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INTRODUCTION
The filamentous brown algal genus Acinetospora is described as plants that possess sparsely branched uniseriate filaments forming entangled tufts, plurilocular sporangia (acinetosporangia) which produce acinetospores, monosporangia, scattered meristematic zones, and crampons (Bornet 1892; Sauvageau 1899). Currently, four Acinetospora species are recognized: A. crinita (Carmichael in Harvey) Sauvageau (1899), A. nicholsoniae Hollenberg (1971), A. filamentosa (Noda) Yaegashi, Uwai et Kogame (2015), and A. asiatica Yaegashi, Yamagishi et Kogame (2015). Of them, only A. crinita has been reported in Korea (Kim 2010).
The genus Botrytella is characterized by heterotrichous thallus organization arising from a basal disc, the erect filaments with sympodial branching, diffuse intercalary meristem, and sub-globular to irregular larger plurilocular sporangia arising terminally or serially on the adaxial side of the laterals (Womersley 1987; Kim 1996; Guiry and Guiry 2018). Currently, four Botrytella species are recognized around the world; Botrytella micromora Bory (1822), B. parva (Takamatsu) H. S. Kim (1996), B. pacifica (Hollenberg) G. I. Hansen (1997) and B. reinboldii (Reinke) Kornmann & Sahling (1988). Of them, the three species have been reported in Korea except B. reinboldii (Kim 2010).
We collected two filamentous brown algae from coast of Korea that fit the description of Acinetospora asiatica and Botrytella reinboldii. We observed their detailed morphology and analysed molecular data based on rbcL gene for their phylogenetic relationships with others in their respective genera and a few selected species within the Ectocarpales. In this study, we add Acinetospora asiatica and Botrytella reinboldii to the Korean marine algal inventory.
MATERIALS AND METHODS
1. Morphology
Plants were collected from coasts of Korea and sorted into voucher herbarium specimens, silica gel samples for molecular analysis, and formalin samples for morphological analysis. Formalin samples were preserved in 4-5% formalin/seawater. Photomicrographs taken using an Olympus BX51TRF microscope (Olympus, Tokyo, Japan) and an Olympus DP71 camera. Permanent slides were mounted in 70% karo syrup. Representative specimens examined in this study were deposited in the herbarium of Chosun University (CUK) and National Institute of Biological Resources (NIBR), Korea.
2. Molecular study
Genomic DNA was manually extracted from silica-gel samples using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). The extracted DNA was stored at -20°C and used to amplify rbcL. Polymerase chain reaction (PCR) was conducted in 30 μL reactions that contained 3.2 μL genomic DNA or 3.2 μL diluted genomic DNA (1 : 10), 1 μL 10 pmols forward and reverse primers, and 15 μL HelixAmp Ready-2x-Go Series (NanoHelix Co., Ltd., Daejeon, Korea). The rbcL gene was amplified using the primer combinations NDrbcL2-DRL1R and DRL2F-R3A (Kogame et al. 1999; Hwang et al. 2005). The PCR product was purified with PCR quick-spinTM PCR product purification kit (iNtRON Biotechnology, Inc. Seongnam, Korea). Determination of the nucleotide sequence or sequencing performed by Macrogen Inc., Seoul, South Korea.
RbcL sequence data were compiled by the present study and obtained from GenBank and aligned with ClustalW (Thompson et al. 1994). New rbcL sequences obtained from Acinetospora asiatica have been deposited in EMBL/Gen- Bank under the accession numbers MH727658, MH727659, MH727660, and MH727661. Also Botrytella reinboldii sequence data are deposited in EMBL/GenBank under accession number MH727662 for rbcL. Phylogenetic analyses were conducted using MEGA version 6.06 (Tamura et al. 2013). Maximum likelihood analyses were conducted using the GTR+G model, with 1,000 bootstrap replicates. A bayesian inference was performed using MrBayes 3.2.6 (Huelsenbeck and Ronguist 2001; Ronguist 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
Acinetospora asiatica Yaegashi, Yamagishi et Kogame, 2015
Type locality: Oshoro, Otaru, Hokkaido, Japan.
Material examined: MBRB0063TC13380 (voucher specimens deposited in the National Marine Biodiversity Institute of Korea for MH727658 GenBank accession number), Byeonsan-myeon, Buan-gun, Jeollabuk-do, Korea (35°37ʹ 41.87ʺN, 126°28ʹ04.60ʺE), January 09, 2015, T. O. Cho and B. Y. Won, at 1 m depth by hand; MBRB0063TC14114 (voucher specimens deposited in the National Marine Biodiversity Institute of Korea for MH727659 GenBank accession number), Hupo-hang, Hupo-myeon, Uljin-gun, Gyeongsangbuk- do, Korea (36°40ʹ42.90ʺN, 129°27ʹ02.96ʺE), January 23, 2015, T. O. Cho, S. Y. Jeong, J. G. Lee, S. Y. Park, J. B. Chai and H. U. Jany, at 1 m depth by hand; MBRB0063TC17307 (voucher specimens deposited in the National Marine Biodiversity Institute of Korea for MH727660 GenBank accession number), Hongdo-hang, Hongdo-ri, Heuksan-myeon, Sinan-gun, Jellanam-do, Korea (34°41ʹ02.15ʺN, 125°11ʹ 35.50ʺE), May 21, 2016, T. O. Cho, S. Y. Jeong, D. B. Mostajo and A. Jose, at 1 m depth by hand; NIBROR0000001359 (deposited in the National Institute of Biological Resources), MBRB0063TC18922 (voucher specimens deposited in the National Marine Biodiversity Institute of Korea for MH727 661 GenBank accession number), Jaranman-ro, Hai-myeon, Goseong-gun, Gyoungsangnam-do, Korea (34°54ʹ19.08ʺN, 128°09ʹ08.43ʺE), May 05, 2018, T. O. Cho and B. Y. Won, at 1 m depth by hand.
Description: Plants forming entangled tufts (Fig. 1A) on rocks and other seaweeds (e.g. Sargassum spp.), sparsely branched uniseriate filaments (Fig. 1B), and up to 30 cm high. Erect filaments have meristematic zones composed of small sized cells (Fig. 1C) and crampons formed on erect filaments at right angles (Fig. 1D). Cells of erect filaments 20-77 μm in length, 18-30 μm in width, and contain many discoid chloroplasts (Fig. 1E). Erect filaments produced from prostrate filaments (Fig. 1F), and tapered slightly in upper part to form pseudohairs (Fig. 1G). Plurilocular zoidangia produced on both prostrate and erect filaments, ectocarpoid, 90-135 μm in length, 25-40 μm in width, with one- or two-celled pedicels (Fig. 1H) or sessile (Fig. 1I). Unilocular sporangia were not found.
Habitat: Epiphytic and saxicolous at the tide pool in intertidal zone.
World distribution: Korea, Japan.
Identifier: Tae Oh Cho.
Phylogenetic analyses: The 1326-nucleotide portion of rbcL was aligned for Acinetosproa asiatica. The phylogenetic trees were obtained from the alignment of the rbcL sequences newly generated and downloaded from GenBank. Asterocladon rhodochortonoides and A. interjectum were selected as outgroup. Phylogenetic analyses revealed that Acinetosproa collected from South Korea was placed within a clade of Acinetospora asiatica (Fig. 2). In addition, Acinetospora asiatica collected from South Korea differs from A. crinita by 4.2-5.2% and from A. filamentosa by 4.7-4.9% gene sequence divergence respectively. However, there was only 0- 0.02% gene sequence divergence between Genbank and our collection of Acinetospora asiatica.
Remarks: Acinetospora asiatica was recently described as a new species from Japan (Yaegashi et al. 2015). Our collections match the diagnosis of Acinetospora asiatica as described from the type locality. Molecular data based on rbcL gene match with the Japanese samples. In this study, we report A. asiatica as a new record from Korea and add this species in the list of Korean macroalgal flora.
Botrytella reinboldii (Reinke) Kornmann & Sahling, 1988 가지바다오디 (신칭) (Figs. 3-4)
Type locality: Helgoland, Germany.
Material examined: MBRB0061TC10574, Beakpo village, Dolsan-eup, Yeosu-si, Jeollanam-do, Korea (34°37ʹ15.03ʺN, 127°47ʹ25.70ʺE), January 18, 2014, T. O. Cho, at 1 m depth by hand; NIBROR0000001360 (deposited in the National Institute of Biological Resources), MBRB0061TC18903 (voucher specimens deposited in the National Marine Biodiversity Institute of Korea for MH727662 GenBank accession number), Aninjin hang, Gangdong-myeon, Gangneungsi, Gangwon-do, Korea (37°44ʹ15.81ʺN, 128°59ʹ19.55ʺE), January 05, 2018, T. O. Cho and B. Y. Won, at 1 m depth by hand.
Description: Plant yellowish brown, epiphytic (e.g. on Sargassum spp.), forming tufts (Fig. 3A), up to 10 cm in length, 8 μm in diameter, irregularly alternating branched uniseriate filaments (Fig. 3B), and attached by rhizoids (Fig. 3C). Cells cylindrical, slightly constricted at the septum, and with numerous discoid chloroplasts (Fig. 3D). Filaments 30 μm in diameter, tapered slightly in upper part, and bear diffuse meristem (Fig. 3E). Plurilocular sporangia sessile or stalked with various shapes and sizes, single on the lower thalli, grapelike clusters on laterals of upper thalli (Fig. 3F), release spores through one opening located anteriorly on the plurilocular sporangium (Fig. 3G). Unilocular sporangia not observed.
Habitat: Epiphytic on other seaweeds (e.g. on Sargassum spp.) at the tide pool in intertidal zone.
World distribution: Korea, Japan (Guiry and Guiry 2018).
Identifier: Tae Oh Cho and So Young Jeong.
Phylogenetic analyses: The 1122-bp portion of rbcL was aligned for Botrytella reinboldii. Ectocarpus fasciculatus and E. crouaniorum were selected as outgroup. Phylogenetic analyses revealed that Botrytella reinboldii from South Korea was nested in a clade of Botrytella reinboldii (Fig. 4). The gene sequence divergence between Botrytella reinboldii and B. micromora is 3% and there is no difference within Botrytella reinboldii.
Remarks: Morphologically, our samples closely match the description of B. reinboldii described by Kornmann and Sahling (1988). Also, molecular data based on rbcL gene revealed that our samples are placed within the clade of the Botrytella and specifically very close to the Japanese B. reinboldii (GenBank accession number: AB302290). In this study, we report B. reinboldii as a new record from Korea and add this species to the list of Korean macroalgal flora.
