INTRODUCTION

The genus Chroomonas is a group of biflagellate algae in Cryptophyceae, and most species inhabit marine ecosystems globally, while a few species are restricted to freshwater (Hansgirg 1885;Bucher et al. 1967;Novarino 2003). Chroomonas species are blue-green due to the chloroplast containing the phycobiliprotein Cr-phycocyanin (Novarino 2003). The matrix of the pyrenoid is traversed by thylakoid membranes (Dodge 1969;Clay and Kugrens 1999; Deane et al. 2002).

Although Chroomonas species can be recognized easily by their color due to phycobiliprotein, the characters that can be used to define this genus are still disputed (Hill 1991). The periplast plate is one of the necessary characters for describing cryptophycean genera (Novarino 2003). The periplast plate in the genus Chroomonas is characterized as rectangular. However, the original type material of C. nordstedtii, a type species of Chroomonas, possesses periplasts with hexagonal plates (Novarino and Oliva 1998). An eyespot was reported only in Chroomonas and Hemiselmis among the genera of Cryptophyceae by transmission electron microscopy. The eyespot, however, was described in only some species: Chroomonas ligulata (Novarino and Lucas 1993), Chroomonas mesostigmatica (Dodge 1969;Lucas 1982), Chroomonas africana (Meyer and Pienaar 1984), and Hemiselmis amylosa (Clay and Kugrens 1999). The phycobiliprotein of Chroomonas species is commonly Cr-phycocyanin 645, whereas a few species rarely possess Cr-phycocyanin 630 (Hill and Rowan 1989).

Approximately 200 cryptophycean species have been reported (Guiry and Guiry 2021), and their species diversity was estimated to range from 300 (Novarino 2003) to 1,200 (Andersen 1992) species. However, only 11 species have been reported in Korea, and most species belong to the genus Cryptomonas (NIBR 2020).

In this study, we provide taxonomic information on C. nordstedtii and C. coerulea based on morphology, ultrastructure, and phylogenetic data and report the genus Chroomonas and two previously unrecorded species in Korea.

MATERIALS AND METHODS

1. Sampling and clonal culture of Chroomonas

Cultures of C. nordstedtii CR40 and C. coerulea CR41 were established by single-cell isolation from freshwater samples collected at Pungho pond (37°44ʹ18.0ʺN, 128°57ʹ13.0ʺE) and Ichonji pond (35°58ʹ47.4ʺN, 126° 46ʹ14.6ʺE) in January 2017 and November 2016, respectively. The cultures were grown in AF-6 medium at 25°C under a 14 : 10 light : dark cycle and a light intensity of 4,000 lux provided by cool-white fluorescent lamps.

2. Light microscopy

Living C. nordstedtii and C. coerulea cells were studied using a Nikon ECLIPSE Ni-U microscope (Nikon, Japan) equipped with differential interference contrast optics. Images were captured using a digital camera (DS-Ri2, Nikon). The length and width of the cells were measured using the NIS-Elements BR 4.50.00 program.

3. Scanning electron microscopy

For scanning electron microscopy, cultures of the two species were preserved in 2% (v/v) glutaraldehyde with AF-6 culture for 1 h. The fixed cells were collected on 5-μm polycarbonate filters (Isopore^TM Membrane filters, Millipore Ltd., Billerica, MA), rinsed three times with the same culture media, postfixed with 1% (w/v) osmium tetroxide in distilled water for 1 h, and then rinsed three times with distilled water. Filters with specimens were serially dehydrated in an ethanol series (50%, 60%, 70%, 80%, 90% and 100%) and dried using an E3100 Critical Point Dryer (Quorum Technologies Ltd., Laughton, UK). The dried filters were mounted on stubs and coated with platinum. Cells were viewed with a Mira-3 FE-SEM (Tescan Ltd., Brno, Czech) at 5-10 kV.

4. Transmission electron microscopy

For transmission electron microscopy, the cells were prefixed in a 1 : 1 mixture of 5% (V/V) glutaraldehyde and AF-6 culture media for 1 h at 4°C. The glutaraldehyde- fixed cells were washed 3 times in AF-6 culture media and postfixed in 1% (W/V) osmium tetroxide for 1 h at 4°C. The fixed cells were rinsed three times with distilled water. Dehydration was carried out at 4°C using a graded ethanol series of 50, 60, 70, 80, and 90% for 10 min each and three times with 10 min changes of pure ethanol. Pellets were then brought to room temperature and transferred to propylene oxide two times for 20 min each, immersed in 50% and 75% Spurr’s embedding resin (Spurr 1969) in propylene oxide for 1 h each, and immersed in 100% Spurr’s embedding resin overnight. The following day, the pellets were moved to new pure resin and polymerized at 70°C. Blocks were thin-sectioned on a PT-X ultramicrotome (RMC Products, Boeckeler Instruments, Tucson, AZ). Sections of 70 nm thickness were collected on slot copper grids, stained with UranyLess solution (Electron Microscopy Sciences, Philadelphia, USA) and Reynold’s lead citrate (Reynolds 1963), and observed and photographed using a JEM-1400 Plus transmission electron microscope, Bio HVEM system in KBSI operated at 120 kV (JEOL, Tokyo, Japan).

5. DNA extraction, amplification, and sequencing

Approximately 10 mL aliquots of culture media were obtained in the exponential growth phase. Cells were harvested by centrifugation (1,330 g; model 5415, Eppendorf, Hamburg, Germany) for 1 min at room temperature, followed by washing three times with sterilized distilled water. According to the manufacturer’s protocol, total genomic DNA was extracted from the pellet using Insta- Genetm Matrix (BIO-RAD, CA, USA). Polymerase chain reaction (PCR) was performed for the nuclear SSU rRNA and chloroplast rbcL genes using specific primers (Table 1). PCR amplification was performed with a total volume of 30 μL containing EF-Taq (SolGent, Daejeon, Korea), each dNTP, 10× Ex Taq buffer, each primer, and 20 ng of template DNA. The SSU rDNA gene was amplified using a DNA Engine Tetrad 2 Peltier Thermal Cycler (BIORAD, CA, USA) with the following conditions: initial denaturation at 95°C for 2 min; 35 cycles each of 95°C for 2 min, 55°C for 1 min, and 72°C for 1 min; a final extension at 72°C for 10 min; and holding at 4°C. According to the manufacturer’s protocol, the PCR products were purified using a Multiscreen filter plate (Millipore Corp., MA, USA). The purified template was sequenced with the PRISM BigDye^TM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). The sequence alignment was edited using the Genetic Data Environment program (Smith et al. 1994), and the aligned sequence was registered in GenBank (Table 2).

6. Phylogenetic analyses

Sequence data of 37 strains (Table 2) were used for MODELTEST v.3.7 (Posada and Cradall 1998) and maximum likelihood (ML) analyses. ML analysis was carried out by RAxML v8.2.4 (Stamatakis 2014) using the general time-reversible plus gamma (GTR+G) model with 1,000 rounds of random sequence addition, followed by a heuristic search using tree-bisection reconnection (TRB) branch swapping. Bayesian analysis was performed using MrBayes v3.2 (Ronquist et al. 2012) to construct random inference trees with the GTR+G+I model 2,000,000 times. A phylogenetic tree was constructed every 1,000 cycles, and the burn-in point was graphically identified based on the likelihood score of the phylogenetic tree (Tracer v1.5; http:// tree.bio.ed.ac.uk/software/tracer/). Nucleotide sequences were examined using MEGA X software to check for similarity with reference NCBI strains.

RESULTS AND DISCUSSION

1. Taxonomic summary

Phylum Cryptophyta Cavalier-Smith, 1986

Class Cryptophyceae Fritsch, 1927

Order Cryptomonadales Pacher, 1913

Family Hemiselmidaceae Butcher ex Silva, 1980

Genus ChroomonasHansgirg, 1885

1) Chroomonas nordstedtiiHansgirg 1885

Material examined. Freshwater was collected from Pungho pond, Hasidong-ri, Gangdong-myeon, Gangneung-si, Gangwon-do, Republic of Korea (N 37°44ʹ18.0ʺ, E 128° 57ʹ13.0ʺ), on January 25, 2017.

Synonym.

Cryptomonas nordstedtii (Hansgirg) Senn, 1885.

Description.Chroomonas nordstedtii cells were biflagellate (Figs. 1A, 2A). Cells were blue-green colored and ovalshaped (Fig. 1A, B). The size was 10.51-16.21 μm (n=61, mean=12.82±1.26 μm) long and 5.24-10.16 μm (n=59, mean=7.17±1.04 μm) wide. Two Maupas ovals were observed in the center of the cell (Fig. 1B). The periplast was composed of hexagonal plates (Fig. 2A, B). The cells had a peripheral chloroplast with the pyrenoid penetrated by several thylakoid membranes (Fig. 3A-D). The nucleus was located at the posterior end of the cell and contained many chromatin areas (Fig. 3A, C). Several ejectosomes were observed in five rows around the gullet (Fig. 3A, B). The Golgi body was located ventrally and anteriorly near the gullet (Fig. 3A, B). Several starch grains were located in the periplastidal compartment (Fig. 3A-C). The nucleomorph was free of the pyrenoid and contained a number of electron-dense granules (Fig. 3E). Among the flagellar apparatus components, the rhizostyle was arranged perpendicular to the striated fibrous and associated microtubular roots (Fig. 3F). The striated fibrous root extended parallel to the associated microtubular root, which comprised three microtubules (Fig. 3G, H).

Distribution. Great Britain (Wołowski 2002;Whitton et al. 2003;John et al. 2011), Czech Republic (Hasler et al. 2007), Germany (Täuscher 2014, 2016;Stutz et al. 2018), the Netherlands (Veen et al. 2015), Romania (Caraus 2017), Scandinavia (Karlason et al. 2018), Slovakia (Hindák and Hindáková 2016), Spain (Alvárez Cobelas 1984), Sweden (Skuja 1948, 1956), Laurentian Great Lakes (Prescott 1962), Northwest Territories (Sheath and Steinman 1982), Québec (Poulin et al. 1995), Brazil (Menezes 2010;Dunck et al. 2018), Iraq (Maulood et al. 2013), Bangladesh (Ahmed et al. 2009), New Zealand (Chapman et al. 1957;Chang and Broady 2012) and Republic of Korea.

Voucher specimens. Four stubs for scanning electron microscopy (NNIBRPR20139-NNIBRPR20142) were deposited at the Nakdonggang National Institute of Biological Resources, Korea.

2) Chroomonas coerulea (Geitler) Skuja 1948

Material examined. Freshwater was collected from the Ichonji pond, 76-1, Ichon-ri, Oeseo-myeon, Sangju-si, Gyeongsangbuk-do, Republic of Korea (N36°29ʹ57.9ʺ, E128°3ʹ45.0ʺ), on November 3, 2016.

Synonyms.

Cryptomonas coerulea Geitler, 1922.

Chroomonas rosenbergii Huber-Pestalozzi, 1950.

Description.Chroomonas coerulea cells were biflagellate (Fig. 2C). Cells were blue-green colored and ovalshaped (Fig. 1C, D). The size was 7.11-10.56 μm (n=32, mean=8.99±0.97 μm) long and 4.98-7.63 μm (n=34, mean=6.16±0.54 μm) wide. The pale-yellow colored eyespot was located in the center of the cell (Fig. 1C). Cells had rectangular periplast plates (Fig. 2C, D). The cells had a laterally positioned chloroplast with a pyrenoid, with the pyrenoidal matrix penetrated by two pairs of thylakoid membranes (Fig. 4A-D). The pyrenoid was surrounded by starch grains (Fig. 4C, D). The nucleus was positioned in the posterior part of the cell and contained many chromatin areas (Fig. 4A, C). Several ejectosomes were observed in two rows around the gullet (Fig. 4A, B). The Golgi body was observed at the gullet level (Fig. 4A, B). Mitochondria had plate-like cristae (Fig. 4A). The eyespot was located at a small lobe end of the chloroplast and composed of several pigment granules (Fig. 4A, E). The nucleomorph was located near the eyespot granules and contained several electron-dense granules and a nucleolus (Fig. 4E, F).

Distribution. Great Britain (Wołowski 2002;Whitton et al. 2003), Germany (Täuscher 2013), the Netherlands (Veen et al. 2015), Romania (Caraus 2017), Scandinavia (Karlason et al. 2018), Sweden (Skuja 1948, 1956), Brazil (Menezes 2010), Bangladesh (Ahmed et al. 2009), Victoria (Hill 1991) and Republic of Korea.

Voucher specimens. Four stubs for scanning electron microscopy (NNIBRPR20143-NNIBRPR20146) were deposited at the Nakdonggang National Institute of Biological Resources, Korea.

2. Phylogeny

The partial nuclear SSU rRNA gene sequences obtained for Korean C. nordstedtii and C. coerulea showed a pairwise similarity of 99.93% to C. nordstedtii NIES-708, NIES- 1004, UTEX 2779 and Chroomonas sp. M1312 and 99.93%-100% to C. coerulea UTEX 2780 and ACOI 1366 and Chroomonas sp. M1481, respectively (Table 3). The partial chloroplast rbcL gene sequence from C. nordstedtii had 100% similarity with that from C. nordstedtii (NIES- 708). Molecular phylogenetic analysis, based on the combined nuclear SSU rRNA gene and chloroplast rbcL gene dataset, also clustered C. nordstedtii and C. coerulea with the C. nordstedtii and C. coerulea clades with bootstrap values and posterior probabilities of 100%/1 and 79%/1, respectively, revealing monophyletic clades (Fig. 6). Although C. coerulea showed slightly lower bootstrap values and a slightly shorter branch length distance from C. coerulea UTEX 2780, this was due to a lack of chloroplast rbcL gene sequence information in other C. coerulea species (Table 2).