The red algal order Bangiales Nägeli (Nägeli 1847) has traditionally been divided into two genera: Porphyra C.Agardh (Agardh 1824), which includes species with a foliose gametophyte, and Bangia Lyngbye (Lyngbye 1819), which encompasses species with a filamentous gametophyte morphology (Boedeker et al. 2008;Zheng and Li 2009). The foliose form of the order Bangiales has been composed of nine genera: Boreophyllum, Clymene, Fuscifolium, Lysithea, Miuraea, Neothemis, Porphyra, Pyropia, and Wildemania (Sanchez et al. 2014;Yang et al. 2017;Kikuchi et al. 2018). In 2020, Yang et al. redefined the genus Pyropia based on a molecular phylogenetic study of foliose Bangiales from China. They resurrected Porphyrella and proposed four additional genera: Calidia, Neoporphyra, Neopyropia, and Uedaea. However, recently, Zuccarello et al. (2022) synonymized all five of these newly proposed genera back into Pyropia. Currently there are nine genera with a foliose gametophyte: Boreophyllum, Clymene, Fuscifolium, Lysithea, Neomiuraea (formerly Miuracea), Neothemis, Porphyra, Pyropia, and Wildemania.
Pyropia (‘Gim’ in Korean) is an economically significant seaweed in Korea and has witnessed significant progress in maximizing production through advancements in cultivation methods and selective breeding of high-quality varieties (Hwang and Park 2020;Hwang et al. 2020). Recently, seaweed cultivation is facing a direct threat from the increasing seawater temperatures caused by global warming (Kim et al. 2007;Hwang et al. 2020). It is important to find or develop high-temperature- tolerant species/strains of Pyropia (Choi et al. 2013;Xing et al. 2023).
“Neoporphyra kitoi” was initially described by Abe et al. in 2021 based on specimens collected from Isumi, Chiba, Japan. They noted that “N. kitoi” exhibited significant molecular differences in rbcL and SSU genes, as well as a distinct morphological characteristic of new blades emerging from rhizoidal cells even if foliose thalli “N. kitoi” were similar to Pyropia pseudolinearis in morphology. According to Niwa et al. (2022) “N. kitoi” show high potential as marine crop due to its tolerance to high temperature.
We collected 13 unidentified foliose red algae from the Korean coastline and recognized their taxonomic position based on both molecular and morphological analyses. Of them, one sample collected from Ulleungdo island has been added as Pyropia kitoi com. nov. to the marine algal inventory of Korea.
2. MATERIALS AND METHODS
2.1. Molecular analyses
Samples were collected from the Korean coastline. Genomic DNA was manually extracted from silica-gel samples using a NucleoSpin Plant II Kit (Macherey- Nagel, Düren, Germany). The rbcL DNA amplifications were performed using primer sets F57-R753, F577-R1150, and F993-Rrbcst. PCR amplifications were carried out using a Veriti 96 well Thermal cycler (Applied Biosystem, Waltham, MA). The PCR products were then purified using a PCRquick-spinTM PCR product purification kit (iNtRON Biotechnology, Inc., Seongnam, Korea). The thirteen rbcL sequence obtained in this study had been deposited in GenBank. The rbcL sequence that we generated, as well as those obtained from GenBank, were aligned using ClustalW (Thompson et al. 1994).
Phylogenetic analyses were performed using raxml GUI1.5b2 (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 Ronguist 2001;Ronguist and Huelsenbeck 2003). Markov chain Monte Carlo runs were conducted for 2,000,000 generations, with one cold chain and three heated chains, using the GTR+Γ+I evolutionary model. Trees were sampled and printed every 1,000 generations and summary trees were generated using a burn-in value of 25%.
2.2. Morphological analyses
Samples were subsequently classified into voucher herbarium specimens, silica gel samples, and formalin samples. Formalin samples were preserved in a solution of 4-5% formalin mixed with seawater. Photomicrographs of the specimens were captured using an Olympus BX51TRF microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP71 camera. For longterm preservation, permanent slides were prepared using 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) in Korea.
3. RESULTS AND DISCUSSION
Phylogenetic analyses: The 1247 rbcL base pair (bp) portion of the 1467-bp rbcL gene (85% sequenced) was sequenced from samples from Korea. The phylogenetic trees were obtained from the alignment of the rbcL sequences newly generated and downloaded from Gen Bank (Fig. 1). Smithora naiadum (C. L. Anderson) Hollenb. and Chlidophyllon kaspar (W. A. Nelson et N. M. Adams) W. A. Nelson were included as outgroups. Phylogenetic analyses inferred from rbcL indicated that all thirteen samples from Korea were nested within Pyropia clade of the Bangiaceae, with 93% bootstrap support in the Maximum likelihood analysis and Bayesian posterior probabilities of 1 in the Bayesian tree. Our sequences cluster with one sequence belonging to Py. kinositae, one sequence belonging to Py. Kuniedae, one sequence belonging to Py. pseudolinearis, one sequence belonging to Py. retorta, five sequences belonging to Py. seriata, one sequence belonging to Py. suborbiculata, and two sequeces belonging to Py. yezoensis (Fig. 1). One collection from Ulleungdo island, Korea falls within the same clade as “Neoporphyra kitoi”, with only 3 base pair differences in rbcL squences (1247 bp). Pyropia kitoi com. nov was found to be closely related to P. meridionalis (Fig. 1).
Pyropia kitoi (Ma.Abe, N.Kikuchi, Tamaki, Tom.
Sato, Murase, Fujiyoshi & Mas.Kobayashi) D.J. Kim, T.O. Cho & B.Y. Won com. nov. (길쭉돌김) (Fig. 2)
Basionym: Neoporphyra kitoi Ma. Abe, N. Kikuchi, Tamaki, Tom. Sato, Murase, Fujiyoshi & Mas. Kobayashi 2021.
Holotype. SAP (the Herbarium of Faculty of Science, Hokkaido University, Sapporo, Japan) 115 574, collected on 11 March 2019 by N. Kikuchi from Iwafune (35° 12′36″N, 140°23′32″E), Isumi, Chiba, Japan.
Material examined. CUK21236 (Herbarium of Chosun University) & NIBRRD0000010448 (National Institute of Biological Resources), Cheonbu-ri, Buk-myeon, Ulleung-gun, Gyeongsangbuk-do, Korea (37°32′ 40.7″N, 130°54′31.5″E), April 09, 2023, T.O. Cho & B.Y. Won, at 1m depth by hand.
Habitat. Thalli grow on rocks or on Scytosiphon lomentaria in intertidal zone.
GenBank accession number. OR233063 (rbcL).
Morphological observation. Thalli are dark red purple or reddish brown (Fig. 2A, B). Thalli are membranous, monostromatic, linear and narrow elliptic, 10-15 cm long, 1.0-1.5 cm broad, with cuneate, obtuse or rotund base, and with entire margin (Fig. 2A-E). Vegetative cells are oblong or irregular tetragonal to polygonal with rounded angles (Fig. 2F, G). Basal cells capitate with projected rhizoidal filaments (Fig. 2E). Rhizoidal filaments are arranged in both directions in sectional view (Fig. 2H). Thalli mixed monoecious. Spermatangia are formed in parallel with zygotosporangial patches along margins (Fig. 2I, J). They are small (Fig. 2K, L). Zygotosporangia are large (Fig. 2M-P) and prototrichogyne are conspicuous and acute to obtuse (Fig. 2N).
Remarks.Pyropia, the most species-rich genus in Bangiales, comprises approximately 76 taxonomically accepted species worldwide (Guiry and Guiry 2023). While Yang et al. (2020) divided Pyropia into several genera within Bangiaceae, Zuccarello et al. (2022) synonymized these genera including Neoporphyra back into Pyropia. Zuccarello et al. in 2022 mentioned that it is essential to ensure that phylogenetic analyses integrate the most current reconstruction techniques, model selection methodologies, and a variety of support measures when proposing new genera without alternative morphological diagnostic features (Simon 2022;Zuccarello et al. 2022). Our phylogenetic tree, based on Maximum Likelihood analysis, also indicated weak support for the clades proposed as genera by Yang et al. (2020) (Fig. 1). In our study, Pyropia kitoi is proposed as a new combination with N. kitoi Ma. Abe, N. Kikuchi, Tamaki, Tom. Sato, Murase, Fujiyoshi & Mas. Kobayashi 2021 based on the detailed molecular and morphology analyses. We also report P. kitoi as a new record in the list of Korean macroalgal flora. Pyropia kitoi may be one of beneficial seaweed species to have a characteristic of having potential for marine crop with high temperature tolerance (Niwa et al. 2022).