Hidden diversity of Chlorococcum (Chlorophyta) in a shallow temporary freshwater lake: description of Chlorococcum szentendrense sp. nov

ABSTRACT Chlorococcum is a coccoid green algal genus, which contains almost 50 species. The genus is polyphyletic, but in a traditional sense, the cells are coccoid and non-motile with a cup-shape chloroplast in the vegetative stage, while the spores have chlamydomonad appearance. They are distributed worldwide mainly in terrestrial habitats, and the biotechnological potential of several strains has been reported. In this study, three new green algal strains from a shallow, temporary freshwater lake in Hungary are characterized using microscopic (light and transmission electron microscopy) and DNA-based methods (phylogenetic analysis of the ribosomal ITS region, the 18S ribosomal RNA and rbcL genes, and ITS secondary structure comparison). Based on the obtained results, one of the three new isolates is considered to represent a new species, which is described here as Chlorococcum szentendrense sp. nov.


Introduction
Microalgae represent a great and yet unravelled diversity of the biosphere and have important roles in the carbon and nitrogen cycles, in the marine and freshwater food webs as well as in the global production of oxygen and in CO 2 fixation (Bhola et al., 2014;Castro & Huber, 2019;Felföldi, 2020;Somogyi et al., 2022). In addition, they also have great potential in many kinds of industrial applications including biofuel production (Mahapatra & Ramachandra, 2013;Selvarajan et al., 2015;Khan et al., 2018;Nagy et al., 2018), application as biostimulant and biofertilizer in the agriculture (Abdel-Raouf et al., 2012;County & County, 2015), and especially in the health, cosmetic and food industries (reviewed by Mimouni et al., 2012;Heydarizadeh et al., 2013;Gateau et al., 2017). In spite of their obvious global importance, out of the 700 000 microalgal species that are expected to exist only ~10 000 have been described so far (Guiry, 2012). This clearly outlines the importance of the identification and description of novel species.
Chlorococcum (Chlamydomonadales, Chlorophyceae) was first described by Meneghini in 1842 (Guiry & Guiry, 2020), and an amended description of this genus was provided recently by Watanabe & Lewis (2017) and by Nakada et al. (2019). The 47 species accepted taxonomically to date (Guiry & Guiry, 2020) represent a polyphyletic group, the type species of the genus (Chlorococcum infusionum (Schrank) Meneghini) with others belong to the Moewusinia macroclade, while several other species belong to the Stephanosphaerinia macroclade (Nakada et al., 2008;Watanabe & Lewis, 2017). Members of the genus Chlorococcum are mainly edaphic (i.e. terrestrial) algae, but they were also reported from diverse aquatic habitats and subaerial surfaces (e.g. Klochkova et al., 2006;Wehr et al., 2015;Temraleeva & Moslalenko, 2019), and even symbiotic forms have been described within the genus (e.g. Feng et al., 2016). Traditional members of this genus, similarly to several other green algal species, exist in two morphological forms. The longer period of the lifespan is spent as a spherical, non-motile form as round cells with a rigid, thick cell wall. These vegetative cells are constantly growing and, under given conditions, are divided into a number of oval, biflagellate zoospores that are morphologically very similar to the vegetative cells of certain Chlamydomonas Ehrenberg species (Miller, 1978).
In this work, our aim was to investigate the hidden diversity of microalgae in Hungarian freshwater habitats. As a result, three new Chlorococcum strains were isolated from a temporary lake, and analysis of molecular phylogenies and internal transcribed spacer (ITS) structures, as well as light and electron microscopy have been applied to determine their taxonomic status. Based on morphological and molecular data, the examined strains represented two species, and one of them is described here as Chlorococcum szentendrense sp. nov.
For cryopreservation at −80°C, 0.1 ml methanol was added to 0.9 ml liquid culture.

Light microscopy
Light microscopy was performed using a Nikon Eclipse 80i microscope (Tokyo, Japan) and an Image-ProPlus 6.0 software package (Media Cybernetics, Rockville, Maryland, USA).
In order to visualize the lipid droplets in algal cells, 2-week-old cultures were investigated by nile red (9diethylamino-5H-benzo[alpha]phenoxazine-5-one; Sigma, St. Louis, Missouri, USA) staining following the protocol of Alemán-Nava et al. (2016). Cells were suspended in 1 ml of 4.5 g l -1 Na 2 EDTA (Molar Chemicals, Halásztelek, Hungary) solution, and then 5 µl of 0.25 mg ml -1 nile red (dissolved in acetone) was added. After this, the samples were incubated for 10 min in the dark. Fluorescence microscopic investigations were carried out with the same instrument using 510-550 nm excitation light and 575-640 nm emission filter.

Electron microscopy
Transmission electron microscopic (TEM) sample preparation was carried out as in Somogyi et al. (2011Somogyi et al. ( , 2013. For this purpose, 2.5% glutaraldehyde (Sigma; dissolved in 70 mM K-Na phosphate buffer, pH 7.2) was pipetted to the algal colonies grown on the agar plate. Cells were suspended in the fixative, fixed for 3 h at room temperature, and then centrifuged at 20°C with 5000× g for 5 min. The pellet was resuspended into solidifying agar (2%), and the solid agar plates were processed further. In another set of experiments solid plaques of algal culture were immersed in the 2.5% glutaraldehyde fixative for 3 h. After fixation both the agar plates and the algal plaques were treated similarly. The samples were rinsed with the phosphate buffer described above three times for 15 min, post-fixed in 1% OsO 4 (Sigma; dissolved in the same buffer) for 2 h, and then rinsed three times for 15 min with the same buffer. After dehydration in mounting ethanol series (25%, 50%, 70% and 90% ethanol, 15 min each; then 2 × 20 min in 96% and finally 20 min in absolute ethanol), the samples were embedded in Durcupan ACM epoxy resin (Sigma). Ultrathin (70 nm) sections were cut with a Ultracut E microtome (Reichert-Jung, Vienna, Austria) using a Diatome diamond knife (Nidau, Switzerland). The sections were stained with 5% uranyl acetate dissolved in methanol for 5 min and by Reynold's lead citrate for 5 min. Samples were investigated using a Hitachi 7100 TEM at 75 kV accelerating voltage and a JEM 1011 (Jeol, Tokyo, Japan) at 80 kV accelerating voltage. Images were taken using Morada digital camera (Olympus, Tokyo, Japan) and were analysed using ImageJ software (Schneider et al., 2012) when necessary.

DNA-based studies
Total genomic DNA was extracted from the strains using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany) according to the instructions given by the manufacturer. PCR amplification of the whole 18S rRNA gene was completed with two separate Chlorophyta-specific reactions using the primer pairs Euk328f-Chlo02R (Moon- Van der Staay et al., 2000;Zhu et al., 2005) and ChloroF-Euk329r (Moon- Van der Staay et al., 2000;Moro et al., 2009) as described in detail by Somogyi et al. (2013). The ribosomal ITS region (containing ITS-1, the 5.8S rRNA gene, ITS-2 and a short region from the 28S rRNA gene) was amplified with primers ITS_f and ITS_r (Liu et al., 2014), while the rbcL gene was amplified with primers rbcL-F1 and rbcL-R1 (Fawley et al., 2005). Purification of PCR products and Sanger sequencing were carried out by the Biomi Ltd (Gödöllő, Hungary). The obtained sequences were deposited in the GenBank database under the accession codes MG784550-MG784555 and MG977017-MG977019.
In the case of the 18S rRNA gene, sequences were aligned with the SINA Alignment Service (Pruesse et al., 2012), while in the case of other regions the ClustalW incorporated in the MEGA 7 software (Kumar et al., 2016) was used. For concatenation, the SequenceMatrix v1.8 software (Vaidya et al., 2011) was used. Maximum likelihood analysis (including the search for the best-fit model) was conducted using the MEGA 7 software. Bayesian analysis for phylogenetic reconstruction was performed with the Markov Chain Monte Carlo algorithm in two simultaneous, completely independent analyses running for 1 000 000 generations (sampled every 100 generations) using MrBayes version 3.1 (Huelsenbeck & Ronquist, 2001). The first 25% of the calculated trees were discarded and posterior probabilities were calculated after the two independent runs had reached convergence.

Taxonomic results
Chlorococcum szentendrense Greipel, Kutasi, Solymosi & Felföldi, sp. nov. (Figs 1-4, Figs 11-17) DIAGNOSIS: Vegetative cells single, spherical, diameter 5.9-13.5 µm when young and up to 30 µm in old cultures. Zoospores 9.0 µm long and 4.0 µm wide on average. Cell wall smooth, bilayered and thin in zoospores and in young vegetative cells, but thicker in older vegetative cells. Single nucleus, one cupshaped chloroplast with in general one large pyrenoid and small plastoglobuli and few starch grains in young cells. In older vegetative cells, diffuse or lobed chloroplasts with two pyrenoids and more starch grains. Pyrenoid matrix surrounded by a continuous, smooth starch sheath, traversed by few intrapyrenoidal channels lined with double membranes, and penetrating the pyrenoid matrix as intramatricial channels. Thylakoids grouped into lamellae (5-10 thylakoids). Zoospores oval with thin bilayered cell wall, parietal chloroplast, one pyrenoid similar to that in vegetative cells, and intraplastidial eyespot. Stigma elliptical. No sexual reproduction observed. Unequivocal distinction from related Chlorococcum species based on molecular taxonomic methods (18S rRNA, rbcL and ITS sequence analysis). HOLOTYPE: Lyophilized material from strain K2/9 has been deposited at the Algological Collection of the Hungarian Natural History Museum (Budapest, Hungary) under HNHM-ALG-H7965. TYPE LOCALITY: Kőhegyi Lake, near Szentendre, Hungary. ETYMOLOGY: The specific epithet 'szentendrense' (neut. adj.) means 'belonging to Szentendre' and refers to the town which is close to the isolation source of the new species. LIVING CULTURE: Strain K2/9 is available at the Sammlung von Algenkulturen der Universität Göttingen (Culture Collection of Algae at Göttingen University, Göttingen, Germany) under the code SAG 2595. DNA SEQUENCES: MG784551 (18S rRNA gene), MG784555 (ITS region) and MG977018 (rbcL gene).

Description
The vegetative cells are green, spherical (Figs 1-2, 11-12 with a diameter of 5.9-13.5 µm when young and up to 30 µm in old cultures. Cells have single nucleus with nucleolus ( Fig. 12), one cup-shaped chloroplast (Figs 7, 11) that becomes diffuse or lobed in older cells (Fig.  12). Regular mitochondria with cristae, normal Golgi apparatus, and some vacuoles sometimes accumulating an electron-dense deposit are observed (Figs 11-12). The chloroplast of young cells has in general one large pyrenoid grain surrounded by a continuous starch sheath (Fig. 13), in older cells two pyrenoids are also sometimes observed within the same chloroplast (Fig. 15). Pyrenoids are surrounded by a continuous starch sheath that is traversed by intrapyrenoidal channels lined with double membranes which penetrate the pyrenoid matrix as intramatricial channels (Figs 13, 15). Individual smaller starch grains are also observed in the plastids (Figs 12, 13, 15), as well as small electron-dense plastoglobuli (Figs 11-13). Thylakoids are grouped into bands (lamellae) consisting in general of 5-10 thylakoids (Figs 13-15). The cell wall is 150-250 nm thick, with thinner cell walls typical in younger cells and the cell wall thickness slightly increasing with cell age (up to 650 nm).
In addition, small electron-dense plastoglobuli were observed in the plastids, and the thylakoids were grouped into bands (lamellae) consisting in general of 2-4 thylakoids (Figs 18-19).
Aggregates (Figs 9 and 10) or 'microcolonies', i.e. an irregular group of individual vegetative cells (some of them at least seemingly empty) are sometimes formed (Figs 6,(9)(10). In some cells of these 'microcolonies' zoospores are surrounded by a common sheath, their mother cell wall (Fig. 20). Zoospores are 7.5 µm long and 3.5 µm wide on average, have a thin but peculiarly striated cell wall (26-42 nm) (Figs 20, 21), intraplastidial eyespot (Fig.  22) and two anterior flagella (data not shown). Ultrastructural features of the zoospores are in other respects very similar to that of vegetative cells . No sexual reproduction has been observed. Unequivocal distinction from other Chlorococcum species based on molecular taxonomic methods (18S rRNA, rbcL and ITS sequence analysis).

Results of DNA-based analyses
The 18S rRNA gene sequence of strains K2/5 and K2/ 11 was identical based on 1750 nucleotide positions and shared high similarity values (99.8-99.9%, only 1-2 nucleotide difference) with other Chlorococcum isolates (RK261 from an oligotrophic lake in Japan and GRK7WB5 from karstwater stream biofilm in Germany; Fujii et al., 2010;Hodač et al., 2015) and with 'Chlamydomonas debaryana' CCAP 11/1 (which was isolated from a freshwater pool in the Czech Republic). These strains were only distantly related (98.4-98.6% similarities) to Chlorococcum aquaticum P.A. Archibald UTEX 2222 and C. minutum R.C.Starr SAG 213-7   strains formed distinct lineages on the phylogenetic trees (Fig. 25, Supplementary figs S1-S3) from the authentic strains of Chlorococcum species based on all three studied DNA regions (18S rRNA and rbcL genes, ITS region), and their position was supported with high bootstrap and posterior probability values.

Discussion
Molecular studies have revealed that Chlorococcum is a polyphyletic genus (Nakada et al., 2008;Krienitz & Bock, 2012;Kawasaki et al. 2015;Watanabe & Lewis, 2017;Temraleeva & Moslalenko, 2019). Our new strains belonged to the Stephanosphaerinia macroclade but outside the Oleo clade which was proposed by Kawasaki et al. (2015). General morphological features (spherical green vegetative cells, single cells or forming aggregates, an average cellular diameter around 10-20 µm, single parietal chloroplast and chlamydomonad-like zoospores with two equal flagella) and ultrastructure (smooth cell wall and presence of pyrenoid(s) with pyrenoid channels) of the novel species were similar to that described for unicellular green microalgae traditionally belonging to the Chlorococcum genus (Starr, 1955;Archibald & Bold 1970;Miller, 1978;Ettl & Gärtner, 1988Péterfi et al., 1988;Klochkova et al., 2006;Feng et al., 2016;Watanabe & Lewis, 2017). Although we studied cultures of different age, we could not observe and identify sexual reproduction in any of the three studied strains.
One of our new isolates showed remarkable morphological and phylogenetic differences with other strains isolated previously. The cell wall of K2/9 is thinner (150-250 nm) than that of the closest-related species, C. rugosum and C. vacuolatum (0.5-1 µm or thicker; Ettl & Gärtner, 2014;Kawasaki et al. 2015), which distinguishes the new strain from them (note that literature data are based on light microscopy). The cell wall of the zoospores is smooth in the case of K2/9 and vegetative cells sometimes have two pyrenoids which clearly distinguishes them ultrastructurally from C. minutum (Péterfi et al., 1988) and C. oleofaciens (Miller, 1978). The continuous starch sheath surrounding the pyrenoids distinguishes the new strain from species belonging to Chlorococcum sensu Watanabe & Lewis in which the starch sheath is discontinuously covered with starch segments or grains (Watanabe & Lewis, 2017). Archibald & Bold (1970) described that several Chlorococcum species (e.g. C. isabeliense) usually have a single pyrenoid. Therefore, it may be possible that the cells containing two pyrenoids were old cells. Some of the pyrenoids observed in our work had atypical shape with shrunk matrices. It should be noted that pyrenoid structure is also influenced by sampling time (daytime vs. night time), culture conditions and the age of the cells (Meyer et al., 2017).
In addition to these morphological differences, distinction of the new strain was confirmed by DNA sequence analysis of the 18S rRNA gene, the ribosomal ITS region and the rbcL gene, and they showed 1.1-1.6% pairwise dissimilarity values to the closest related authentic strains based on the 18S rRNA gene. According to Temraleeva & Moslalenko (2019)

the average dissimilarity values among closely related
Chlorococcum species within the Stephanosphaerinia macroclade is even lower (0-1.1%), which supported that our strain represents a new species to science, therefore for strain K2/9, the establishment of a new species, Chlorococcum szentendrense sp. nov., is proposed.
The other two new isolates, K2/5 and K2/11, had vegetative cells with similar cell size and ultrastructural features to those of K2/9, but the oval zoospores were slightly larger in these two strains. Some ultrastructural differences were also observed: the number of thylakoid lamellae was 5-10 on average in the case of K2/9, while in K2/5 and K2/11 thylakoids were grouped into only 2-4-layered assemblies; and the thickness of the bilayered cell wall varied with the age of the cells in the case of the K2/9 strain (150-250 nm, in some cases extraordinarily thick, ~450-650 nm), and had a relatively constant thickness (130-160 nm) in the case of the K2/5 and K2/11 strains. These strains belonged to a different phylogenetic group as K2/9. It seems that strain CCAP 11/1, closely related to K2/5 and K2/11, labelled as 'Chlamydomonas debaryana' Goroschankin (reclassified recently as Edaphochlamys debaryana (Goroschankin) Pröschold & Darienko (Pröschold et al., 2018)) is currently misclassified. This could be because motile cell (spores) in the genus Chlorococcum are chlamydomonad in appearance (biflagellar, cup-shaped chloroplast, persistent eyespot, etc.; Wehr et al., 2015), although micrographs available at the homepage of Culture Collection of Algae and Protozoa (www.ccap.ac.uk; downloaded 15 November 2020) show cellular morphology similar to Chlorococcum species (non-flagellar spherical gregarious cells with a cellular diameter around 5-15 µm). To clarify the taxonomic status of strain CCAP 11/1 with direct comparison was not possible, since the strain is no longer available from the culture collection (i.e. it was archived). Morphological features (e.g. the size of vegetative cells and zoospores, the uninterrupted starch shell characteristic for pyrenoids) made the new isolates (K2/5 and K2/11) similar to the closely related species, C. aquaticum and C. minutum (Ettl & Gärtner, 1988;Kawasaki et al., 2015;Temraleeva & Moslalenko, 2019), and unequivocal distinction from them is possible only with molecular taxonomic methods. Since morphological results did not support the description of another new species, we did not propose that for strains K2/5 and K2/11. Additional formal Chlorococcum species descriptions for strains distinguishable solely based on DNA sequence data would probably create an even more complicated taxonomic situation for this polyphyletic genus.
Some Chlorococcum species have been reported to accumulate abundant amounts of lipids (as was observed in the case of strain K2/5) and are thus considered as potential algae for biofuel production (Mahapatra & Ramachandra, 2013;Prabakaran et al., 2019). Chlorococcum minutum produces biological hydrogen (Paramesh et al., 2018), while other strains within this genus could be potential natural sources of B-vitamin (Fujii et al., 2010). Furthermore, with the use of a carotenoid biosynthesis inhibitor, relatively high amounts of phytoene could be accumulated by Chlorococcum strains (Laje et al., 2019). Phytoene is a colourless, highly added value compound with potential UV-protective, anti-ageing and photo-protective cosmetic applications. In addition, some of these species are proliferating in municipal wastewater (Mahapatra & Ramachandra, 2013) and can be effectively used for the treatment of wastewater effluents (Aravantinou et al., 2016). Therefore, the description, cultivation and characterization of novel microalgal species have not only taxonomic importance, but new strains may represent direct links to various kinds of industrial applications.