, Scandinavia, and Spain. Atlantic Islands: Greenland and Iceland. Central America: Mexico. South America: Argentina; Asia: Russia (Far East). Antarctic: King George Islands (Guiry and Guiry 2020).

Phylogenetic analyses. The 1069-nucleotide portion of rbcL was aligned for Petroderma maculiforme. Phylogenetic analyses revealed that our Petroderma sample from Korea was nested in a clade of Petroderma maculiforme (Fig. 2) and it revealed that the gene sequence divergence between California and Korean samples of Petroderma maculiforme is 1.0%.

Remarks. Sample of Petroderma maculiforme from Korea is matched into original description of P. maculiforme (Wollny) Kuckuck. Our P. maculiforme differs from P. steinitzii by having a monostratos basal disc giving rise to assimilatory filaments and uni- or plurilocular sporangia or hairs developed terminally on assimilatory filaments. Petroderma steinitzii has a three-layer basal disc and unilocular sporangia, plurilocular sporangia, or hairs developed from cells of basal disc (Rayss and Dor 1963). Also, although Petroderma maculiforme has more similarities with P. vietnamensis in having a monostratos basal disc giving rise to assimilatory filaments, it differs from P. vietnamensis by being epilithic and having branched assimilatory filaments (Kuckuck 1897;Edelstein and McLachlan 1969;Boraso de Zaixso 2013). Petroderma vietnamensis has unbranched (simple) assimilatory filaments and smaller unilocular sporangia (Pham-Hoàng 1969).

Our molecular analysis based on rbcL gene reveals that sample from Korea is placed in the same clade with Petroderma maculiforme from California, USA (EU579934, Bittner et al. 2008). Although there was no rbcL sequence from the type locality (Helgoland) in GenBank, Our Petroderma sample from Korea is identified as Petroderma maculiforme because P. maculiforme from California was recognized into identical species with type locality based on ITS1 sequences (Peters and Moe 2001). In this study, we report Petroderma maculiforme as a new record from Korea and add this species to the list of Korean macroalgal flora based on morphological and molecular analyses.

Hincksia sordida (Harvey) P.C. Silva, 1987 깃대긴털실말 (신칭) (Figs. 3, 4)

Basionym: Ectocarpus sordidusHarvey 1859: 294 Homotypic synonym(s): Ectocarpus sordidusHarvey 1859;Giffordia sordida (Harvey) M.N. Clayton 1974.

Material examined. CUK18158 (=MBRB0107TC181 58H1) & NIBROR0000001765 (National Institute of Biological Resources); Gampo, Gampo-eup, Gyeongju-si, Gyeongsangbuk-do, Korea (35°48′23.74″N, 129°30′ 22.48″E); July 09, 2017; T.O. Cho and S.Y. Jeong.

Morphological observations. Plants are epiphytic (on larger algae or seagrasses), free-floating, or rarely epilithic, forming more or less extensive, entangled, 5-30 cm long, with sparsely branching laterals (Fig. 3A). Main filaments are spirally or irregularly branched with many short and curved laterals, 31-52 μm wide (Fig. 3B). Rhizoids are occurring throughout the plant (Fig. 3C). Growth is from scattered meristematic regions in axes and main branches (Fig. 3D). Branchlets are tapered and terminated into pseudohairs (Fig. 3E), 8-15 μm wide. Vegetative cells have a few or inconspicuous physodes and numerous discoid plastids (Fig. 3F) with one(-2) pyrenoid. Plurilocular sporangia are rare, scattered or occasionally grouped, sessile (or rarely pedicellate), conical, 52-68 μm long and 22-38 μm wide. Unilocular sporangia are rare, on separate plants, scattered, sessile, ovoid, 39-44 μm long, and 24-32 μm wide.

Habitat. Epiphytic on larger algae (e.g. Sagarssum spp.) or seagrasses and confined to calm rock pools or to sheltered bays and inlets.

World distribution. Asia: China, Australia, and New Zealand (Guiry and Guiry 2020).

Phylogenetic analyses. The 1300-nucleotide portion of rbcL was aligned for Hincksia sordida. Phylogenetic analyses revealed that our sample of Hincksia sordida from Korea was nested within a clade of Hincksia (Fig. 4). Hincksia hincksiae was a sister species with Hincksia sordida and the gene sequence divergence between them was is 3.6%.

Remarks. Morphologically, our Hincksia samples are matched into the description of Hincksia sordida. Hincksia sordida is morphologically distinct from other reported Hincksia species from Korea. Hincksia sordida is mainly distinguished by having many short curved laterals and rhizoids scattered on the main axis and large branches (Womersley 1987). Molecular data based on rbcL gene revealed that Hincksia sordida from Korean is nested in the same clade of the genus Hincksia and distinguished from congeners, H. granulosa, H. sandriana, and H. hincksiae. In this study, we report Hincksia sordida as a new record from Korea and add this species to the list of Korean macroalgal flora based on morphological and molecular analyses.