1. INTRODUCTION

The genus Basicladia, established by Hoffman and Tilden (1930), could be characterized by unbranched or rarely branched filaments, growing from coalesced rhizoidal holdfast, epiphytic on the back of freshwater turtles (Hoffman and Tilden 1930;Van den Hoek 1963). Although Basicladia had been known for occurring on the back of freshwater turtles at first, later it has also been reported on further various substrates including the shell of snail or even as a free-living algal species (Ueda 1932;Normandin and Taft 1959;Van den Hoek 1963).

Basicladia okamurae (Ueda) Garbary 2010 is a freshwater green filamentous alga established based on a Japanese specimen as a basionym of Chaetomorpha okamuraeUeda 1932. Ueda (1932) described that this species has pale to deep green cylindrical, and 50-60 μm thick segments with short barrel-shaped cell, distinguished from other freshwater species by branched and fibrous attachments. Garbary (2010) transferred two species, Cladophora kosterae and C. okamurae, to the genus Basicladia. Basicladia kosterae (C. Hoek) Garbary comb. nov. was taxonomically treated as a synonym of Arnoldiella kosterae (Hoek) Boedeker (Boedeker et al. 2012) Therefore, two species (B. okamurae and B. ramulosa) are listed in the genus Basicladia (Guiry and Guiry 2025). This species reportedly occurs in Japan (Ueda 1932;Hanyuda et al. 2002;Watanabe et al. 2025), China (Boedeker et al. 2012), the Netherlands (Van den Hoek 1963;Boedeker et al. 2012), France (Van den Hoek 1963), and Mexico (Pedroche and Sentíes 2020). Cladophoralean species display high morphological similarity and are frequently misidentified based solely on morphological characteristics (Lee et al. 2023). Therefore, DNA barcoding is an essential tool for the species identification of filamentous green algae (e.g. Taylor et al. 2017;Lee et al. 2023).

In this study, we investigated filaments of B. okamurae isolated from a lump of threads and mucilage, and conducted morphological and DNA barcoding methods to identify the species. Boedeker et al. (2012) and Watanabe et al. (2025) analyzed the phylogenetic relationship among species of the family Pithophoraceae including Basicladia okamurae with SSU rDNA and LSU rDNA from Netherlands, China and Japan. Therefore, we used those LSU rDNA as DNA references to identify Korean samples because of LSU rDNA sequence showing appropriate genetic variation on the species level of the family Pithophoraceae.

2. MATERIALS AND METHODS

During the examination of an unidentified sample collected from a river, Sanhocheon, Changwon-si, Gyeongsangnam-do, Korea (35.228140N, 128.584652E; 21 Nov. 2024) for species identification, we isolated an algal specimen morphologically similar to Cladophoralean algae. The sample was predominantly composed of grayish-white fungal mycelial aggregates, along with a few strands of green filamentous algae (Fig. 1). For the morphological observations, we used a light microscope (Olympus BX50; Japan) and a stereo-microscope (Nikon, Japan). We deposited all specimens in the algal herbarium of the National Institute of Biological Resources (NIBR) in Incheon, Korea (NIBRCL0000118 031, NIBRCL0000118090).

To minimize potential contamination during the genetic analysis, we isolated a single filament (<0.5 cm) from the field samples. We used the DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) to extract total genomic DNAs, according to previously described protocols (Lee et al. 2024). LSU rDNA sequences were used in previous taxonomic studies for Basicladia okamurae (e.g. Leliaert et al. 2007;Boedeker et al. 2012;Watanabe et al. 2025). Therefore, we used the partial LSU rDNA sequence as the reference sequence for the species identification. And, LSU rDNA sequences were amplified using the primer pair “C1FL/D2FL” (Leliaert et al. 2007).

The cycling conditions of the polymerase chain reaction were the following initial denaturation at 94°C for 3 min, 40 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, final extension at 72°C for 7 min using a Gene Amp-PCR-System-9700 (Applied Biosystems, Foster City, CA, USA). We used the AmfiXpand PCR Master Mix (GenDEPOT, Katy, TX, USA) for amplification, and sequenced the PCR products, using a commercial sequencing service (Macrogen, Seoul, Korea). We analyzed and assembled the chromatograms using the Sequencher 5.4.6 software (Gene Codes, Ann Arbor, MI, USA). For species identification, we compared the LSU rDNA sequences to the public database using BLAST search in GenBank at the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih. gov/Blast.cgi), and conducted molecular analyses using the MEGA version 6 software (Kumar et al. 2016). We applied the maximum likelihood method based on the Kimura 2-parameter model, placing Pseudocladophora horii on the outgroup. A bootstrap analysis was conducted with 1,000 replicates and numbers on branches indicate bootstrap values.

3. RESULTS

3.1. Morphological description of B. okamurae

Ulvophyceae Mattox and Stewart, 1984 갈파래강

Cladophorales Haeckel, 1894 대마디말목

Pithophoraceae Wittrock, 1877 알마디말과

BasicladiaHoffmann and Tilden, 1930

녹색실말속 (국명신칭)

Basicladia okamurae (Ueda) Garbary, 2010 녹색실말 (국명신칭)

Basionym: Chaetomorpha okamuraeUeda 1932.

The thalli are deep green, uniseriate, displaying unbranched filaments (Fig. 3A) with cylindrical to barrelshaped cells (Fig. 3B). Each cell is 67-144 μm and 216- 1,421 μm in width and length, at the basal and middle parts of the filament and 45-50 and 150-200 μm in width and length, at the upper part of the filament. In the upper part, the apical cell exhibits a round end, tapering toward the end (Fig. 3C). In the middle to the upper part, moniliform fertilized cells of the filament form a pointed protrusion without any cytoplasm (Fig. 3D, E) and the tip bursts, releasing spores through the opening (Fig. 3F, G). The released spores are pear-shaped at first (Fig. 3H), swimming for a while, then spinning around, and becoming round and motionless (Fig. 3I). The zoospores are 10-15, 13-20 μm in width and length, respectively, and 13.3-14.9 μm in diameter. The cells are filled with round granules.

Specimens examined. NIBRCL0000118031, NIBRCL 0000118090; 21 Nov. 2024; Sanhocheon, Yangdeokdong, Masanhoewon-gu, Changwon-si, Gyeongsangnam- do, Korea (35.228140N, 128.584652E).

Accession numbers. PV546362 (LSU rDNA) (DNA sequence derived from NIBRCL0000118031).

3.2. Basicladia okamurae species identification using LSU rDNA sequences analyses

For species identification, we sequenced a total of 584 bp LSU rDNA sequence of a freshwater green alga B. okamurae (GenBank accession number PV5463 62) collected from Sanhocheon, Changwon-si, Gyeongsangnam- do, Korea. The DNA sequence exhibited 99.8-100% similarity with those of B. okamurae deposited in GenBank (NCBI) and under 91.2% similarity with those of other species of the order Cladophorales from the BLAST search. The pairwise distance by Kimura-2 parameter model showed 0-0.2% on the intraspecific level of B. okamurae. Excepting one sample of B. okamurae from China (KU727274, 2.4%), other LSU rDNA sequences (10 samples) of B. okamurae from Korea, China, Japan and Netherlands showed 99.8-100% similarity on the intraspecific level. Moreover, LSU rDNA sequence of B. okamurae showed below 91.2% similarity with species of the order Cladophorales. In this study, we successfully identified Korean B. okamurae using LSU rDNA sequence and could add a new report on the distribution of this species in Korea for worldwide distribution of B. okamurae (Figs. 1- 3).

4. DISCUSSION

Basicladia okamurae (Ueda) Garbary displays pale to deep green, filamentous morphological features, which is consistent with the original description by Ueda (1932). Basicladia okamurae has long been taxonomically placed in the genus Chaetomorpha (Ueda 1932). Recent molecular phylogenetic studies have revised the taxonomic position of C. okamurae among the cladophoralean species, transferring it as a new combination, identified as B. okamurae (Hanyuda et al. 2002;Garbary 2010;Watanabe et al. 2025).

The morphological similarity among cladophoralean species has previously led to the misidentification of these taxonomic groups (e.g. Taylor et al. 2017). DNA barcoding provides useful genetic information for species identification. Taxonomic studies, including new records and species, have contributed to revising cladophoracean algal biodiversity (Taylor et al. 2017;Lee et al. 2023). In this study, we identified a green alga B. okamurae in a Korean stream, demonstrating that an integrative approach combining morphological and molecular characteristics could allow for important taxonomic insights into the green algal flora on Korea.

Currently, the cladophoralean algal flora of Korea included 11 genera such as Cladophora, Pithophora, Ulothrix, and Chaetomorpha (NIBR 2025). However, Basicladia species records have not been previously reported in Korea, making this finding a notable addition to the list, thereby increasing regional diversity.