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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.43 No.3 pp.275-293
DOI : https://doi.org/10.11626/KJEB.2025.43.3.275

First report of two Pleurastrum species (Chlorophyceae) from Korean freshwater: Pleurastrum insigne and P. microstigmatum

Yu Ho Kim1, Bok Yeon Jo1, Jae Hak Lee1, KwangHuem Hong2,3, Seung Won Nam1*, Woongghi Shin4*
1Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea
2Department of Medicine, Graduate School, Wonkwang University, Iksan 54538, Republic of Korea
3Sarcopenia Total Solution Center, Wonkwang University, Iksan 54538, Republic of Korea
4Department of Biology, Chungnam National University, Daejeon 34134, Republic of Korea
*Co-corresponding authors Seung Won Nam Tel. 054-530-0882 E-mail. seungwon1007@gmail.com
Woongghi Shin Tel. 042-821-6409 E-mail. shinw@cnu.ac.kr

Contribution to Environmental Biology


▪ This study presents the first records of Pleurastrum insigne and Pleurastrum microstigmatum in Korean freshwater habitats, thereby enhancing our understanding of algal diversity and distribution in the region.


▪ By integrating morphological, ultrastructural, and multigene phylogenetic analyses with comparisons of ITS2 secondary structures, this study provides robust taxonomic evidence to support biodiversity assessment and conservation in freshwater ecosystems.


09/01/2025 01/09/2025 04/09/2025

Abstract


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.


ITS2 secondary structure , morphology , phylogeny , ultrastructure

초록

    1. INTRODUCTION

    The genus Pleurastrum Chodat is one of the green algal genera and its species are found in various habitats, including aquatic and terrestrial ecosystems (Chodat 1894;Sciuto et al. 2023). Pleurastrum have diverse forms, ranging from having unicellular coccoids, biflagellated motile cells (zoospores), and sarcinoid stages to elaborate branched filaments (Chodat 1894;Visher 1933;Sciuto et al. 2023). The growth form of Pleurastrum species can change depending on the environmental conditions (culture or field).

    Since the first description of P. insigne, the type species of genus Pleurastrum, the several species were described based on morphological characters (Visher 1933;Deason and Bold 1960;Tupa 1974;Sluiman and Gärtner 1990). Although culture-based observations and comparative morphological studies have enhanced our understanding of Pleurastrum diversity, unavailable type specimen of P. insigne and phenotypic plasticity due to the environmental conditions leads to taxonomic confusion (Deason and Bold 1960;Tupa 1974;Sluiman and Gärtner 1990;Lukešová 1991). For example, Vischer (1933) observed only the P. insigne filamentous form and consequently proposed a revision of the description of this genus. Tupa (1974) conducted a comparative study of the morphology of all available Pleurastrum cultures and revealed that they had a branched filamentous form, a characteristic of common green algae. Sluiman and Gärtner (1990) described a Pleurastrum lectotype in a culture-based study and revealed Pleurastrum morphological diversity ranges from coccoid to filamentous forms. In contrast, Sciuto et al. (2023) revealed that they have a filamentous (-like) form in the early stages of culture. Moreover, Kawasaki et al. (2015) did not observe the filamentous form at all.

    In contrast to morphological studies, phylogenetic analyses using molecular markers and internal transcribed sequence 2 (ITS2) secondary structure have enabled a comprehensive understanding of their diversity. Previous phylogenetic analysis based on 18S rDNA and ITS gene sequences suggested that genus Pleurastrum is a polyphyletic group originating from Ulvophyceae, Trebouxiophyceae, and Chlorophyceae lineages (Frield 1996), and indicated that the lectotype P. insigne matches with Chlorococcum oleofaciens (Kawasaki et al. 2015). Recently, a phylogenetic analysis using molecular markers such as rbcL, tufA, and ITS, along with ITS2 secondary structure studies, have defined the molecular boundaries of the genus Pleurastrum (Sciuto et al. 2023). Following these results, several species previously classified under Chlorococcum, including C. oleofaciens, have been transferred to P. insigne, while C. microstigmatum has been transferred to P. microstigmatum. Through several revisions of this genus, 10 species have been reported worldwide (Sciuto et al. 2023); however, no species have been documented in Korea, and there are no diversity studies on this genus.

    Due to its complex and confused taxonomy history, the species diversity of Pleurastrum and Chlorococcum in Korea is also expected to undergo revision. Accordingly, this study investigated the morphological and ultrastructural characteristics of Korean strains using light, confocal, and transmission electron microscopy, along with molecular characteristics based on the ITS2 secondary structure. We also conducted phylogenetic analysis using multigene sequences, including nuclear SSU rDNA, 5.8S, ITS2, and plastid-encoded rbcL and tufA genes. Through these approaches, we aim to clarify the taxonomic boundaries between the two genera and contribute to a better understanding of species diversity in Korea.

    2. MATERIALS AND METHODS

    2.1. Sampling and cultivation

    Pleurastrum cultures were collected from freshwater habitats in Korea and maintained in BG-11 culture medium (Cat. No. C3061; Sigma-Aldrich, USA) under a 14 : 10 light : dark cycle and a light intensity of 67±10 μmol photons m-2 s-1 at 20°C (Table 1).

    2.2. Light microscopy

    Each cultured strain was observed under an Eclipse Ni-U microscope (Nikon, Tokyo, Japan) equipped with an oil immersion object (Plan Apo lambda 100×/1.45 oil). Cell images were obtained using a DS-Ri2 photomicrographic system (Nikon, Tokyo, Japan). Images were acquired using NIS-Elements software (Nikon, Tokyo, Japan). Numerical values of the morphological characteristics were determined by measuring 20-30 cells of each species from the photographic images.

    2.3. Confocal microscopy

    Confocal microscopy images were taken using a ZEISS LSM 980 (Carl Zeiss AG, Oberkochen, Germany) at the Core Facility for Supporting Analysis & Imaging of Biomedical materials at Wonkwang University. The autofluorescence of chlorophyll was used to visualize chloroplast structure.

    2.4. Transmission electron microscopy

    Aliquots of the culture were pelleted by centrifugation for 2 min at 2,300×g (5,000 rpm) in a Varispin L15R (Cryste, Bucheon, Korea). After removing the supernatant, the pelleted cells were fixed in 2.5% (v/v) glutaraldehyde mixed with BG-11 culture medium for 1 h at 4°C. The glutaraldehyde-fixed cell pellets were washed three times in BG-11 culture medium and postfixed in 4% (w/v) OsO4 for 1 h at 4°C. Dehydration was conducted at 4°C using a graded ethanol series of 50, 60, 70, 80, and 90% for 10 min each time and three times with 10 min changes of absolute ethanol. The pellets were brought to room temperature, transferred to propylene oxide twice for 20 min each time, and infiltrated with 50 and 75% Spurr’s embedding resin (Spurr 1969) in propylene oxide for 1 h and 100% overnight. The next day, the pellets were transferred to a new pure resin and polymerized at 71°C. The blocks were thin sectioned using an ultramicrotome (Leica, Wetzlar, Germany). Sections 70 nm thick were collected using slot copper grids, stained with 3% (w/v) uranyl acetate and Reynold’s lead citrate (Reynolds 1963), and observed and imaged using a JEM-1400 Plus transmission electron microscope in Korean Basic Science Institute operated at 120 kV (Jeol, Tokyo, Japan).

    2.5. DNA isolation, polymerase chain reaction, and sequencing

    All cultured cells were centrifuged during the exponential growth phase using a Varispin L15R centrifuge (Cryste, Bucheon, Korea). Genomic DNA was extracted from the cultures using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The nuclear 18S rRNA and ITS, and plastid rbcL and tufA genes were amplified using a combination of forward and reverse primers in a Nexus X2 thermal cycler (Eppendorf, Hamburg, Germany). SSU rDNA, 5.8S, and ITS2 region, and plastid rbcL and tufA genes were amplified using the following primers: ALG1F, ALG8R, ITS1, ITS4, ScenRub_F1, ScenRub_R1, tufAF, and tufAR (Table 2). The four genes were amplified by using 20 μL reaction mixture containing 5 μL of DNA, 1.0 μL of each primer (10 pmol), AccuPower Taq PCR PreMix (Bioneer, Daejeon, Korea) and enough distilled water to reach a total reaction volume of 20 μL. The 18S rRNA amplifications were performed using the following program: 2 min of denaturation at 95°C; 30 cycles of 95°C for 45 s, 58°C for 30 s, and 72°C for 45 s; a final extension at 72°C for 10 min. The ITS amplifications were performed using the following program: denaturation for 5 min at 95°C; 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min; a final extension at 72°C for 5 min. The rbcL amplifications were performed using the following program: denaturation for 2 min at 95°C; 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min; a final extension at 72°C for 5 min. The tufA amplifications were performed using the following program: denaturation for 2 min at 95°C; 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min; a final extension at 72°C for 5min. The PCR products were loaded onto an agarose gel (Agarose Bead Technologies, Alachua, FL, USA). Purified PCR products were sequenced using specific PCR primers (Macrogen Corp., Seoul, Korea), and 23 new sequences were generated: 7 sequences of the nuclear ITS1, 5.8S, and ITS2 rDNA regions; 7 of the nuclear SSU rDNA, 5 of the tufA, and 4 of the rbcL gene. Sequences were aligned using ClustalW (GitHub, San Francisco, CA, USA), integrated into Molecular Evolutionary Genetics Analysis Version 11 software (Kumar et al. 2012), and aligned visually. The conserved regions of the four loci were aligned across taxa and used for phylogenetic analysis. Additionally, we excluded unaligned parts of the sequence from the analysis.

    2.6. Phylogenetic analysis

    Forty-one strains used for phylogenetic analysis are listed in Table 1. The combined dataset consisted of published and newly generated sequences. Published sequences were downloaded from the National Center for Biotechnology Information database. Phylogenetic analysis was performed using a combined dataset of nuclear SSU rDNA, 5.8S, and ITS2 regions, and plastid rbcL and tufA gene sequences using the maximum likelihood (ML) method. Nuclear ITS2 sequences were determined and used to examine groups of genetically identical strains and as barcodes for species identification. The sequences of two species of Trebouxiophyceae (Trebouxia arboricola and Chlorella vulgaris) were used as outgroups to root the trees. Primer regions and ambiguously aligned regions were removed before phylogenetic analyses. Prior to the ML analysis, the best-fit model for the concatenated datasets was traced under the Bayesian information criterion (BIC) using Modeltest 3.7 (Posada and Crandall 1998). ML analyses were performed using RAxML version 8.2.12 (Stamatakis 2014) with a GTR+I+G model. We performed 1,000 tree inferences using the # option of the program to identify the best tree. Bootstrap values were computed based on 1,000 pseudoreplicates with the same substitution model. The trees were visualized using the FigTree v.1.4.4 program (http://tree. bioed.ac.uk/software/figtree/).

    2.7. ITS2 secondary structure prediction

    Secondary structures of the nuclear ITS sequences were predicted using the Mfold v.2.3 program (Walter et al. 1994;Zuker 2003), and the folding temperature was used as the default value. The secondary structures were compared with the published ITS2 structures of P. insigne SAG 30.93 (Sciuto et al. 2023), and common secondary structures were drawn using PseudoViewer3 (http://pseudoviewer.inha.ac.kr/). This typically results in several alternating folds for the same ITS2 sequences. The ‘true’ folding pattern corresponded to the P. insigne SAG 30.93 secondary structure (Sciuto et al. 2023). Secondary structures were used to identify single-base changes and were manually displayed as an ITS2 secondary structure diagram (Adobe Illustrator v28.7.1).

    2.8. CBC analysis

    A dataset comprising the ITS2 sequences and corresponding secondary structure of 27 strains of the genus Pleurastrum was constructed. The sequences in this dataset were aligned based on the data from Sciuto et al. (2023). The aligned sequences of Pleuatsrum strains were used to detect compensatory base change (CBCs) among species, and this analysis was performed using 4SALE version 1.7.1.

    3. RESULTS

    3.1. Morphology and ultrastructure

    P. insigne and P. microstigmatum, have similar morphologies. The P. insigne vegetative cells were primarily spherical (Fig. 1A) or sometimes surrounded by an extracellular layer (Fig. 1B). Unlike P. microstigmatum, oil droplets were not observed in the BBM medium under the light microscopy. Cup-shaped green chloroplasts occupied most of the cytoplasm with one pyrenoid (Fig. 1A-C). Vegetative cells ranged from 8 to 15 μm in diameter. In the matured phase, two (dyad), four (tetrad, sarcinoid), eight and many daughter cells were observed in each sporangium of 30 μm (Fig. 1D-F). As the number of daughter cells in the sporangia increased, they assumed an irregular shape (Fig. 1F). The P. insigne zoospores are ellipsoidal and contain two flagella and eyespots (Fig. 1G). There were several oil droplets and a chloroplast with a pyrenoid surrounded by a large starch in the cytoplasm. The pyrenoid was penetrated by the thylakoid. Several starch granules were interspersed within the thylakoids (Fig. 1H).

    P. microstigmatum vegetative cells were primarily spherical, as confirmed by several oil droplets observed in the BBM medium under the light microscope (Fig. 2A), were sometimes surrounded by an extracellular layer (Fig. 2B), and the cup-shaped green chloroplasts occupied most of the cytoplasm within the pyrenoid (Fig. 2A-C). The vegetative cells ranged from 7 to 20 μm. In the mature phase, there were two (dyad), four (tetra, sarcinoid), and eight daughter cells in each sporangium (35 μm; Fig. 2D-F). Several zoospores were formed, and they formed irregular colonies as they matured (Fig. 2G, H). P. microstigmatum zoospores were ellipsoidal and had two flagella (Fig. 2I). In the cytoplasm, there were several oil droplets and a chloroplast with a pyrenoid surrounded by large starch molecules. Thylakoids penetrated the pyrenoid. Several starch granules were interspersed within the thylakoids (Fig. 2J, K).

    3.2. Phylogenetic analysis

    Phylogenetic analysis was conducted using a dataset of nuclear SSU rDNA, 5.8S rDNA, the ITS2 region, and plastid rbcL and tufA genes encompassing 41 strains (Table 1). The ML phylogenetic tree was rooted with T. arboricola SAG 219-1a and C. vulgaris SAG 211-1b from the Trebouxiophyceae family as outgroups (Fig. 3). Among the 10 Pleurastrum species, eight-P. insigne, P. diplobionticum, P. aquaticum, P. minutum, P. rubrioleum, P. vacuolatum, P. microstigmatum, and P. isabeliense- were included in the analysis, forming a strongly supported monophyletic clade (Fig. 3).

    P. rubrioleum was a sister group to the clade containing P. microstigmatum and P. insigne (ML=100), with P. microstigmatum and P. insigne subsequently diverging from each other. The 10 P. insigne strains formed a monophyletic lineage with high bootstrap support (ML=100), revealing intraspecific sequence similarity in the ITS2 region ranging from 97.015 to 100% (Table 3). Similarly, the six P. microstigmatum strains formed a monophyletic lineage with strong support (ML=95), and their intraspecific ITS2 sequence similarity ranged from 96.255 to 100% (Table 4). These results provide robust molecular evidence supporting the distinctiveness of P. insigne and P. microstigmatum, with the Korean strains grouped consistently within their respective species. P. aquaticum, P. minutum, Chlorococcum szentendrense, and P. vacuolatum formed a well-supported monophyletic group (ML=99). A single strain of P. diplobionticum was the earliest diverging lineage, followed by P. isabeliense.

    3.3. Secondary structures prediction of nuclear ITS2

    The secondary structures of the ITS2 region in the domestic strains were predicted. The structure consisted of four helices and five spacers (Fig. 4). In ITS2 secondary structure, core structure of eukaryotes was confirmed in both species. (1) It consists of four helices, (2) with helix III being the longest (Fig. 4). (3) In helix II, an unpaired ‘U-U’ base pair was confirmed (Fig. 6). (4) In helix III, the ‘UGGU’ motif with conserved positions across species was confirmed (Fig. 7). Helices I and II of P. insigne and P. microstigmatum, respectively, differed in their final portions, with a conserved basal portion (Figs. 5, 6). The basal portion of each helix was identical between the two species, whereas base differences were primarily found in the final portion. In helix III, no differences were observed in the bases of the stems, excluding the loops (Fig. 7). Helix IV had an extremely short stem structure and composed a loop (Fig. 8). Among the five spacers, the spacer separation of helix IV from the 5.8S/LSU stem was the longest (eight bases), whereas the spacer between helices I and II was the shortest (three bases).

    A comparison of ITS2 sequences between the strains analyzed by Sciuto et al. (2023) and those from this study revealed significant intra- and inter-specific variations. Despite being conspecific, multiple sequences were identified in each species. In helix I, ‘C-GU’ and ‘G-A’ base changes were confirmed in the loop and stem between two species (Fig. 5). In helix II, the stem of P. insigne was longer than P. microstigmatum due to the base insertions of stem (Fig. 6). Additionally, ‘A-G’ base change and insertion were confirmed in the stem and loop of P. microstigmatum (Fig. 6). In helix III, ‘A-U’ base change was confirmed in the loop between two species (Fig. 7). In helix IV, the base insertions were confirmed in the loop of P. microstigmatum (Fig. 8). Further analysis revealed four distinct ITS2 sequence variants among the P. insigne strains: (1) SAG 30.93, SAG 213-11, and SAG 62.80; (2) SAG 66.80; (3) UTEX 2227; and (4) the FBCC strains (A16, A22, A23, A24, and A444) (Table 3). Similarly, three distinct ITS2 sequence types were identified among P. microstigmatum strains: (1) UTEX 1777; (2) CCAP 11/52 and SAG 11.43; (3) TKAC 1035; (4) the FBCC strains (A1226 and A1335) (Table 4).

    3.4. Molecular signatures

    The sequences of the ITS2 gene were selected as molecular signatures for the species delimitation of the genus Pleurastrum. Each of the eight species included in the phylogenetic analysis exhibited unique molecular signatures. In helix I, the P. insigne had a different base pair, ‘C : G’ (No. 1 in Fig. 9), compared to the ‘U : A’ base pair of the closely related species P. microstigmatum. In the loop region of helix I, the P. insigne had unique sequence, ‘C’ (No. 2 in Fig. 9), compared to the ‘U’ base of other species. The P. rubrioleum had two different base in loop, ‘C and U’ (No. 3, 4 in Fig. 9), compared to other species. The P. isabeliense had unique sequences in loop, ‘UCU’ (No. 5 in Fig. 9), compared to other species. In the stem region, the P. isabeliense had a different base pair, ‘A : U’ (No. 6 in Fig. 9), compared to the ‘C : G’ base pair of the P. diplobionticum. The P. diplobionticum had unique sequence in loop, ‘A, C and C’ (No. 7, 9, 10 in Fig. 9), compared to other species. Also, the P. diplobiontium had a different base pair, ‘U : A’ (No. 8 in Fig. 9), compared to the ‘A : U’ base pair of other species. The P. vacuolatum had unique sequence in loop, ‘A and C’ (No. 11, 13 in Fig. 9), compared to other species. The P. vacuolatum had a different base pair, ‘U : A’ (No. 12 in Fig. 9), compared to the ‘C : G’ base of the P. minutum. Also, the P. minutum had a different base pair, ‘G : C’ (No. 14 in Fig. 9), compared to the ‘A : U’ base pair of the closely related species P. aquaticum. And the P. minutum had a different base in loop, ‘U’ (No. 15 in Fig. 9), compared to other species.

    In helix II, the P. insigne had two different base in loop, ‘CG’ (No. 1 in Fig. 10), compared to other species. The P. microstigmatum had a different base pair, ‘C : G’ (No. 2 in Fig. 10), compared to the ‘U : A’ base pair of the P. rubrioleum. In the loop region, the P. microstigmatum had a different base, ‘A’ (No. 4 in Fig. 10), compared to other species. The P. rubrioleum had unique sequences in loop, ‘UACC and C’ (No. 5, 6 in Fig. 10), compared to other species. The P. isabeliense had unique sequences in loop, ‘UAAUA’ (No. 7 in Fig. 10), compared to other species. The P. diplobionticum had a different base pair, ‘A : U’ (No. 8 in Fig. 10), compared to the ‘G : C’ base pair of the P. isabeliense. Also, the P. diplobionticum had unique sequences in loop, ‘AAC and A’ (No. 9, 10 in Fig. 10), compared to other species. The P. vacuolatum had a different base pair, ‘A : U’ (No. 11 in Fig. 10), compared to the ‘G : C’ base pair of the P. diplobionticum. The P. minutum had unique sequences in the loop, ‘G and ACUUUC’ (No. 12, 14 in Fig. 10), compared to other species. Also, the P. minutum had a different base pair, ‘G : C’ (No. 13 in Fig. 10), compared to the ‘A : U’ base pair of the P. vacuolatum. The P. aquaticum had unique sequences in the loop, ‘AAA’ (No. 15 in Fig. 10), compared to other species.

    The ITS2 sequences were also used to detect compensatory base change (CBCs) among the Pleruatsrum species (Table 5). In this study, no CBCs were detected within the same species for all Pleuratsrum species except for P. microstigmatum, while CBCs ranging from one to eight were detected between different species. One CBC was detected between the P. microstigmatum TKAC 1035 strain and P. microstigmatum FBCCA1226, FBCC-A1335 and UTEX 1777 (No. 3 in Fig. 10).

    3.5. Taxonomic descriptions

    Pleurastrum insigneChodat 1894

    Kawasaki et al. 2015, Fig. 5.

    Basionym.Pleurastrum insigneChodat 1894.

    Homotypic synonym.Leptosira insignis (Chodat)

    Sprung & Wujek 1971.

    Heterotypic synonyms.

    Chlorococcum oleofaciensTrainor & Bold 1953.

    Chlorococcum citriforme Archibald & Bold 1970.

    Chlorococcum sphacosum Archibald & Bold 1970.

    Chlorococcum tatrense Archibald & Bold 1979.

    Reference strain. SAG 30.93.

    Material examined. FBCC-A16 (Wolsong pond, 423- 23, Wolsong-ri, Pyeonghae-eup, Uljin-gun, Gyeongsangbuk- do, Korea, 36°44ʹ38.9ʺN, 129°27ʹ48.9ʺE, 02. 15.2019).

    Molecular vouchers. SSU rRNA: PV866813; ITS2: PV866814; rbcL: PV797563; tufA: PV797564.

    Other molecularly verified strains. SAG 62.80, SAG 213-11, SAG 66.80, UTEX 2227.

    Description. The cells were mostly spherical. Vegetative cells were approximately 8-15 μm in diameter, and that of mature cells ranged from 20-30 μm in diameter. The cup-shaped chloroplasts contained one pyrenoid. Colonies, which are irregular groups of individual vegetative cells, are sometimes formed. Up to 64 daughter cells were observed at the sporangia stage. When they were four cells in each of the sporangia, cells formed a ‘sarcinoid’ shape, and oil droplets were observed. The pyrenoid matrix was infiltrated by the thylakoids and was surrounded by a continuous starch plate. Biflagellate zoospores were ellipsoidal and motile.

    Distribution. Britain and Ireland (John et al. 2011), Germany (Sluiman and Gärtner 1990;Mikhailyuk et al. 2019), Switzerland (Sluiman and Gärtner 1990), India (Kargupta and Keshri 2022), Czech Republic (Archibald 1979;Ettl and Gärtner 1995), Slovakia (Ettl and Gärtner 1995), Romania (Cărăuş 2017), Russia (Black Sea) (Ettl and Gärtner 1995), Spain (Cambra Sánchez et al. 1998), New Zealand (Broady et al. 2012), USA (Ettl and Gärtner 1995), and Korea (this study).

    Pleurastrum microstigmatum (P.A. Archibald & Bold) K. Sciuto, M.A. Wolf, M. Mistri & I. Moro 2023

    Kawasaki et al. 2015, Fig. 6.

    Basionym.Chlorococcum microstigmatum Archibald & H.C. Bold 1970.

    Synonyms.Chlorococcum microstigmatum Archibald & H.C. Bold 1970.

    Reference strain. UTEX-1777.

    Material examined. FBCC-A1335 (Daewon reservoir, 106-1, Deokchon-ri, Okseong-myeon, Gumi-si, Gyeong sangbuk-do, Korea, 36°16ʹ9.9ʺN, 128°15ʹ2.5ʺE, 01.22. 2020).

    Molecular vouchers. SSU rRNA: PV866815; ITS2: PV866816; rbcL: PV797566; tufA: PV797565.

    Other molecularly verified strains. CCAP 11/52, TKAC 1035, SAG 11.43.

    Description. The cells were mostly spherical. Vegetative cells were approximately 7-20 μm in diameter, and that of mature cells was approximately 35 μm. The chloroplasts were cup-shaped with one pyrenoid. Colonies, which are irregular groups of individual vegetative cells, were sometimes formed. Up to 16 daughter cells were observed at the sporangia stage. Oil droplets were observed when four cells in each of the sporangia, and cells formed a ‘sarcinoid’ shape. Pyrenoid matrix was infiltrated by a thylakoid and were surrounded by a continuous starch plate. Biflagellate zoospores were ellipsoidal and motile.

    Distribution. Ukraine (Cherevko 1993), USA (Komáek and Fott 1983), Finland (SAG 2025), Japan (Kawasaki et al. 2015), and the Korea (this study).

    4. DISCUSSION

    The genus Pleurastrum has undergone numerous taxonomic revisions due to its historical reliance on morphological traits for classification. Chodat (1894) first described the Pleurastrum filamentous form but without comprehensible details, leading to subsequent revisions (Chodat 1894;Sciuto et al. 2023). Visher (1933), Tupa (1974), and Sluiman and Gärtner (1990) attempted to re-examine this genus in challenges of morphological characteristic. Notably, Sluiman and Gärtner (1990) observed a filamentous-like form in cultures grown in BBM medium, but only after the production of numerous zoospores. Similarly, Sciuto et al. (2023) noted a transient filamentous-like form upon an isolate from Antartic Pleurastrum samlple; however, this characteristic disappeared after prolonged culture, suggesting that the filamentous form can only be observed under specific environmental conditions. In the present study, although cell elongation was observed during rapid division, the filamentous form was not identified. This could be because our material was cultured for several years, consistent with the findings reported by Sciuto et al. (2023), or because it suggests that a true filamentous form does not exist.

    All of the Pleurastrum morphological traits described by Chodat (1894)-except for the filamentous-like form-were observed in this study, including coccoid cells, sporangia containing daughter cells, sarcinoid forms, and biflagellate zoospores. However, light microscopy and ultrastructural observations did not reveal any significant morphological differences that could definitively distinguish the two species under study. Variations in physiological characteristics such as the presence of oil droplets have been observed in different growth media. For instance, oil droplets were visible under a light microscope (Fig. 2A), and Kawasaki (2015) reported species-specific coloration of oil droplets after three months of culture in agar-AF6 medium. The results revealed that the color ranged from colorless to brownish or orange red, considering the species (Kawasaki et al. 2015). Therefore, to clarify the species-specific morphological traits within Pleurastrum, a more extensive comparative analysis involving multiple species is necessary.

    Molecular analyses have provided robust evidence for distinguishing species. Sciuto et al. (2023) identified well-supported clades of P. insigne and P. microstigmatum in phylogenetic trees based on 18S rRNA and ITS gene sequences. In our phylogenetic trees based on the 18S rRNA, ITS2 gene, plastid rbcL, and tufA genes, the Pleurastrum strains of the two species formed monophyletic groups and our analysis is consistent with the finding of Sciuto et al. (2023).

    Chlorococcum szentendrense K2-9 formed a monophyletic group within Pleurastrum, and Tetracystis aeria SAG 89.90 formed a monophyletic group within Chlorococcum. These results are consistent with the previous studies, but the evidence remains insufficient. Therefore, further study is needed to determine their precise taxonomic position (Greipel et al. 2023;Sciuto et al. 2023).

    The secondary structure analysis of the ITS2 region further reinforced the distinction between P. insigne and P. microstigmatum. Typically, the ITS2 region is characterized by four helices conserved across eukaryotic lineages (Schultz et al. 2005). In this study, the notable base differences between the two species were predominantly localized in the terminal portions of helices I and II, indicating clear molecular differentiation. Moreover, intraspecific sequence differences in P. insigne and P. microstigmatum was highlighting the genetic diversity within each species. These sequence variations, particularly the conserved base substitutions and insertions in the helices, supported the phylogenetic clustering of these strains into well-defined monophyletic clades, underscoring the genetic distinction between P. insigne and P. microstigmatum.

    CBC analysis based on the ITS2 secondary structure successfully distinguished Pleurastrum species and supported the phylogenetic results. It has been demonstrated that the presence of a CBC between two organisms within the same genus indicates a 93% probability that they belong to different species (Müller et al. 2007). No CBCs were detected within monophyletic Pleurastrum species, whereas between species, CBCs ranging from one to eight were detected (Table 5). Notably, one CBC was detected within P. microstigmatum TKAC 1035 when compared to other P. microstigmatum strains. However, consistent with the phylogenetic analysis, P. microstigmatum TKAC 1035 formed a monophyletic group, in agreement with previous study (Kawasaki et al. 2015). This CBC may reflect genetic diversity within the species, and is considered to represent a possible early stage of speciation. In such cases, species delimitation methods such as Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) could be applied to more precisely define species boundaries.

    ACKNOWLEDGEMENTS

    This research was supported by a grant (No. NNIBR 20251108) from the Nakdonggang National Institute of Biological Resources (NNIBR), and by the Korea Environment Industry & Technology Institute (KEITI) through the project to make multi-ministerial national biological research resources a more advanced program funded by the MOE (RS-2021-KE001788).

    CRediT authorship contribution statement

    YH Kim: Investigation, Resources, Data Curation, Writing-Original draft. BY Jo: Investigation, Resources, Data Curation. JH Lee: Investigation, Resources. K Hong: Investigation. SW Nam: Conceptualization, Methodology, Supervision, Project administration. W Shin: Supervision, Writing-Review and editing.

    Declaration of Competing Interest

    The authors declare no conflicts of interest.

    Figure

    KJEB-43-3-275_F1.jpg

    Light, confocal, and transmission electron micrographs of Pleurastrum insigne. (A, B) A vegetative cell indicating pyrenoid. (C) Cupshaped chloroplast under confocal micrographs. (D) A sporangium forming a dyad. (E) A sporangium forming a tetrad (sarcinoid). (F) A sporangium containing eight cells. (G) Biflagellate zoospore. (H-K) Transmission electron micrographs of P. insigne. Py, pyrenoid; e, eyespot; od, oil droplet; S, starch; ty, thylakoid. Scale bars represent: A-G, 10 μm; H-J, 2 μm; K, 5 μm.

    KJEB-43-3-275_F2.jpg

    Light, confocal, and transmission electron micrographs of Pleurastrum microstigmatum. (A, B) A vegetative cell indicating oil droplet and pyrenoid. (C) Cup-shaped chloroplast under confocal micrographs. (D) A sporangium forming a dyad. (E) A sporangium forming a tetrad (sarcinoid). (F) A sporangium containing eight cells. (G, H) Early vegetative cells with many maturing zoospores forming a colony. (I) Biflagellate zoospore. (J, K) Transmission electron micrographs of P. microstigmatum. od, oil droplet; Py, pyrenoid; N, nucleus; S, starch; ty, thylakoid. Scale bars represent: A-I, 10 μm; J, 2 μm; K, 2 μm.

    KJEB-43-3-275_F3.jpg

    Phylogenetic tree based on a combined nuclear SSU rDNA, 5.8S, ITS2 region, and plastid rbcL and tufA gene sequences data. Maximum- likelihood (ML) bootstrap values are presented above or below the branches. The bold branches indicate strongly supported values (ML=100). Scale bar, 0.05 substitutions/site.

    KJEB-43-3-275_F4.jpg

    Predicted secondary structures of the internal transcribed spacer 2 transcripts of Pleurastrum insigne. The secondary structure of Pleurastrum was composed of four helices, with helix III being the longest.

    KJEB-43-3-275_F5.jpg

    Predicted secondary structures of the internal transcribed spacer 2 helix I. (A) Pleurastrum insigne. (B) Pleurastrum microstigmatum. The changes and insertions in the base between the two species are indicated in red.

    KJEB-43-3-275_F6.jpg

    Predicted secondary structures of the internal transcribed spacer 2 helix II. (A) Pleurastrum insigne. (B) Pleurastrum microstigmatum. The changes and insertions in the base between the two species are indicated in red.

    KJEB-43-3-275_F7.jpg

    Predicted secondary structures of the internal transcribed spacer 2 helix III. (A) Pleurastrum insigne. (B) Pleurastrum microstigmatum. The changes and insertions in the base between the two species are indicated in red. The ‘UGGU’ motif, one of the core structures in eukaryotes, was indicated.

    KJEB-43-3-275_F8.jpg

    Predicted secondary structures of the internal transcribed spacer 2 helix IV. (A) Pleurastrum insigne. (B) Pleurastrum microstigmatum. It was confirmed that helix IV of both species consists almost entirely of a loop structure. The changes and insertions in the base between the two species are indicated in red.

    KJEB-43-3-275_F9.jpg

    Molecular signatures of helix I in the ITS2 gene. Base differences in the loop regions were indicated using different colors (A-red, U-green, G-yellow, C-blue), while differences in base pairs were highlighted with a different background color.

    KJEB-43-3-275_F10.jpg

    Molecular signatures of helix II in the ITS2 gene. Base differences in the loop regions were indicated using different colors (A-red, U-green, G-yellow, C-blue), while differences in base pairs were highlighted with a different background color.

    Table

    Strains used in this study listed in alphabetical order and GenBank accession numbers for their SSU rDNA, ITS, rbc L, and tuf A sequences

    The strains used in this study have been indicated in bold

    PCR and sequencing primers

    Pairwise similarities (in %) of the ITS2 gene sequences among 10 Pleurastrum insigne strains

    Pairwise similarities (in %) of the ITS2 gene sequences among six Pleurastrum microstigmatum strains

    Matrix showing the compensatory base change (CBCs) among Pleurastrum ITS2 secondary structures

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    Vol. 40 No. 4 (2022.12)

    Journal Abbreviation 'Korean J. Environ. Biol.'
    Frequency quarterly
    Doi Prefix 10.11626/KJEB.
    Year of Launching 1983
    Publisher Korean Society of Environmental Biology
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