Chloroidium saccharophilum (Chlorophyta) from the Lake Baikal shore (Republic of Buryatia, Russia)

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.

Full text

https://doi.org/10.31111/nsnr/2022.56.2.255
Chloroidium saccharophilum (Chlorophyta) from the Lake Baikal shore
(Republic of Buryatia, Russia)
I. N. Egorova1, N. V. Kulakova1, Ye. D. Bedoshvili2
1Siberian 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, Rus-
sia). 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 microsco-
py, molecular phylogeny, Buryatia, Russia.
Chloroidium saccharophilum (Chlorophyta) с побережья озера Байкал
(Республика Бурятия, Россия)
И. Н. Егорова1, Н. В. Кулакова1, Е. Д. Бедошвили2
1Сибирский 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 saccharo-
philum (Trebouxiophyceae). История исследований этого вида насчитывает более 100 лет.
Нами при помощи световой и электронной микроскопии и методов молекулярной филоге-
нии был изучен штамм C. saccharophilum IRK–A 230, изолированный из небольшой лужи на
юго-восточном побережье оз. Байкал (Республика Бурятия, Россия). Водоросль характери-
зуется эллипсоидной формой клеток, пристенным хлоропластом, имеет пиреноид с обклад-
кой 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 культу-
ры 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. saccha-
rophilum.
255
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Ключевые слова: Chloroidium, внутривидовая изменчивость, зеленые микроводоросли, мо-
лекулярная 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 dierent 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 fea-
tures, 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, humidi ty,
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 dierent 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 lo-
cations 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
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
256

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 dierent terrestrial localities of
the world there are algae, morphologically similar to Chloroidium, but belonging to
other Watanabeales genera, such as Calidiella Darienko et Pröschold, 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 wide-
spread (Darienko et al., 2010). Molecular methods are an important tool for establi-
shing the taxonomic aliation of such algae. Moreover, with the accumulation of data,
the problem of studying the intraspecific genetic, morphological, and biochemical di-
versity of the green microalgae species, including Chloroidium, becomes increasingly
urgent. In the Baikal Region, the study of morphologically similar algae using mole-
cular 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 natu-
ral 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°26ʹN, 104°51ʹE; 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 µl 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 collec-
tion 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 morphologi-
cal 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 me-
dium (Stanier et al., 1971). Detailed cultivation conditions of IRK–A 230 were pub-
lished earlier (Egorova et al., 2018). Additionally, the strain was grown on liquid me-
dia N BBM with glucose, lactose, maltose, rhamnose, ranose, in the light and the
dark, as described by Andreyeva (1975).
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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 identifica-
tion 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% parafor-
maldehyde (Sigma-Aldrich, Germany) in the 0.1 M phosphate buer (pH 7.4) for
24 hours, washed twice in the buer and postfixed with 1% osmium tetroxide solution
(Sigma-Aldrich, Germany) in the same buer for two hours. Cells were embedded in
a 0.5% agar and dehydrated in a series of ethanol solutions with ascending concent-
rations. Samples were embedded in mounting resin Araldite 502 Kit (EMS, UK) ac-
cording to the manufacturerʼs 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 manufacturer`s instructions.
The PCR amplification was performed in a 15 µl reaction mixture using BioMaster
HS-Taq PCR Kit (Biolabmix, Russia), with 0.3 µM (final concentration) forward and
reverse primers (Syntol, Russia), nuclease-free water and 1 µl 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 OM870406,
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
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
258

positions. The 18S-ITS1-5.8S-ITS2 dataset consisted of 63 representatives of the ge-
nus. 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 substitu-
tion model for each dataset was calculated by JModelTest 2.1.10 (Darriba et al., 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 parame-
ters 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 param-
eter (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 fre-
quencies: 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 propor-
tion of invariable sites (I = 0.5580) and gamma shape parameter (G = 0.9370).
The evolutionary history was inferred using the Bayesian reconstruction, Neigh-
bor-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 × 106 and 4 × 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 repli-
cates, 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 suit-
able 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 as-
signed 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 Pröschold,
though all other Chloroidium species diered from C. saccharophilum by 1–7 substitu-
tions. 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
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259

at position 286 of the rbcL gene. This insertion (AAA or AAG) resulted in the inser-
tion of a lysine residue in the encoded protein (Neustupa et al., 2013; Procházková et
al., 2015). The studied rbcL gene fragment did not cover this site. However, in com-
parison 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 con-
sistent 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. an tarcticum 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 loca-
lized 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) × 1.2–7.3(8.9) µm. In cultures grown at ele-
vated 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 rela-
tively thick. It does not become slimy. A granular cell wall is not registered. Unilateral
thicke ning 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 o and penetrate the pyrenoid, they are undulate and irre gularly 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
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
260

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.
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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 µm 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.
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.
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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 simi-
lar 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. 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.
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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 µm.
distribution, biology and ecology has been accumulated. Extended studies on several
species of the genus allowed researchers to consider the issues of intraspecific variabi-
lity. 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 dierentiation of Chloroidium species may dier significantly between strains of
the same species (Darienko et al., 2010).
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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 re-
peatedly examined by light microscopy. Subsequently, the description of the species
was supplemented by studies of other strains. Several studies have noted that the ellip-
soidal 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 (Komárek, 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 dierent strain of this species or in the same strain under dierent culti-
vation conditions.
Andreyeva (1975) noted that the cells retained their ellipsoid species-specific shape
in a medium with dierent salt concentrations. In the liquid Tamiya medium and in
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 µm; B — 500 nm; D, E — 200 nm.
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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 re-
gistered as Jaagia 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 cul-
tures grown at elevated temperatures and round-the-clock lighting, as well as in mi-
neral medium in aging cultures.
Ikeda, Takeda (1995) showed that there are clear dierences in the pyrenoid in-
ner structure among Chlorella-like species, which included Chloroidium representa-
tives. 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 na-
ked, which is confirmed by the light microscopy data. Subsequent studies on several
C. saccharophi lum 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 in-
dividual 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) exami-
ned 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. saccha-
rophilum strain IRK-A 230 formed autospores in even and odd numbers which is con-
sistent 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 variabi-
lity 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 se-
veral 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
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
266

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 diers 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 substitu-
tions 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.saccha-
rophilum 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 addi-
tion, some dierences in the ability to hydrolyse starch were found within the genus
and among strains of C.saccharophilum (Kessler, 1978). Kessler, Huss (1992) estab-
lished 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 representa-
tives 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 ta-
king 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).
Новости систематики низших растений — Novosti sistematiki nizshikh rastenii 56(2): 255–272. 2022
267

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 dierent strains dier in shape, from cylindrical, ellip-
soidal to almost spherical or slightly pyriform, 3.4–13.6(14.5) × 1.2–7.3(8.9) µm. Cell
wall thin over most of cells or they thickness varies only slightly with age, but some-
times 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
vi sible under light microscope, with starch sheath. Pyrenoid is surrounded by thyla-
koids of the chloroplast stroma. Solitary thylakoids of the stroma branch o and pen-
etrate 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 µm in diameter. Their con-
tents 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 Facili-
ties ‘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.
References / Литература
Andreyeva V. M. 1970. On the variability of taxonomic characters in unicellular green algae under
the conditions of culture. III. The concentration of the medium components and the cell size in
certain species of the genus Chlorella Beijer. Botanicheskii zhurnal 55(11): 1619–1630. [Андрее-
ваВ. М. 1970. Изменчивость систематических признаков зеленых одноклеточных водоро-
слей в условиях культуры. III. Концентрация среды и размер клеток некоторых видов рода
Chlorella Beijer. Ботанический журнал 55(11): 1619–1630].
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
268

Andreyeva V. M. 1975. Rod Chlorella. Morphologiya, sistematika, printsipy klassifikatsii [The genus
Chlorella. Morphology, systematics, classification principles]. Leningrad: 86 p. [Андреева В. М.
1975. Род Chlorella. Морфология, систематика, принципы классификации. Л.: 86 с.].
Andreyeva V. M. 1998. Pochvennye i aerophil’nye zelenye vodorosli (Chlorophyta: Tetrasporales, Chlo-
rococcales, Chlorosarcinales) [Terrestrial and aerophilic green algae (Chlorophyta: Tetrasporales,
Chlorococcales, Chlorosarcinales)]. St. Petersburg: 351 p. [Андреева В. М. 1998. Почвенные и
аэрофильные зеленые водоросли (Chlorophyta: Tetrasporales, Chlorococcales, Chlorosarcinales).
СПб.: 351 с.].
Ankenbrand M. J., Keller A., Wolf M., Schultz J., Förster F. 2015. ITS2 database V: Twice as much.
Molecular Biology and Evolution 32(11): 3030–3032. https://doi.org/10.1093/molbev/msv174
Darienko T., Gustavs L., Mudimu O., Menendez C. R., Schumann R., Karsten U., Friedl T., Prö-
schold T. 2010. Chloroidium, a common terrestrial coccoid green alga previously assigned to
Chlorella (Trebouxiophyceae, Chlorophyta). European Journal of Phycology 45(1): 79–95.
https://doi.org/10.1080/09670260903362820
Darienko T., Lukešová A., Pröschold T. 2018. The polyphasic approach revealed new species of
Chloroidium (Trebouxiophyceae, Chlorophyta). Phytotaxa 372(1): 51–66.
https://doi.org/10.11646/phytotaxa.372.1.4
Darriba D., Taboada G. L., Doallo R., Posada D. 2012. jModelTest 2: more models, new heuristics and
parallel computing. Nature Methods 9(8): 772. https://doi.org/10.1038/nmeth.2109
Dillon K. P., Correa F., Judon C., Sancelme M., Fennell D. E., Delort A.-M., Amato P. 2021. Cyano-
bacteria and algae in clouds and rain in the area of puy de Dôme, Central France. Applied and
Environmental Microbiology 87(1): e01850-20. https://doi.org/10.1128/AEM.01850-20
Egorova I. N. 2006. Epifitnye vodorosli Pribaikalʼya: vidovoe raznoobrazie i ekologicheskie oso-
bennosti. Kand. Diss. [Epiphytic algae of Baikal area: species diversity and ecological pecu-
liarities. PhD thesis]. Ulan-Ude: 337 p. [Егорова И. Н. 2006. Эпифитные водоросли При-
байкалья: видовое разнообразие и экологические особенности. Дисс. … канд. биол. наук.
Улан- Удэ: 337с.].
Egorova I. N., Mincheva E. V., Boldina O. N. 2018. Ataktogamous green microalgae of the genus
Chlorosarcinopsis Herndon (Chlorophyceae, Chlorophyta) from Zabaikalskiy region (Russia).
Phytotaxa 343(1): 1–19. https://doi.org/10.11646/phytotaxa.343.1.1
Egorova I. N., Sudakova E. A., Maksimova E. N., Tupikova G. S. 2020. Terrestrial algae of the
mountains of South Siberia and North Mongolia. Botanicheskii zhurnal 105(2): 107–132. [Его-
рова И. Н., Судакова Е. А., Максимова Е. Н., Тупикова Г. С. 2020. Наземные водоросли
гор Южной Сибири и Северной Монголии. Ботанический журнал 105(2): 107–132].
https://doi.org/10.31857/S0006813620020027
Ettl H., Gärtner G. 2014. Syllabus der Boden-, Luft- und Flechtenalgen. 2 Aufl. Berlin, Heidelberg:
773 p. https://doi.org/10.1007/978-3-642-39462-1
Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG).
2022. http://www.uni-goettingen.de. (Date of access: 02 III 2022).
González M. A., Pröschold T., Palacios Y., Aguayo P., Inostroza I., Gómez P. I. 2013. Taxonomic iden-
tification and lipid production of two Chilean Chlorella-like strains isolated from a marine and an
estuarine coastal environment. AoB PLANTS 5: plt020. https://doi.org/10.1093/aobpla/plt020
Guiry M. D., Guiry G. M. 2022. AlgaeBase. World-wide electronic publication, National University
of Ireland, Galway. https://www.algaebase.org (Date of access: 10 III 2022).
Gustavs L., Görs M., Karsten U. 2011. Polyol patterns in biofilm-forming aeroterrestrial green algae
(Trebouxiophyceae, Chlorophyta). Journal of Phycology 47(3): 533–537.
https://doi.org/10.1111/j.1529-8817.2011.00979.x
Gvozdetskii N. A., Mikhailov N. I. 1978. Fizicheskaya geografiya SSSR. Aziatskaya chastʼ [Physical
Geography of the USSR. Asian part]. Moscow: 448 p. [Гвоздецкий Н. А., Михайлов Н. И. 1978.
Физическая география СССР. Азиатская часть. М.: 448 с.].
Новости систематики низших растений — Novosti sistematiki nizshikh rastenii 56(2): 255–272. 2022
269

Hildreth K. C., Ahmadjian V. 1981. A study of Trebouxia and Pseudotrebouxia isolates from dierent
lichens. Lichenologist 13(1): 65–86. https://doi.org/10.1017/S0024282981000078
Huelsenbeck J. P., Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinfor-
matics 17(8): 754–755. https://doi.org/10.1093/bioinformatics/17.8.754
Huss V. A. R., Frank C., Hartmann E. C., Hirmer M., Kloboucek A., Seidel B. M., Wenzeler P.,
Kessler E. 1999. Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu
lato (Chlorophyta). Journal of Phycology 35(3): 587–598.
https://doi.org/10.1046/j.1529-8817.1999.3530587.x
Ikeda T., Takeda H. 1995. Species-specific dierences of pyrenoids in Chlorella (Chlorophyta). Jour-
nal of Phycology 31: 813–818. https://doi.org/10.1111/j.0022-3646.1995.00813.x
Katana A., Kwiatowski J., Spalik K., Zakryś B., Szalacha E., Szymańska H. 2001. Phylogenetic po-
sition of Koliella (Chlorophyta) as inferred from nuclear and chloroplast small subunit rDNA.
Journal of Phycology 37: 443–451. https://doi.org/10.1046/j.1529-8817.2001.037003443.x
Kessler E. 1978. Physiological and biochemical contributions to the taxonomy of the genus Chlorella.
XII. Starch hydrolysis and a key for the identification of 13 species. Archiv für Microbiologie 119:
13–16. https://doi.org/10.1007/BF00407921
Kessler E., Huss V. A. R. 1992. Comparative physiology and biochemistry and taxonomic assignment
of the Chlorella (Chlorophyceae) strains of the culture collection of the University of Texas at
Austin. Journal of Phycology 28: 550–553. https://doi.org/10.1111/j.0022-3646.1992.00550.x
Kessler E., Soeder C. J. 1962. Biochemical contributions to the taxonomy of the genus Chlorella.
Nature 4833: 1096–1097. https://doi.org/10.1038/1941096b0
Komárek J., Fott B. 1983. Das Phytoplankton des Süβwassers. Bd XVI, Teil 7, Hälfte 1. Chlorophyceae
(Grünalgen). Ordnung Chlorococcales. Stuttgart: 1044 p.
Krüger W. 1894. Beiträge zur Kentnis der Organismen des Saftflusses (sog. Scheimflusses) der Laub-
bäume. II. Über zweiaus Saftflussen rein gezüchtete Algen. Beiträge zur Physiologie und Morpho-
logie niederer Organismen 4: 69–116.
Kumar S., Stecher G., Li M., Knyaz C., Tamura K. 2018. MEGA X: Molecular Evolutionary Ge-
netics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549.
https://doi.org/10.1093/molbev/msy096
Lee J. J., Reidy J., Kessler E. 1982. Symbiotic Chlorella species from larger foraminifera. Botanica
Marina 25: 171–176. https://doi.org/10.1515/botm.1982.25.4.171
Li S., Tan H., Liu B., Zhu H., Hu Z., Liu G. 2021. Watanabeales ord. nov. and twelve novel species of
Trebouxiophyceae (Chlorophyta). Journal of Phycology 57: 1167–1186.
https://doi.org/10.1111/jpy.13165
Lindgren H., Moncada B., Lücking R., Magain N., Simon A., Gonet B., Sérusiaux E., Nelsen M.
P., Mercado-Díaz J. A., Widhelm T. J. et al. 2020. Cophylogenetic patterns in algal symbionts
correlate with repeated symbiont switches during diversification and geographic expansion of
lichen-forming fungi in the genus Sticta (Ascomycota: Peltigeraceae). Molecular Phylogenetics
and Evolution 150: 106860. https://doi.org/10.1016/j.ympev.2020.106860
Maksimova E. N. 2004. Vidovoe raznoobrazie i ekologiya pochvennykh vodoroslei mineral‘nykh istoch-
nikov Baikal‘skoi Sibiri. Kand. Diss. [Species diversity and ecology of soil algae of mineral springs
of the Baikal Siberia. PhD thesis]. Ulan-Ude: 177 p. [Максимова Е. Н. 2004. Видовое разно-
образие и экология почвенных водорослей минеральных источников Байкальской Сибири.
Дисс. … канд. биол. наук. Улан-Удэ: 177 с.].
Metz S., Singer D., Domaizon I., Unrein F., Lara E. 2019. Global distribution of Trebouxiophyceae
diversity explored by high-throughput sequencing and phylogenetic approaches. Environmental
Microbiology 21(10): 3885–3895. https://doi.org/10.1111/1462-2920.14738
Nadson G. 1906. Zur Morphologie der niederen Algen. Izvestiya Imperatorskogo Sankt-Peterburgs-
kogo Botanicheskogo Sada 6: 184–194. [Надсон Г. 1906. К морфологии низших водорослей.
Известия Санкт-Петербургского ботанического сада 6: 184–194].
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
270

Nelson D. R., Khraiwesh B., Fu W., Alseekh S., Jaiswal A., Chaiboonchoe A., Hazzouri K. M., O’Con-
nor M. J., Butterfoss G. L., Drou N. et al. 2017. The genome and phenome of the green alga
Chloroidium sp. UTEX 3007 reveal adaptive traits for desert acclimatization. eLife (Plant Biolo-
gy) 6: e25783. https://doi.org/10.7554/eLife.25783
Neustupa J., Němcová Y., Veselá J., Steinová J., Škaloud P. 2013. Parachloroidium gen. nov. (Tre-
bouxiophyceae, Chlorophyta), a novel genus of coccoid green algae from subaerial corticolous
biofilms. Phycologia 52: 411–421. https://doi.org/10.2216/13-142.2
Ouyang L., Du D., Yu S., Li C., Zhang Ch., Gao H., Zhou Z. 2012. Expressed sequence tags ana-
lysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green mic-
roalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta). Chinese Science Bulletin 57:
3342–3352. https://doi.org/10.1007/s11434-012-5159-2
Patova E. N., Novakovskaya I. V. 2018. Soil algae of the Northeastern European Russia. Novosti siste-
matiki nizshikh rastenii 52(2): 311–353. [Патова Е. Н., Новаковская И. В. 2018. Почвенные
водоросли северо-востока европейской части России. Новости систематики низших
растений 52(2): 311–353]. https://doi.org/10.31111/nsnr/2018.52.2.311
Perminova G. N., Gutishvili I. S., Kitaev E. V. 1985. Pochvennye vodorosli fitotsenozov Baikalʼs-
kogo zapovednika. Sbornik nauchnykh trudov TsNIL RSFSR: Vodorosli, lishainiki, griby i mokho-
obraznye v zapovednikakh RSFSR. [Collection of scientific papers: “Algae, lichens, fungus and
mosses in nature reserves of RSFSR”]. Moscow: 17–26. [Перминова Г. Н., Гутишвили И. С.,
КитаевЕ.В. 1985. Почвенные водоросли фитоценозов Байкальского заповедника. Сборник
научных трудов ЦНИЛ РСФСР: «Водоросли, лишайники, грибы и мохообразные в заповед-
никах РСФСР». Москва: 17–26].
Peshkova G. A. 1985. Rastitelnostʼ Sibiri (Predbaikalʼye i Zabaikalʼye) [Vegetation of Siberia (Pri-
baikalʼe and Zabaikalʼe)]. Novosibirsk: 145 p. [Пешкова Г. А. 1985. Растительность Сибири
(Прибайкалье и Забайкалье). Новосибирск: 145 с.].
Pleshanov A. S., Pleshanova G. I., Shamanova S. I. 2002. Landscape-climatic patterns of spatial dis-
tribution of refugia in the Baikal region. Contemporary Problems of Ecology 5: 603–610.
Procházková K., Němcová Y., Kulichová J., Neustupa J. 2015. Morphology and phylogeny of para-
sitic and free-living members of the genus Phyllosiphon (Trebouxiophyceae, Chlorophyta). Nova
Hedwigia 101(3–4): 501–518. https://doi.org/10.1127/nova_hedwigia/2015/0288
Sanders W. B., Masumoto H. 2021. Lichen algae: the photosynthetic partners in lichen symbioses.
The Lichenologist 53(5): 347–393. https://doi.org/10.1017/S0024282921000335
Seibel P. N., Müller T., Dandekar T., Schultz J., Wolf M. 2006. 4SALE – A tool for synchronous
RNA sequence and secondary structure alignment and editing. BMC Bioinformatics 7: 498.
https://doi.org/10.1186/1471-2105-7-498
Seibel P. N., Müller T., Dandekar T., Wolf M. 2008. Synchronous visual analysis and editing of
RNA sequence and secondary structure alignments using 4SALE. BMC Research Notes 1: 91.
https://doi.org/10.1186/1756-0500-1-91
Sharma S. K., Nelson D. R., Abdrabu R., Khraiwesh B., Jijakli K., Arnoux M., O’Connor M.J., Bah-
mani T., Cai H., Khapli S., et al. 2015. An integrative Raman microscopy-based workflow for
rapid in situ analysis of microalgal lipid bodies. Biotechnology for Biofuels 8: 164.
https://doi.org/10.1186/s13068-015-0349-1
Shihira I., Krauss K. K. 1965. Chlorella, Physiology and Taxonomy of Forty-one Isolates. Maryland:
xiii + 1–97.
Solovchenko A. E., Khozin-Goldberg I., Didi-Cohen S., Cohen Z., Merzlyak M. N. 2008. Eects of
light and nitrogen starvation on the content and composition of carotenoids of the green microal-
ga Parietochloris incisa. Russian Journal of Plant Physiology 55(4): 455–462.
https://doi.org/10.1134/S1021443708040043
Stanier R. Y., Kunisawa R., Mandel M., Cohen-Bazire G. 1971. Purification and properties of unicel-
lular blue-green algae (order Chroococcales). Bacteriological Reviews 35(2): 171–205.
https://doi.org/10.1128/br.35.2.171-205.1971
Новости систематики низших растений — Novosti sistematiki nizshikh rastenii 56(2): 255–272. 2022
271

Starr R. C., Zeikus J. A. 1993. UTEX—The Culture Collection of Algae at the University of Texas at
Austin 1993 List of cultures. Journal of Phycology 29(2): 1–106.
https://doi.org/10.1111/j.0022-3646.1993.00001.x
Takeda H. 1991. Sugar composition of the cell wall and the taxonomy of Chlorella (Chlorophyceae).
Journal of Phycology 27(2): 224–332. https://doi.org/10.1111/j.0022-3646.1991.00224.x
Temraleeva A. D., Dronova S. A., Moskalenko S. V., Vagapov I. M., Ovchinnikov A. Yu. 2015. Addi-
tions to the algaflora (Chlorophyta) of gray forest soil. Novosti sistematiki nizshikh rastenii 49: 92–
109. [Темралеева А. Д., Дронова С. А., Москаленко С. В., Вагапов И. М., ОвчинниковА.Ю.
2015. Дополнения к флоре зеленых водорослей серой лесной почвы. Новости систематики
низших растений 49: 92–109]. https://doi.org/10.31111/nsnr/2015.49.92
Uchino Y., Yokota A. 2003. “Green-like” and “red-like” RubisCO cbbL genes in Rhodobacter azotofor-
mans. Molecular Biology and Evolution 20(5): 821–830. https://doi.org/10.1093/molbev/msg100
Vinayakumar M., Kessler E. 1975. Physiological and biochemical contributions to the taxonomy of
the genus Chlorella. X. Products of glucose fermentation. Archives of Microbiology 103: 13–19.
https://doi.org/10.1007/BF00436324
White T. J., Bruns T. D., Lee S. B., Taylor J. W. 1990. Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. M. A. Innisʼs PCR protocols: A Guide to Methods and
Applications. San Diego: 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
Zuppini A., Andreoli C., Baldan B. 2007. Heat stress: an inducer of programmed cell death in Chlo-
rella saccharophila. Plant and Cell Physiology 48(7): 1000–1009.
https://doi.org/10.1093/pcp/pcm070
Zuppini A., Gerotto C., Moscatiello R., Bergantino E., Baldan B. 2009. Chlorella saccharophila cyto-
chrome f and its involvement in the heat shock response. Journal of Experimental Botany 60(14):
4189–4200. https://doi.org/10.1093/jxb/erp264
Egorova et al. Chloroidium saccharophilum from the Lake Baikal shore
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