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50Chloroidium saccharophilum (Chlorophyta) from the Lake Baikal shore (Republic of Buryatia, Russia)
I. N. Egorova1, N. V. Kulakova1, Ye. D. Bedoshvili2
'Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia
2Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia Corresponding author. I. N. Egorova, egorova@sifibr.irk.ru
Abstract. The article provides information about green microalga Chloroidium saccharophilum (Trebouxiophyceae) whose history of study dates back to more than 100 years. The issues of its intraspecific variability are considered. We also studied the strain of C. saccharophilum IRK-A 230 isolated from a small puddle on the southeastern shore of Lake Baikal (Republic of Buryatia, Russia). Light and electron microscopy and molecular phylogeny methods establish the species identity of the strain. The alga has an ellipsoidal cell shape, parietal chloroplast and visible pyrenoid with starch sheath; reproduction by equal and unequal autospores in even and odd numbers; the ability to form spherical cells; and accumulated yellow pigments in old non-heterotrophic cultures. The data obtained by us complement the species characteristics, and its biology and geography. The amended description of C. saccharophilum is provided.
Keywords: Chloroidium, green microalgae, intraspecies variability, light and electron microscopy, molecular phylogeny, Buryatia, Russia.
Chloroidium saccharophilum (Chlorophyta) с побережья озера Байкал (Республика Бурятия, Россия)
И. Н. Егорова1, Н. В. Кулакова1, Е. Д. Бедошвили2
'Сибирский институт физиологии и биохимии растений СО РАН, Иркутск, Россия 2Лимнологический институт СО РАН, Иркутск, Россия Автор для переписки. И. Н. Егорова, egorova@sifibr.irk.ru
Резюме. В статье приводятся сведения о зеленой микроводоросли Chloroidium saccharophilum (Trebouxiophyceae). История исследований этого вида насчитывает более 100 лет. Нами при помощи световой и электронной микроскопии и методов молекулярной филогении был изучен штамм C. saccharophilum IRK-A 230, изолированный из небольшой лужи на юго-восточном побережье оз. Байкал (Республика Бурятия, Россия). Водоросль характеризуется эллипсоидной формой клеток, пристенным хлоропластом, имеет пиреноид с обкладкой из зерен крахмала, размножается автоспорами равной и неравной величины в четном и нечетном числе, способна формировать клетки шаровидной формы в старых культурах и в условиях роста при повышенной температуре и круглосуточном освещении; старые культуры могут приобретать желтую окраску. Штамм растет на минеральных средах на свету, на средах с добавлением органических веществ на свету и в темноте. Полученные нами данные дополняют характеристики вида, сведения о его биологии и географии. С учетом авторских результатов исследований и имеющихся в литературе данных расширено описание C. saccharophilum.
https://doi.org/1031111/nsnr/2022.562.255
255
Ключевые слова: Chloroidium, внутривидовая изменчивость, зеленые микроводоросли, молекулярная филогения, световая и электронная микроскопия, Бурятия, Россия.
The Chloroidium Nadson emend. Darienko et Pröschold representatives (Wata-nabeales, Trebouxiophyceae) are unicellular green microalgae widely distributed in aquatic and especially terrestrial ecosystems in different natural biomes as symbiotic and free-living organisms (Krüger, 1894; Lee et al, 1982; Darienko et al, 2010; Metz et al, 2019; Lindgren et al, 2020; Dillon et al, 2021; Li et al, 2021; Sanders, Misumoto, 2021; etc.). The species of this genus have biochemical, physiological and genetic features, which allow them to inhabit a variety of ecological niches successfully. These algae can grow heterotrophically on distinct carbon sources, at salinities from zero to several dozen g^L-1. They can show resistance to heavy metals, temperature, humidity, nutrients, and acidity changes (Shihira, Krauss, 1965; Andreyeva, 1970; Kessler, Huss, 1992; Huss et al., 1999; Zuppini et al., 2007; etc.). The Chloroidium cells contain po-lyols, which participate in maintaining their homeostasis (Vinayakumar, Kessler, 1975; Gustavs et al., 2011). They have unique protein families involved in osmotic stress tolerance. Chloroidium accumulated oil with a similar composition to palm oil, which may help these organisms to survive in extremal environments (González et al., 2013; Sharma et al., 2015; Nelson et al., 2017). The genus representatives are potential candidates for industrially relevant lipids production. A process of programmed cell death and signification in its cytochrome f has been detected in unicellular photosyn-thetic Chloroidium. This process could have a widespread role in the control of cell survival (Zuppini et al., 2009). Despite a great interest in studying these green mic-roalgae, information on their ecology, geography and biology cannot be considered complete.
The genus Chloroidium was recognized by Nadson (1906). His taxonomic history is analyzed in detail in the works of Darienko with coauthors (Darienko et al., 2010, 2018). Currently, the genus comprises 10 species (Darienko et al., 2010, 2018; Guiry, Guiry, 2022), the type species is C. saccharophilum (W. Krüger) Darienko et al. The type Chloroidium morphospecies belongs to the common microalgae (Komárek, Fott, 1983). In Russia, C. saccharophilum morphospecies was found in different terrestrial and aquatic ecotopes of arctic and temperate regions (Andreyeva, 1975; Temraleeva et al., 2015; Patova, Novakovskaya, 2018; etc.). The Baikal Region is a major biogeo-graphic node of North Asia (Pleshanov et al., 2002). For many years, research has been conducted here to study algae in terrestrial ecosystems and adjacent territories (Egorova et al., 2020). Currently, the number of C. saccharophilum morphospecies locations in this region is insignificant. It was registered on the east shore of Lake Baikal (the exact location is not known; Andreyeva, 1975), in soils of Khamar-Daban Range (Perminova et al., 1985) and of thermal springs (Maksimova, 2004), bark of trees in some localities (Egorova, 2006), artificial substrates and lichens of the Eastern Sayan Mountains (data obtained by I. N. Egorova). Perhaps, an insignificant number of the morphospecies locations in the Baikal Region are due to poor knowledge of algae
inhabiting various terrestrial substrates, the low number of aquatic green microalgae studies which apply culture-dependent methods, and/or molecular genetics.
According to the molecular phylogenetic data, in different terrestrial localities of the world there are algae, morphologically similar to Chloroidium, but belonging to other Watanabeales genera, such as Calidiella Darienko et Proschold, Jaagichlorella Reisigl, Mysteriochloris H.Y. Song et al., Polulichloris H.Y. Song et al. On the other hand, it is shown that some Chloroidium species, including C. saccharophilum, are widespread (Darienko et al., 2010). Molecular methods are an important tool for establishing the taxonomic affiliation of such algae. Moreover, with the accumulation of data, the problem of studying the intraspecific genetic, morphological, and biochemical diversity of the green microalgae species, including Chloroidium, becomes increasingly urgent. In the Baikal Region, the study of morphologically similar algae using molecular methods has so far been limited to only one epilithic strain, SAG 2197 from the city of Irkutsk (Irkutsk Region), which was assigned to C. saccharophilum (Darienko et al., 2010; Gustavs et al, 2011).
Our research aimed to characterize freshwater Chloroidium-like strain IRK-A 230 from Buryatia by methods of light and transmission electron microscopy and molecular genetics, to clarify its taxonomic position and features of microalga biology.
Materials and Methods
Locality, sampling, strain isolation and cultivation
The strain IRK-A 230 was isolated from a water sample taken from a small natural water pool located four kilometers from the southeastern shore of Lake Baikal (Republic of Buryatia) at the foot of the Khamar-Daban Range, on its northwestern macroslope (51°26'N, 104°51'E; altitude near 470 m a. s. l.) in August 2018. This area is characterized by increased humidity (800-1000 mm-year-1), since Khamar-Daban stands in the way of the western transfer of moist atmospheric masses creating humid and excessively humid conditions. The average annual air temperature is -0.9 °C. The predominant type of vegetation is mountain taiga, where swamps are common (Gvoz-detskii, Mikhailov, 1978; Peshkova, 1985).
The water sample was collected by N. V. Kulakova. It was put into a sterile 1.5 ml Eppendorf tube and delivered to the laboratory. There 50 |il of the sample was placed onto agar-solidified medium (N BBM; Starr, Zeikuss, 1993). A green algal colony of Chloroidium morphology was isolated, studied and deposited in the culture collection of algae of the Siberian Institute of Plant Physiology and Biochemistry SB RAS (IRK-A collection; Irkutsk, Russia) under the number IRK-A 230. For morphological evaluation, the strain was cultivated in the liquid and agar-solidified (0.1N, N, 3N BBM) medium, with or without vitamins (B1 and B12), as well as the BG-11 medium (Stanier et al., 1971). Detailed cultivation conditions of IRK-A 230 were published earlier (Egorova et al., 2018). Additionally, the strain was grown on liquid media N BBM with glucose, lactose, maltose, rhamnose, raffinose, in the light and the dark, as described by Andreyeva (1975).
Light and electron microscopy, identification
Microphotographs of the studied alga were taken with an Axio Scope A1 (Carl Zeiss, Germany) light microscope (LM) equipped with a color camera ICc5. The cultures were monitored for two-six months, a maximum up to three years. About 5000 cells were measured during the period of observation. Morphological identification of the IRK-A 230 was based mainly on Andreyeva (1975, 1998) and Darienko et al. (2010).
For transmission electron microscopy (TEM), cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Germany) in the 0.1 M phosphate buffer (pH 7.4) for 24 hours, washed twice in the buffer and postfixed with 1% osmium tetroxide solution (Sigma-Aldrich, Germany) in the same buffer for two hours. Cells were embedded in a 0.5% agar and dehydrated in a series of ethanol solutions with ascending concentrations. Samples were embedded in mounting resin Araldite 502 Kit (EMS, UK) according to the manufacturer's instructions and polymerized at 60 °C for 3 days. Slices were taken with ultramicrotome Leica Ultracut R (Leica, Austria) and stained with Reynolds' lead citrate. The slices were examined in a Leo 906 E transmission electron microscope (Zeiss, Germany) at 80 kV and imaged with a MegaView II digital camera (Olympus Soft Imaging Solutions, Germany).
DNA extraction, PCR amplification, and sequencing
DNA was extracted from living cells of the algal strain IRK-A 230 using the FastDNASpinKit (QBioGene, Canada) following the manufacturers instructions. The PCR amplification was performed in a 15 |il reaction mixture using BioMaster HS-Taq PCR Kit (Biolabmix, Russia), with 0.3 |iM (final concentration) forward and reverse primers (Syntol, Russia), nuclease-free water and 1 |il of DNA (80-100 ng).
The 18S rRNA gene and the ITS1-5.8S-ITS2 region were amplified by PCR and sequenced with primers 402-23F, 895-916F, 898-919R, 1308-39R (Katana et al., 2001), ITS5, ITS4 (White et al., 1990). Conservative primers RubiLF1 and Ru-biLR1 (Uchino, Yokota, 2003) were used for amplification of the rbcL gene fragment. The setting of PCR amplification conditions was similar to those recommended by referred authors. The PCR amplification was carried out using T100 Thermal Cycler (Bio-Rad, USA). The sequencing was performed from forward and reverse primers (Syntol, Russia).
The nucleotide sequences of rbcL, 18S rRNA gene, and ITS1-ITS2 obtained in this study were submitted to the GenBank® database (www.ncbi.nlm.nih.gov/genbank/) and available under the accession numbers OM865395, OM867679, and 0M870406, respectively.
Phylogenetic and ITS2 secondary structure analyses
The molecular phylogeny of the genus Chloroidium was based on the datasets of rbcL and 18S rDNA-ITS1-5.8S rDNA-ITS2 sequences. The rbcL dataset included 18 representatives of Chloroidium with the alignment consisting of 565 nucleotide
positions. The 18S-ITS1-5.8S-ITS2 dataset consisted of 63 representatives of the genus. The alignment included hypervariable 18S rRNA gene region V4-V6 and ITS1-5.8S-ITS2 region with a total length of 1064 nucleotide positions. The best substitution model for each dataset was calculated by JModelTest 2.1.10 (Darriba etal., 2012).
For the rbcL gene, the parameters of the best model were given as follows: GTR+I+G (base frequencies: A 0.2560, C 0.1841, G 0.2482, T 0.3117; rate parameters A-C: 1.1107, A-G: 3.1545, A-T: 3.4599, C-G: 0.0014, C-T: 7.6016, and G-T: 1.0000) with the proportion of invariable sites (I = 0.5660) and gamma shape parameter (G = 1.0670).
The parameters of the best model for 18S rDNA were given as follows: TrN+I (base frequencies: A 0.2319, C 0.2009, G 0.3024, T 0.2648; rate parameters A-C: 1.0000, A-G: 0.4221, A-T: 1.0000, C-G: 1.0000, C-T: 3.7138, and G-T: 1.0000) with the proportion of invariable sites (I = 0.9440), and for ITS1-ITS2: GTR+I+G (base frequencies: A 0.2025, C 0.2840, G 0.2990, T 0.2146; rate parameters A-C: 3.2026, A-G: 3.7397, A-T: 2.3854, C-G: 1.3802, C-T: 8.1430, and G-T: 1.0000) with the proportion of invariable sites (I = 0.5580) and gamma shape parameter (G = 0.9370).
The evolutionary history was inferred using the Bayesian reconstruction, Neighbor-Joining (NJ), and Maximum Likelihood (ML) methods. The Markov chain Monte Carlo (MCMC) calculations were carried out in triplicates using MrBayes 3.2.6 (Huelsenbeck, Ronquist, 2001). Analysis was run until a stable value of ESS statistics (300 units) was achieved. For the rbcL and 18S rDNA-ITS1-ITS2 datasets MCMC analysis was run for 1 x 106 and 4 x 106 generations with final sampling of 1500 and 1602 trees in each of two runs, respectively. NJ and ML trees were calculated using Mega X (Kumar et al., 2018). Bootstrap tests were run for 1000 and 500 replicates, respectively.
The ITS2 secondary structure of the strain IRK-A 230 was predicted using the ITS2 Database (Ankenbrand et. al., 2015) and homology modeling with the most suitable GI model 631798724 of Chloroidium saccharophilum SAG 211-9a. Available in the ITS2 database sequences of Chloroidium with their secondary structures were aligned with 4SALE (Seibel et al., 2006, 2008) and analyzed for compensatory base changes: hemi-CBC and CBC.
Results
Molecular data
Molecular identification was based on the analysis of the chloroplast rbcL gene, nuclear 18S rDNA and ITS1-ITS2 regions. The studied strain was unambiguously assigned as Chloroidium saccharophilum. The 18S rRNA gene fragment showed 100% nucleotide identity in all analyzed sequences of C. saccharophilum. This marker also was identical in C. saccharophilum and C. viscosum (Chodat) Darienko et Proschold, though all other Chloroidium species differed from C. saccharophilum by 1-7 substitutions. ITS2 has the highest level of nucleotide variability followed by ITS1 and rbcL gene. Earlier it was found that the Chloroidium sequences have a single codon insertion
at position 286 of the rbcL gene. This insertion (AAA or AAG) resulted in the insertion of a lysine residue in the encoded protein (Neustupa et al., 2013; Prochazkova et al., 2015). The studied rbcL gene fragment did not cover this site. However, in comparison with other species of the genus Chloroidium, sequences of C. saccharophilum including the studied strain shared the specific pattern of amino acid substitutions.
The 18S rDNA-ITS phylogenetic tree of the genus Chloroidium was generally consistent with the topology of the tree previously presented by Darienko et al. (2018), except the position of C. antarcticum Darienko et al., strain ISBAL-1013 (Fig. 1). In our study, C. antarcticum forms a single well supported clade which is a sister lineage to C. engadinensis SAG 812-1. According to Darienko et al. (2018), ISBAL-1013 formed a separate unsupported branch on the SSU-ITS tree. However, molecular phylo-geny of Chloroidium members based on rbcL and 18S rDNA sequences revealed that C. antarcticum ISBAL-1013 was clustered together with C. engadinensis (Vischer) Darienko et al. (Darienko et al., 2018).
The strain IRK-A 230 was found in a strongly supported Chloroidium clade (1/100/100) including C. saccharophilum representatives. No CBC or hemi-CBC was detected in the ITS2 secondary structure of IRK-A 230 in comparison with strains of C. saccharophilum. Five nucleotide substitutions in ITS2 distinguished the studied strain from the authentic strain SAG 211-9a (Fig. 2). These substitutions were localized in non-helical regions and the loop structure of the helix III.
The phylogenetic analysis of the rbcL gene (Fig. 3) corroborates the clustering of the studied strain IRK-A 230 within the Chloroidium saccharophilum clade.
Morphology and ultrastructure
Vegetative cells of IRK-A 230 are solitary, ellipsoidal, cylindrical, oviform, and rarely pyriform (Fig. 4), 3.4-11.6(14.5) x 1.2-7.3(8.9) jm. In cultures grown at elevated temperatures (about 30 °C) and round-the-clock lighting, many vegetative cells are broadly ellipsoid or almost spherical (Fig. 4). Cell wall is smooth, thin or relatively thick. It does not become slimy. A granular cell wall is not registered. Unilateral thickening of the cell wall, similar to that of Coccomyxa/Pseudococcomyxa-represen-tatives, was rarely noted. One nucleus is sometimes clearly visible in large vegetative cells under a LM. Chloroplast is parietal, band-shaped, or girdle-shaped, or in the form of a plate, with a smooth or slightly wavy to slightly lobed margin. It can cover up to 2/3 of the cell surface. Pyrenoid is often distinctly visible under a LM, with starch sheath, sometimes naked and indistinct. According to TEM data, the pyrenoid is surrounded by thylakoids of the chloroplast stroma (Fig. 5A-C). Solitary thylakoids of the stroma branch off and penetrate the pyrenoid, they are undulate and irregularly loop-shaped. Many pyrenoglobuli are present on the pyrenoid periphery close to the thylakoids of chloroplast stroma (Fig. 5B). There are small starch grains around the pyrenoid and in the chloroplast. However, they are not present in all cells (Fig. 4, 5). Vacuoles can be found in the free cell space, especially in aging cultures. Due to the large number of vacuoles in the cell cytoplasm, they may look net-like (Fig. 5). Colorless oil droplets
Fig. 1. Phylogenetic reconstruction of the genus Chloroidium based on the concatenated alignment
of 18S rDNA-ITS1-ITS2 regions. The Bayesian tree was inferred from 1064 aligned nucleotide positions. Bayesian probability support and bootstrap values (PP/ML/NJ) are given next to the branch nodes. The studied strain IRK-A 230 is marked with bold font. The scale bar value indicates the number of substitutions
per site.
Fig. 2. Predicted ITS2 secondary structure of the strain IRK-A 230. Characters in the enlarged bold font indicate substitutions in the IRK-A 230 strain regarding authentic Chloroidium saccharophilum strain SAG 211-9a.
may present in the cells. Old cultures are colored yellow or green. Old cultures have a lot of spherical cells, up to 11.2-12.8 |im in diameter. Their contents are granular (Fig. 4Q).
Reproduction occurs asexually by autospores. They are formed in an even (2-4-8(16)) and odd number (3, 5 and probably more) or only in an even number (Fig. 4). Autospores have equal and unequal sizes. They are released by rupturing the mature cell wall. Remains of sporangial cell wall usually are preserved in cultures for some time. Autospores are narrowly ellipsoidal, ovoid, cylindrical and sometimes tetrahedral. The chloroplast of spores is parietal, band-shaped, or a plate, with an even, smooth margin. Pyrenoid is not always clearly visible. Autospores can linger in sporangia, and grow to the size of adult cells. One of the spore cells can begin dividing inside the sporangium.
C. saccharophilum SAG 211-9a C. saccharophilum CAUP<CZH>:H191i - C. saccharophilum SAG 2055 ■ C. saccharophilum FACHB-2420 'Chlorella' ellipsoidea Ce C. saccharophilum FACHB-1796 IRK-A 230 — C- saccharophilum FACHB-1795
0.99/99/98
1/96/95
1/100/100
C. antarcticum IS BAL-1013 — С. viscosum SAG 56.87
1/97/99
1/100/100
— С. engadinense SAG 812-1
г C. ellipsoideum CAUP<CZH>:H1904 г C. ellipsoideum SAG 3.95 L C. lichenum SAG 2115
- C. lichenum FACHB-2421
- C. laureanum CAÜP H8501
- C. arboriculum MG-3
C. lobatum CAUP H8502
0.03
Fig. 3. Phylogenetic reconstruction of the genus Chloroidium based on the rbcL gene fragment. The Bayesian tree was inferred from 565 aligned positions. Bayesian probability support and bootstrap values (PP/ML/NJ) are given next to the branch nodes. The studied strain is marked with bold font. The scale bar value indicates the number of substitutions per site.
The strain IRK-A 230 can grow in both mixotrophic and heterotrophic conditions. On media with the addition of organic matter, cells mainly retain their ellipsoid shape. The cell content in such cultures is filled with oil (?) droplets, it is granular. The chloro-plast takes the form of a small plate, sometimes not noticeable. The cell structure is similar to that of cells in old cultures growing on a mineral medium (for example, Fig. 4Q).
Discussion
The studied strain IRK-A 230 is similar to some representatives of the genus Cali-diella, Polulichloris, Mysteriochloris, as well as Chloroidium, by the shape of cells, chlo-roplast, and pyrenoid. Molecular and ultrastructural data showed strong evidence that the strain IRK-A 230 belongs to Chloroidium saccharophilum.
The history of research of the Chloroidium genus representatives is more than 100 years old. Over the past time, a massive amount of data on the genus diversity,
Fig. 4. Light micrographs of the strain IRK-A 230 cells. A-G, I, J, L-P — chloroplast and pyrenoid with starch sheath in cells. G, J — unilateral thickening of the cell wall. A, H, I, K, L-P — sporangia, autospores. H, M, P — remains of sporangial cells. M, N, P — vacuoles in vegetative cells and autospores. N — liberation of autospores. Q — old cells in 3-years culture. Designation: asp — autospores; ch — chloroplast; mcw — mature cell wall; py — pyrenoid; s — starch; sp - sporangia; ut — unilateral thickening; v — vacuole. Scale bars: 10 |im.
distribution, biology and ecology has been accumulated. Extended studies on several species of the genus allowed researchers to consider the issues of intraspecific variability. It was shown that such features as the shape of cells, chloroplast morphology, and presence or absence of pyrenoid with or without starch grains which were considered for the differentiation of Chloroidium species may differ significantly between strains of the same species (Darienko et al, 2010).
Fig. 5. Transmission electron micrographs of the strain IRK-A 230 cells.
A-E — cells ultrastructure. Designation: ch — chloroplast; mcw — mature cell wall; n — nucleus;
Pg — pyrenoglobuli; py — pyrenoid; s — starch; t — thylakoid; v — vacuole.
Scale bars: A, C —1 |m; B — 500 nm; D, E — 200 nm.
Variability of morphological features of Chloroidium saccharophilum The description of Chloroidium saccharophilum initially was based on the study of the strain Camb 211-9a/SAG 211-9a (authentic), which was isolated from the sap of wounded Populus alba in Germany (Krüger, 1894). The authentic strain has been repeatedly examined by light microscopy. Subsequently, the description of the species was supplemented by studies of other strains. Several studies have noted that the ellipsoidal shape of cells, band-shaped or girdle-shaped chloroplast with a naked pyrenoid, reproduction by equal and unequal autospores, and number of autospores are some of the distinctive features of this alga (Komarek, Fott, 1983; Andreyeva, 1998; Darienko et al., 2010; Ettl, Gärtner, 2014). However, in some studies, including the current one, it was shown that such features as the ellipsoid shape of cells, and naked pyrenoid are variable in a different strain of this species or in the same strain under different cultivation conditions.
Andreyeva (1975) noted that the cells retained their ellipsoid species-specific shape in a medium with different salt concentrations. In the liquid Tamiya medium and in
heterotrophic cultures, there is some rounding of mainly adult cells, and the largest of them can take a shape close to spherical. Shihira, Krauss (1965) showed that the strain UTEX 27 with ellipsoid cells registered as C. saccharophilum formed spherical cells on a medium containing glucose. The strain C. saccharophilum SAG 2149 (initially registered asJaagia aquatica Vischer; = Pseudochlorella aquatica (Vischer) Vischer) has spherical cells along with ellipsoidal (Darienko et al., 2010). The strain IRK-A 230 has ellipsoid cells, but it produces broadly ellipsoid or spherical cells in agar-solidified cultures grown at elevated temperatures and round-the-clock lighting, as well as in mineral medium in aging cultures.
Ikeda, Takeda (1995) showed that there are clear differences in the pyrenoid inner structure among Chlorella-like species, which included Chloroidium representatives. They studied the authentic strain C. saccharophilum SAG 211-9a and another strain SAG 211-1d using TEM, and established features of the pyrenoid structure (undulate, irregularly loop-shaped thylakoids penetrate the pyrenoid; around the pyrenoid are small pyrenoglobuli). These authors also noted that the pyrenoid is naked, which is confirmed by the light microscopy data. Subsequent studies on several C. saccharophilum strains and the current study confirmed the conclusions of Ikeda and Takeda on the pyrenoid ultrastructure (Zuppini et al., 2007, 2009; González et al., 2013). However, other light and electron microscopy studies found that the individual cells of some C. saccharophilum strains, as well as IRK-A 230, have pyrenoid with starch grains (Andreyeva, 1975; González et al., 2013; Experimental..., 2022). The results of this and other studies point to the previously unrecognized feature and indicate that the absence of starch grains around the pyrenoid is not a reliable characteristic, as commonly believed. For example, Ouyang et al. (2012) examined trebouxiophycean alga Lobosphaera incisa (Reisigl) U. Karst. et al. with naked pyrenoid and showed the formation of starch grains around pyrenoid under nitrogen starvation conditions.
The reproduction of Chloroidium members occurs asexually by autospores. Only an even number of autospores was shown for this species in a few studies (Komárek, Fott, 1983; Darienko et al., 2010; Ettl, Gärtner, 2014). In the current study the C. saccharophilum strain IRK-A 230 formed autospores in even and odd numbers which is consistent with data obtained by Andreyeva (Andreyeva, 1975, 1998). An odd number of autospores distinguishes these Chloroidium members from other Chlorella-like algae.
Variability of biochemical and molecular features of Chloroidium
saccharophilum
The wide environmental plasticity of Chloroidium saccharophilum leads to variability of its morphological, physiological, biochemical, and genetic traits. The nucle-otide alignment of the V4-V6 in the studied strain and other analyzed members of C. saccharophilum showed 100% nucleotide identity. It was previously shown that several Chloroidium representatives have a group I introns in 18S rDNA, but the strains of C. saccharophilum SAG 211-9a and CCAP 211/42 lacked introns. Perhaps, the
presence/absence of introns and their sequence information can be used to distinguish Chloroidium species at the population level (Darienko et al., 2010).
Based on cell morphology, the strain Chloroidium saccharophilum SAG 2149 was originally described as the species of another genus (see above). It differs from the authentic strain SAG 211-9a by only two substitutions in their ITS-2 sequences (Darienko et al., 2010). The strain IRK-A 230 is distinguished from SAG 211-9a and SAG 2197 (epilithic strain isolated in Irkutsk) by five and two nucleotide substitutions in ITS-2, respectively. In C. saccharophilum, the ITS1-ITS2 region showed up to 4% nucleotide variability due to substitutions and indels (see electronic supplement1). The analysis of this region revealed that the studied strain has the highest similarity to strains of C. saccharophilum CCAP 211/27 (marine environment, UK) and OD-1-1-C (biocrust on saline soil, Germany). Within ITS1-ITS2, several other C. saccharophilum strains had about 98% nucleotide similarity with IRK-A 230. The highest nucleotide similarity of the rbcL gene fragment was found between IRK-A 230 and the strain FACHB-1796 isolated from a tree stump in China (Hubei Province) (Supp. 1).
It was revealed that Chloroidium algae have glucose-mannose rigid cell walls (Takeda, 1991), which distinguish them from other Chlorella-like algae. In addition, some differences in the ability to hydrolyse starch were found within the genus and among strains of C. saccharophilum (Kessler, 1978). Kessler, Huss (1992) established that several strains of C. saccharophilum have a growth limit at pH=2, and some other strains at pH=3, from 3 to 6% NaCl in medium, at the temperature from 26 to 30°C. Kessler, Soeder (1962) demonstrated that studied Chloroidium representatives do not contain hydrogenase, and after four weeks of growth in the nitrogen-deficient cultures they turn pale (pale greenish-yellow?) due to the loss of both chlorophylls and carotenoids. Andreyeva (1975) reported that C. saccharophilum SAG 211-9a, SAG 211-1c and LABIK 257-1 cultures had a yellow-green color in heterotrophic conditions. At the same time, she also noted the ability of Chlorella vulgaris Beij. to turn yellow in heterotrophic cultures. The ability of Trebouxiophyceae representatives to accumulate yellow pigment has been reported in several papers (Hildreth, Ahmadjian, 1981; Solovchenko et al., 2008; etc.). The same features, which we have repeatedly observed in studied cultures of Trebouxiophyceae, allow concluding that the trait is quite common in these algae and Chloroidium also.
We propose to amend the description of the species based on adding data and taking into account the published information.
Chloroidium saccharophilum (W. Krüger) Darienko, Gustavs, Mudimu, C.R. Me-nendez, R. Schumann, U. Karst., Friedl et Pröschold, 2010, Eur. J. Phycol. 45(1): 92, emend. I. N. Egorova, Kulakova et Ye. D. Bedoshvili
= Chlorothecium saccharophilum W. Krüger, 1894, Beitr. Physiologie Morphol. Nied. Organismen 4: 94, pl. V: figs 2-30 (authentic strain: SAG 211-9a).
1 Electronic supplement is available at the end of the article page on the journal website (https://doi. org/10.31111/nsnr/2022.56.2.255).
Amended diagnosis. Young cells narrowly ellipsoidal, ovoid, sometimes almost cylindrical and tetrahedral. Cell wall is thin. Chloroplast is parietal, band-shaped, with even, smooth margin. Pyrenoid is indistinct, naked, without starch sheath or with starch grains.
Mature vegetative cells of different strains differ in shape, from cylindrical, ellipsoidal to almost spherical or slightly pyriform, 3.4-13.6(14.5) x 1.2-7.3(8.9) jm. Cell wall thin over most of cells or they thickness varies only slightly with age, but sometimes cultured cells have a warted (granular) cell wall (probably remains of parent cells). Granular cell wall also presents in autosporangia. Unilateral thickening of the cell wall can be noted. Single nucleus is large, readily visible without staining. Chlo-roplast has very variable form, from parietal, band- or girdle-shaped, sometimes with slightly wavy margin, to slightly lobed. Pyrenoid is naked and indistinct or clearly visible under light microscope, with starch sheath. Pyrenoid is surrounded by thyla-koids of the chloroplast stroma. Solitary thylakoids of the stroma branch off and penetrate into the pyrenoid, they undulate and irregularly loop-shaped. Many pyrenoglo-buli are present on the pyrenoid periphery close to thylakoids of chloroplast stroma. Small starch grains can be presented in the stroma. Aging cells contain many colorless vacuoles and lipid droplets, which can displace chloroplast from cell wall. Old cultures are colored yellow or green. Strains with ellipsoidal shape of cells in old cultures may have a lot of cells with spherical shape, size up to 11.2-12.8 jm in diameter. Their contents are granular.
Asexual reproduction occurs via 2-4-8-16(32) or 3, 5, 7, 9, and more autospores. All strains have equal and unequal autospores. Liberation of autospores occurs through apical rupturing of sporangial wall. Remains of sporangial cell wall are usually sac-like. Liberation of autospores continues for some time after rupturing. One autospore often remains within sporangial wall and develops into a mature vegetative cell.
Acknowledgments
The transmission electron microscopy was made in the Shared Research Facilities 'Ultramicroanalysis' (Limnological Institute of the Siberian Branch of Russian Academy of Sciences). The research was carried out as part of the Scientific Project of the State Order of the Government of Russian Federation to Siberian Institute of Plant Physiology and Biochemistry Siberian Branch of the Russian Academy of Sciences No 122041100045-2. We thank O. N. Boldina, anonymous reviewers and the editor R. M. Gogorev for valuable comments during the preparation of the manuscript, G. S. Tupikova for the preparation of nutrient media.
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