The genus Pleurastrum is a coccoid green alga comprising 10 species worldwide. Pleurastrum exhibits simple morphology and high polymorphism, which complicates the understanding of its diversity. We examined the morphological and ultrastructural characteristics of Pleurastrum using light, confocal, and transmission electron microscopy. Additionally, we performed phylogenetic analysis based on multigene sequences (nuclear SSU rDNA, 5.8S, internal transcribed spacer (ITS2) region, and plastid rbcL and tufA genes) from Pleurastrum strains to report two previously unrecorded freshwater species (Pleurastrum insigne and Pleurastrum microstigmatum) in Korea. The vegetative cells were predominantly spherical, with a few being ellipsoidal, and each cell contained a chloroplast with one pyrenoid. The sporangia produced several daughter cells, while the biflagellate zoospores were ellipsoidal and motile. Phylogenetic analysis confirmed that P. insigne and P. microstigmatum form well-supported monophyletic clades. Analysis of ITS2 secondary structures revealed similar patterns, with several differences in nucleotide sequences and insertions between the two species. The findings of this study expand the known distribution of Pleurastrum and enhance our understanding of its species diversity in Korea.

Abstract
1. INTRODUCTION
2. MATERIALS AND METHODS
    2.1. Sampling and cultivation
    2.2. Light microscopy
    2.3. Confocal microscopy
    2.4. Transmission electron microscopy
    2.5. DNA isolation, polymerase chain reaction,and sequencing
    2.6. Phylogenetic analysis
    2.7. ITS2 secondary structure prediction
    2.8. CBC analysis
3. RESULTS
    3.1. Morphology and ultrastructure
    3.2. Phylogenetic analysis
    3.3. Secondary structures prediction ofnuclear ITS2
    3.4. Molecular signatures
    3.5. Taxonomic descriptions
4. DISCUSSION
CRediT authorship contribution statement
Declaration of Competing Interest
ACKNOWLEDGEMENTS
REFERENCES

• Yu Ho Kim(Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea)
• Bok Yeon Jo(Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea)
• Jae Hak Lee(Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea)
• Seung Won Nam(Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea) Corresponding author
• KwangHuem Hong(Department of Medicine, Graduate School, Wonkwang University, Iksan 54538, Republic of Korea, Sarcopenia Total Solution Center, Wonkwang University, Iksan 54538, Republic of Korea)
• Woongghi Shin(Department of Biology, Chungnam National University, Daejeon 34134, Republic of Korea) Corresponding author