New Records of Intertidal Marine Algae from Korea

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So Young Jeong, Danilo E. Bustamante, Jin Gyo Lee, Boo Yeon Won, Seung Hee Kim and Tae Oh Cho

354Korean J. Environ. Biol. 35(3) : 354~360(2017) https://doi.org/10.11626/KJEB.2017.35.3.354

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 mono- graphic 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 dis- tinct 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 flo- ristic listings without taxonomic consideration.

Genus Pyropia J. Agardh was resurrected for a large group of Porphyra according to morphological and molec-

ular 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

* Corresponding author: Tae Oh Cho, Tel. 062-230-7161, Fax. 062-230-7161, E-mail. [email protected]

New Records of Intertidal Marine Algae from Korea

So Young Jeong

¹

, Danilo E. Bustamante

^1,2

, Jin Gyo Lee

¹

, Boo Yeon Won

¹

, Seung Hee Kim

³

and Tae Oh Cho

^1,

*

1

Department of Life Science, Chosun University, Gwangju 61452, Republic of Korea

2

Escuela Profesional de Ingieneria Ambiental, Universidad Nacional Toribio Rodriguez de Mendoza (UNTRM), Chachapoyas, Peru

3

Department of Dental Hygiene, Gwangju Health University, Gwangju 62287, Republic of Korea

Abstract - Bryopsis africana and Pyropia kinositae are reported for the first time on the list of Korean marine algal flora based on integrated morphological-molecular study. Bryopsis africana from Korea is recognized with distinct main axes, robust thalli, pinnately and radially branched above, and basally denuded. Phylogenetic analyses revealed that B. africana was placed within a clade of Bryopsis. Bryopsis africana differs from B. corymbosa by 2.7% gene sequence divergence.

Pyropia kinositae is characterized by oblong to lanceolate thalli, entire margin, purplish red in color, twice as long as broad cells in transversal section view. Phylogenetic analyses revealed that P.

kinositae was placed within a clade of Pyropia. 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.

Key words : Bryopsis africana, Chlorophyta, Pyropia kinositae, Rhodophyta

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were preserved in 4-5% formalin/seawater for morpholog- ical examination. Microscope observations were made by using materials stained with 1% aqueous aniline blue acid- ified with 0.1% diluted HCl. Photomicrographs were taken using an Olympus BX51TRF microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP71 camera. Repre- sentative 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, Germa- ny), 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-spin

^TM

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. Por- phyra 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 conduct- ed 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 us- ing 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 africana Areschoug, 1851

^{아프리카깃털말}

(

^신칭

) (Fig. 1A-H)

Synonym: Bryopsis tenuis Levring 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, form- ing 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 pin- nately and spirally, with rounded apex (Fig. 1D-F). Main axes 66-92 μm in diameter, branchlets 24-32 μm in diam- eter. Branches of main axes typically developed into sec- ondary 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

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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

Fig. 1. Bryopsis africana from Bogil Island, Korea. A. Habit of a typical plumose cluster of thalli; B. Thalli attached on coralline algae; C.

Branched rhizoidal system; D. Translucent apical cytoplasm of apex; E. Branching pattern; F. Basal points of lateral branches; G.

Complete plug at base of branch(arrow); H. Chloroplasts distributed along axis. Scale bars: A, B=1cm; C=1mm; D, G=50μm;

E=0.5mm; F, H=100μm.

A B

C D E

F G H

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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 sim- ilar 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, Daepo- dong, 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 substra- tum 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 sur- face 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 sporo- carps 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 anth- erozoids 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.

Fig. 2. Phylogenetic tree of species of Bryopsis based on Maximum-likelihood(ML) analysis with rbcL sequences. Values above branches=ML bootstrap(BS) values in % >50/Bayesian posterior probabilities(BPP) >0.75. Values lower than BS 50% or BPP 0.75 are indicated by hyphens(-). Values of BS 100% or BPP 1.00 are indicated by asterisks(*).

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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 Pyro-

pia kinositae. Phylogenetic analyses revealed that P. kinos- itae 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

Fig. 3. Pyropia kinositae from Sokcho, Korea. A. Specimen of vegetative thalli; B. Lanceolate foliose type of thalli; C. Rhizoid; D. Rhizoidal cells in surface view; E. Entire margin; F. Monostromatic; G. Tetrasporangious zygotosporangia in surface view show; H. Carpospore portion in surface view; I. Spermatangial portion in surface view(arrows); J. Spermatangia in surface view. Scale bars: A, B=1cm;

C=1mm; D, E, H=20μm; F, G, I=10μm.

A B C

D E F G

H I J

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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.

ACKNOWLEDGEMENT

This study was supported by a grant from the Nation- al Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201701204). This research was also sup- ported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2015R1D1A1A01058359) and a grant from Marine Bio- technology Program (20140449 and 20170431) funded by Ministry of Oceans and Fisheries of Korean Government to Tae Oh Cho.

Fig. 4. Phylogenetic tree of species of Pyropia based on Maximum-likelihood(ML) analysis with rbcL sequences. Values above branches=ML bootstrap(BS) values in % >50/Bayesian posterior probabilities(BPP) >0.75. Values lower than BS 50% or BPP 0.75 are indicated by hyphens(-). Values of BS 100% or BPP 1.00 are indicated by asterisks(*).

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Received: 19 August 2017 Revised: 12 September 2017 Revision accepted: 12 September 2017