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Newly Recorded Chlorophytes, Monoraphidium Subclavatum, Deuterostichococcus Epilithicus, and Pseudostichococcus Monallantoides in Korea

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Newly Recorded Chlorophytes, Monoraphidium Subclavatum, Deuterostichococcus Epilithicus, and Pseudostichococcus Monallantoides in Korea Journal of Species Research 9(3):181-190, 2020 Newly recorded chlorophytes, Monoraphidium subclavatum, Deuterostichococcus epilithicus, and Pseudostichococcus monallantoides in Korea Sung Ro Yoon, Do Hyun Kim and Ok-Min Lee* Department of Life Science, College of Natural Science, Kyonggi University, Suwon 16227, Republic of Korea *Correspondent: [email protected] Monoraphidium subclavatum (FBCC-A409) of this study clustered in the same clade as Messastrum gracile strains in the 18S rDNA phylogeny. Compared to Messastrum gracile, Monoraphidium subclavatum did not form a colony, and the curvature of the cell was slightly curved or slightly crescent-shaped. This result means that the genus Monoraphidium is still based on the morphospecies concept, and was not monophyletic and not distinguishable as a separate genus. Two Stichococcus-like strains of this study (NIBRCL0000114567, NIBRCL0000114571) belong to Deuterostichococcus epilithicus and Pseudostichococcus monallantoides respectively in phylogenetic analysis using 18S rDNA sequences. These two species are consistent with recent research in the morphology and the genetic analyses using 18S and ITS rDNA sequences. We reported M. subclavatum, D. epilithicus, and P. monallantoides as newly recorded species in Korea. Keywords: Aerophytic algae, Deuterostichococcus, Monoraphidium, Pseudostichococcus Ⓒ 2020 National Institute of Biological Resources DOI:10.12651/JSR.2020.9.3.181 ble pyrenoid under the light microscope (Hindák, 1970). INTRODUCTION Species are largely recognized by cell shape, size, and characteristics associated with cell curvature. Therefore, Green algae commonly inhabit terrestrial habitats, ma- intermediate forms may be encountered, which are im- rine shorelines, streams, ponds, and lakes. Green algae possible to classify under one or other species (John et al., also participate in wide variety of important biotic asso- 2011). The genus Monoraphidium is not monophyletic ciations with a diverse range of cell organization, repro- based on rbcL and 18S rDNA sequences (Da Silva et al., ductive processes, and morphology compared with other 2017). algae (Bold and Wynne, 1978). They also contain chlo- Stichococcus-like algae are very common and could rophyll tissues associated with terrestrial plants, such as be found in almost all types of habitats from freshwater, pyrenoids that store photosynthetic products, and photo- brackish, marine to soil and snow (Butcher, 1952; Kol, synthetic pigments (Happy-Wood, 1988). 1968; Ettl and Gärtner, 2014). They are characterized by Monoraphidium taxa of the class Chlorophyceae are a simple morphology. Stichococcus-like algae has mostly solitary and mainly planktonic organisms. Monoraphid­ short cylindrical and elongate or spherical to slightly oval ium cells are more or less spindle-shaped, straight, curved shaped cells containing chloroplast without pyrenoid ex- or spirally twisted, gradually or abruptly narrowing to cept in two species, Deuterostichococcus marinus (Dea- acute apices. They are thin-walled and without a mucilag- son) Pröschold and Darienko and Tetratostichococcus inous envelope, with a single chloroplast located in cell jenerensis (Neustupa, Eliás and Sejnohová) Pröschold and periphery and a naked pyrenoid when examined under the Darienko (Neustupa et al., 2007; Pröschold and Darienko, light microscope (Komárek and Fott, 1983; Da Silva et 2020; Pröschold et al., 2020). They reproduce vegeta- al., 2017). Komárková-Legnerová (1969) separated this tively, but only Pseudostichococcus monallantoides was genus from Ankistrodesmus and transferred it to Monora­ reported to have zoospore formation (Moewus, 1951). Re- phidium, those single-celled species of Ankistrodesmus cent studies based on molecular data have shown a high carrying serially arranged autospores and without a visi- biodiversity among these organisms and put into question 182 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 3 the traditional generic and species concept (Nestupa et schoff and Bold (1963) using a Pasteur pipette. The colo- al., 2007; Hodac et al., 2016; Pröschold and Darienko, nies in the solid medium were transferred to liquid Bold’s 2020). Pröschold and Darienko (2020) studied 34 strains basal medium (Bold, 1949). The algal strains were culti- - assigned as Stichococcus, Pseudostichococcus, Diplos­ vated under a light/dark cycle of 16 : 8 with 40 μmol m 2 - phaera and Desmococcus, and revised these genera using s 1 light at 25℃ (Stein, 1973; Bold and Wynne, 1978). 18S and ITS rDNA sequences. They also proposed four One strain of this study was deposited in the Nakdong- new genera and four new species. Many described species gang National Institute of Biological Resources (spec- of Stichococcus-like algae were transferred to new genera imen no. FBCC-A409) and two strains in the National and synonymized using polyphasic studies. Institute of Biological Resources (specimen nos. NI- The generic name Deuterostichococcus Pröschold and BRCL0000114567 and NIBRCL0000114571). Darienko was introduced by Pröschold and Darienko Samples were observed at 100 to 1000× magnification (2020) for four species originally included in Stichococ­ using an optical microscope (Axio Imager A2, Carl Zeiss, cus Nägeli. Stichococcus deasonii Neustupa, Eliás and Germany; Olympus BX53, Olympus, Japan), and images Sejnohová and S. allas Reisigl required further nomencla- were taken using AxioCam HRC camera (Carl Zeiss, Ger- tural clarification (Pröschold et al., 2020). Deuterostichoc­ many) and Olympus UC-90 (Olympus, Japan) at 1000× occus deasonii (Deason) Pröschold and Darienko trans- magnification. ferred from Stichococcus deasonii Neustupa, Eliás and We followed the taxonomic classification from Al- Sejnohová needed to be changed to D. marinus (Deason) gaeBase (Guiry and Guiry, 2020), and carried out the Pröschold and Darienko because the basionym of this is morphological identification of the taxa as described by Hormidium marinum Deason (1969). Deuterostichococ­ Chodat (1913), Skuja (1948), Reisigl (1964), Hirose et cus allas (Reisigl) Pröschold and Darienko was proposed al. (1977), Komárek and Fott (1983), John et al. (2011), to be correct in D. tetrallantoideus (Kol) Pröschold and Hodac et al. (2016), Beck et al. (2019), Pröschold and Darienko because Stichococcus allas Reisigl (1964) was a Darienko (2020), and Pröschold et al. (2020). heterotypic synonym (Pröschold et al., 2020). A total of 51 Stichococcus species names were in the DNA extraction database, but currently only eight species are accepted Cells in the exponential phase were harvested via cen- taxonomically (Guiry and Guiry, 2020). Stichococcus-like trifugation (3000 rpm), reconstituted with 1 ×TE buffer, algae were reorganized into eight genera including Sti­ and stored at -20℃ until further analysis. Total genomic chococcus based primarily on genetic data because these DNA was extracted using the cetyltrimethylammonium taxa are morphologically variable and are not easily dis- bromide (CTAB) method described by Richards et al. tinguished for each other (Pröschold and Darienko, 2020). (2001). In this study, chlorophytes were isolated from the mi- crobial samples of soil surfaces in Suwon-si, Gyeong- DNA amplification and sequencing gi-do and planktonic samples from the Joochun River, Hoengseong-gun, Gangwon-do. We investigated the Nuclear 18S rDNA sequences were amplified using a morphology and sequenced partial 18S rDNA gene of the long PCR technique with primers 18SF (5′-AAC CTG three species. Our results indicate that these organisms GTT GAT CCT GCC AGT-3′) and 18SR (5′-TGA TCC represent three unrecorded species in Korea. TTC TGC AGG TTC ACC TAC G-3′) (Katana et al., 2001). PCR was carried out in 20 μL reaction mixtures containing 17 μL of sterile distilled water, 1 μL of each MATERIALS AND METHODS primer (10 pmoles), and 1 μL extracted DNA. PCR cy- cling was performed on a Mastercycler®Nexus gradient Sampling, culture, and morphological analysis 6331 Model (Eppendorf, Germany) using the following The specimens were collected from Suwon-si, Gyeong- program: 94℃ for 3 min; followed by 30 cycles of 94℃ gi-do in May 2019 and Joochun River, Hoengseong-gun, for 30 sec, 58℃ for 30 sec, and 72℃ for 60 sec; with a fi- Gangwon-do in December 2018. nal extension at 72℃ for 5 min (Katana et al., 2001). The Samples were scraped from soil surface using a soft resulting PCR products were subjected to electrophoresis brush or sterile spatula in Suwon-si (Kiel and Gaylarde, in 1.0% agarose gel (Promega, WI), stained with ethidium 2006). The planktonic algae were netted vertically and bromide, and visualized under ultraviolet light on a tran- horizontally using a plankton net of 25 μm mesh and 30 silluminator. cm diameter from stagnant or flowing freshwater areas of The PCR products were purified using the MEGA the Joochun River (Sournia, 1978). quick-spinTM Plus DNA Purification Kit (iNtRON, Korea Samples were separated into solid medium by addition and sequenced (ABI 3730XL) with the same primers used of 1.5% agar to Bold’s Basal Medium as modified by Bi- for amplification. August 2020 Yoon et al. Monoraphidium subclavatum, Deuterostichococcus epilithicus and Pseudostichococcus monallantoides 183 The SSU rDNA sequence were aligned and compared braunii. The Monoraphidium subclavatum strain (FB- with previously published data in BioEdit (Hall, 1999). CC-A409) of this study grouped into the same clade as All the sequences acquired were deposited in the NCBI Messastrum gracile strains, and the 18S rDNA similar- GenBank database.
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