INTRODUCTION

Kang (1966) reported 403 species in the catalogue of Korean marine algal flora. Since then, many species have been added to the Korean algal inventory though monographic and floristic researches (Lee and Kang 2001; Lee 2008). Until now, approximately 900 species have been recorded in Korean marine algal flora (Kim et al. 2013).

Genus Bryopsis Lamouroux occurs mostly in temperate regions (Womersley 1984). Bryopsis is characterized by erect thalli of several centimeters height and one to several distinct axes with pinnate to radial laterals. Branches and ramuli are basally constricted, cylindrical to slightly tapering (Lee et al. 1991). Twelve species of Bryopsis were reported in Korea. Most of them, however, have been recorded as floristic listings without taxonomic consideration.

Genus Pyropia J. Agardh was resurrected for a large group of Porphyra according to morphological and molecular data by Sutherland et al. (2011). Currently, 14 Pyropia species are recognized in Korea. Pyropia species has been known as Porphyra species in Korea. However, Pyropia is distinguished from Porphyra by cells in the distal part of the blade with small appendages (Sutherland et al. 2011).

We collected one green algal and one red algal species not listed in the Korean marine algal inventory. In this study, we report them as the first records from Korea based on integrated morphological-molecular study: Bryopsis africana Areschoug; Pyropia kinosiate (Yamada and Tanaka) N. Kikuchi, M. Miyata, M.S. Hwang & H.G. Choi.

Of them, Bryopsis africana is an introduced species on the coast of Korea and that its distribution now extends to the Pacific Ocean.

METERIALS AND METHODS

1.Morphology

Plants were collected from coasts of Korea. Samples were preserved in 4-5% formalin/seawater for morphological examination. Microscope observations were made by using materials stained with 1% aqueous aniline blue acidified with 0.1% diluted HCl. Photomicrographs were taken using an Olympus BX51TRF microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP71 camera. Representative specimens examined in this study are deposited in the National Institute of Biological Resources (NIBR) and herbarium of Chosun University (CUK), Korea.

2.Molecular study

Samples were cleaned in autoclaved seawater under a dissecting microscope and were preserved in silica gel for molecular analysis. Genomic DNA was extracted using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany), following manufacturer’s instructions. Polymerase chain reaction (PCR) was conducted in 30 μL reactions that contained 4-8 μL genomic DNA or 10 μL diluted genomic DNA (1 : 10), 1 μL 10 pmols forward and reverse primers, and HelixAmp Ready-2x-Go Series (NanoHelix Co., Ltd., Daejeon, Korea). The rbcL was amplified using primer combinations F57-R1150 and F993-RrbcStart (Freshwater and Rueness 1994) and purified with PCRquick-spinTM PCR product purification kit (iNtRON Biotechnology, Inc. Seongnam, Korea). Cycle sequencing was conducted with amplification primers. Sequences were determined by using an ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA). The rbcL sequences generated in this study were deposited in EMBL/GenBank under accession numbers MF668137 for Bryopsis africana and MF668138 for Pyropia kinosiate. Trichosolen myura (AF212144) was selected as outgroup for Bryopsis. Porphyra umbilicalis (JN847246) was selected as outgroup for Pyropia. These sequences and other nucleotide sequences from GenBank were aligned using Clustal W (Thompson et al. 1994) and manually corrected using MEGA5 (Tamura et al. 2011). Maximum likelihood analyses were conducted in MEGA5, using 1000 bootstrap replications and the GTR+Γ+I model, and Bayesian inference was conducted using MrBayes 3.1.2 (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

Class Ulvophyceae Mattox & Stewart, 1984 갈파래강

Order Bryopsidales J.H. Schaffner, 1922 깃털말목

Family Bryopsidaceae Bory, 1829 깃털말과

Genus Bryopsis J.V. Lamouroux, 1809 깃털말속

Bryopsis africanaAreschoug, 1851 아프리카깃털말 (신칭) (Fig. 1A-H)

Synonym:Bryopsis tenuisLevring 1938.

Material examined: NIBRAL0000148974 (deposited in the National Institute of Biological Resources), CUK12722 (36°09′32.28″N, 126°35′18.34″E), Joong-ri, Bogil-myeon, Bogil Island, Wando-gun, Jeollanam-do, July 27, 2015, Cho and Jeong, at 1 m depth by hand.

Description: Plants light green, robust, caespitose, forming clumps, and 1-3 cm high (Fig. 1A), epiphytic on other algae (Fig. 1B), with few to numerous axes arising from a branched rhizoidal system (Fig. 1C). Axes branched pinnately and spirally, with rounded apex (Fig. 1D-F). Main axes 66-92 μm in diameter, branchlets 24-32 μm in diameter. Branches of main axes typically developed into secondary branched axes as they mature, resulting in a highly branched plant. Plug of lateral branches completely closed, attached on the side of axes (Fig. 1G). Multinucleate cell with thin parietal layer of cytoplasm (Fig. 1H).

Habitat: Epiphytic on other algae at intertidal zone.

World Distribution: Korea, Namibia, South Africa.

Deposition: Collected specimens deposited in the National Institute Biological Resources (NIBR) and herbarium of Chosun University (CUK), Korea.

Identifier: Tae Oh Cho and So Young Jeong.

Phylogenetic analyses: The 550 rbcL bp portion of the 1467-bp rbcL gene (37.4% sequences) sequenced for Bryopsis africana. Phylogenetic analyses revealed that B. africana was placed within a clade of Bryopsis (Fig. 2). Bryopsis africana differs from B. corymbosa by 2.7% gene sequence divergence respectively.

Remarks:Bryopsis africana was first described from South Africa by Areschoug (1851). This is a well-known species in the Indian Ocean. In this study, we report as a new record from Korea and extend its distribution into Northeast Asia. Bryopsis africana was collected on various different algae like Corallina, Gracilaria, and Hypnea species. Bryopsis africana is distinguished from other species within the genus with distinct main axes, robust thalli, pinnately and radially branched above, basally denuded although it is similar with B. plumosa in habit of thalli.

Class Bangiophyceae Wettstein, 1901 김파래홍조강

Order Bangiales Nägeli, 1847 김파래목

Family Bangiaceae Duby, 1830 김파래과

Genus Pyropia J. Agardh, 1899 돌김속

Pyropia kinositae (Yamada & T. Tanaka) N. Kikuchi, M. Miyata, M.S. Hwang & H.G. Choi, 2011 더부살이돌김 (신칭) (Fig. 3A-J)

Synonym:Porphyra yezoensis f. kinositae Y. Yamada & T. Tanaka in Tanaka, 1952.

Material examined: NIBRAL0000152869 (deposited in the National Institute of Biological Resources), CUK9265 (38°09′58.31″N, 128°36′32.50″E), Seolak harbor, Daepodong, Sokcho-si, Gangwon-do, February 1, 2013, Cho and Jeong, at 1 m depth by hand.

Description: Thalli membranous, foliose, erect, oblong, lanceolate to narrow elliptic, 1-4 cm high, 1-3 cm wide, purplish red in color (Fig. 3A, B), attached to the substratum by means of basal small discoid holdfast (Fig. 3C). Holdfast composed of specialized rhizoidal cells. Rhizoidal cell internally developed by transformation of vegetative cells in lower portion of thalli, with long tapering elongate tails (Fig. 3D). Blade margin undulate but not laciniate (Fig. 3E). Vegetative cells angular with rounded angles in surface view, 8-14 μm diameter, irregularly arranged, usually twice as long as broad (Fig. 3E, F). Blades monostromatic, 13-17 μm thick in vegetative portion (Fig. 3F). The sporocarps perpendicular to the surface (Fig. 3G), changed into 8 carpospores after dividing according to the formula 16 (a/2, b/2, c/2) (Fig. 3H). Spermatangia containing 64 antherozoids each after dividing according to the formula 64 (a/2, b/4, c/8) (Fig. 3I, J).

Habitat: Epilithic on the rock at intertidal zone.

World Distribution: Korea, Japan.

Deposition: Collected specimens deposited in the National Institute Biological Resources (NIBR) and herbarium of Chosun University (CUK), Korea.

Identifier: Tae Oh Cho and So Young Jeong.

Phylogenetic analyses: The 1386 rbcL bp portion of the 1467-bp rbcL gene (94.4% sequences) sequenced for Pyropia kinositae. Phylogenetic analyses revealed that P. kinositae was placed within a clade of Pyropia (Fig. 4). Pyropia kinositae differs from P. tenera by 1.5-1.8%, P. ishigecola by 1.2-1.3%, and P. yezoensis by 1.5-1.8% gene sequence divergence respectively.

Remarks: This species was originally described by Yamada et Tanaka (1952) as Porphyra yezoensis f. kinostiae from Utasutsu, Suttsu, Hokkaido of Japan. Sutherland et al. (2011) transferred it into genus Pyropia based on molecular data using nSSU and rbcL genes. Pyropia kinositae is recognized by lanceolate to narrow elliptic cuneate shape of foliose thalli, presence of conchospore and archeospore, absence of neutral spore, and having clear patches of spermatangial sori (Tanaka 1952; Kikuchi et al. 2010). We collected this species growing on rocks at intertidal zones at Sokcho on eastern coasts of Korea. Our morphological observations agreed with the description of Tanaka (1952) and Kikuchi et al. (2010). We add this species as new record species to the list of Korean algal flora.