Microb. Resour. Syst. 35
June
（1）
：13
2019─ 23, 2019Vol. 35, No. 1

Taxonomic re-examination of two NIES strains of“Chlamydomonas” within the Reticulata group of
the genus Chloromonas (Volvocales, Chlorophyceae)
Tomoyo Makino1), Ryo Matsuzaki2), Shigekatsu Suzuki2), Haruyo Yamaguchi2),
Masanobu Kawachi2) and Hisayoshi Nozaki1)＊
Department of Biological Sciences, Graduate School of Science, University of Tokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
2)
Center for Environmental Biology and Ecosystem Studies, National Institutefor Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan1)

Two strains, NIES-2215 and NIES-2243, have been labeled“Chlamydomonas (Cd.) gerloffii”and“Cd. subangulosa,”
respectively. Although these two strains were previously suggested to belong to the Reticulata group of the genus
Chloromonas (Cr.), detailed taxonomic examinations are lacking for both strains. Here we taxonomically re-examined thetwo strains based on light and transmission electron microscopy and molecular phylogenetic analysis using a combined
dataset from 18S and 28S ribosomal RNA gene sequences and genes encoding ATP synthase β-subunit, P700 chlorophyll
a-apoprotein A1 and P700 chlorophyll a-apoprotein A2. The“Cd. gerloffii”strain NIES-2215 differed from all otherspecies of the Reticulata group in terms of phylogeny and ultrastructure of the pyrenoid and eyespot; it wasmorphologically identified as Cd. gerloffii. Thus, we propose Cr. difformis Tomo. Makino, Matsuzaki & Nozaki nom. nov.,
to encompass Cd. gerloffii, because of presence of the previous valid description of Cr. gerloffii for a different species.
The“Cd. subangulosa”strain NIES-2243 was significantly different in morphology from the original description of Cd.
subangulosa. It was identified as Cr. typhlos because NIES-2243 and Cr. typhlos strain SAG 26.86 formed a small robust
clade with essentially the same morphological characteristics except for the presence or absence of an eyespot in thechloroplast.

Key words: C hlamydomonas gerloffii, Chlamydomonas subangulosa, Chloromonas difformis nom. nov.,
Chloromonas typhlos, molecular phylogeny, morphology, taxonomy

INTRODUCTION

Chloromonas (Cr.) is a genus of unicellular green
algae with the type species Cr. reticulata(Goroschankin) Gobi (Pröschold et al., 2001). This
genus was previously distinguished from
Chlamydomonas (Cd.) by the absence of pyrenoids
in the chloroplast (e.g. Ettl, 1983). Based on phylogenetic analysis of 18S ribosomal RNA (rRNA) genes
and light microscopy, Pröschold et al., (2001) classified a clade composed of five pyrenoid-containing“Chlamydomonas”strains and two pyrenoid-lacking“Chloromonas”strains as a single species, Cr. reticu︲
lata. Later, Matsuzaki et al. (2012) re-examined ninestrains of“Cr. reticulata”sensu Pröschold et al.
Corresponding author＊

E-mail: nozaki@bs.s.u-tokyo.ac.jp
Accepted: January 21, 2019(2001), based on light and electron microscopy andphylogenetic analysis of multiple DNA regions.
They found enough morphological and genetic differences among the“Cr. reticulata”strains to divide
them into four species: Cr. reticulata; Cr. rosae (H. &O. Ettl) H. Ettl; Cr. chlorococcoides (H. Ettl & K.
Schwartz) Matsuzaki, Y. Hara & Nozaki; and Cr.
typhlos (Gerloff) Matsuzaki, Y. Hara & Nozaki. They
further classified these species as the“Reticulata
group.”
Subsequently, based on comprehensive analysis of
18S rRNA gene sequences, Yumoto et al. (2013) suggested that the Reticulata group includes the following three additional“Chlamydomonas”strainsdeposited in the Microbial Culture Collection at the
National Institute for Environmental Studies (MCCNIES) (Kawachi et al., 2013; http://mcc.nies.go.jp/
index_en.html): Cd. gerloffii H. Ettl strain NIES-2215,
Cd. subangulosa F.E. Fritsch & R.P. John strain

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Taxonomy of two Chloromonas strains

Makino et al.

NIES-2243, and Cd. subtilis Pringsheim strain NIES2244. Although Cd. subtilis strain NIES-2244 wasresolved to be a mislabeled strain of Cd. gerloffii(our unpublished data), the other two strains are
identical to those listed in the Culture Collection of
Algae and Protozoa (CCAP; https://www.ccap.ac.uk)
as authentic strains of Cd. gerloffii and Cd. subangu︲
losa (George 1976; Kawachi et al., 2013). However,these two strains have not been evaluated by electron microscopy or other phylogenetic methods
except in a recent study by Barcytė et al. (2018a),
who examined the secondary structure of the internal transcribed spacer region-2 of nuclear rDNA
(nuclear rDNA ITS2) in Cd. gerloffii strain CCAP
11/72 (=NIES-2215; Kawachi et al., 2013).
The present study was undertaken to taxonomically re-examine these two strains of“Chlamydomonas”based on light and electron
microscopy and phylogenetic analysis of five DNA
regions. The results indicated that Cd. gerloffii
should be reclassified into the genus Chloromonas
whereas Cd. subangulosa strain NIES-2243 should beclassified as Cr. typhlos. The morphology, phylogeny and taxonomy of Cr. difformis Tomo. Makino,Matsuzaki & Nozaki nom. nov. and Cr. typhlos aredescribed in this report.

MATERIALS AND METHODS

Cultures
Nine strains of four species in the Reticulata group(Matsuzaki et al., 2012) were obtained from theSammlung von Algenkulturen at the University of
Göttingen (SAG; https://www.uni-goettingen.de/en/www.uni-goettingen.de/de/184982.html): Cr. reticula︲ta strains SAG 29.83, SAG 32.86 and SAG 26.90; Cr.
rosae strain SAG 51.72; Cr. chlorococcoides strains
SAG 15.82, SAG 16.82, SAG 12.96 and SAG 72.81; andCr. typhlos strain SAG 26.86. “Cd. gerloffii”strain
NIES-2215 and“Cd. subangulosa”strain NIES-2243
were provided by MCC-NIES. The original strain of“Cd. subangulosa”(CCAP 11/28) was directly sentfrom CCAP. The cultures were grown in screw-cap
tubes (18×150 mm) containing approximately 10 ml
AF-6 medium (Kato 1982; Kawachi et al., 2013) at
20℃ under 110-150 μmol photons m-2 s-1 light fromfluorescent lamps under a 14 h light: 10 h dark photoperiod.
Light microscopy
Light microscopy was performed using BX-53 andB X -60 m i c r o s c o p e s ( O l y m p u s , T o k y o , J a p a n )equipped with Nomarski differential interference
optics. Vegetative cells in 6- to 9-day-old cultures
were observed during the light photoperiod. Cell
divisions were observed during the light or dark
photoperiod.
Transmission electron microscopy
Six- to 9-day-old cultures were fixed during thelight photoperiod for observation by transmission
electron microscopy (TEM). Fixation and observation were performed as described by Matsuzaki etal., (2012).
Gene sequencing
New sequences were determined by direct
sequencing of polymerase chain reaction products as
described previously (Nozaki et al., 2000, 2002;
Matsuzaki et al., 2012, 2014) for genes encoding ATP
synthase β-subunit (atpB), P700 chlorophyll a-apoprotein A1 (psaA) and P700 chlorophyll a-apoprotein A2(psaB) in“Cd. gerloffii”strain NIES-2215 and“Cd.
subangulosa”strain NIES-2243, and for 28S rRNA
sequences in two outgroup species (Table 1) except
for psaB gene of NIES-2215. The“Cd. gerloffii”
strain NIES-2215 psaB gene was sequenced by using
cDNA that was constructed using RNA (see below)
with SuperScript III Reverse Transcriptase (Thermo
Fisher Scientific, Waltham, MA, USA). Sequences ofnuclear rDNA ITS2 of NIES-2244 and CCAP 11/28
were determined as described previously(Matsuzaki et al., 2012).
F o r ne xt -ge ne r a t io n s e que nc ing, R N A wasextracted during the light photoperiod based on themethods of Featherston et al. (2018), and cDNA
library was constructed using the NEBNext Ultra
RNA Library Prep Kit for Illumina (New England
Biolabs, Ipswich, MA, USA). cDNA was sequenced
using the MiSeq platform (Illumina, San Diego, CA,
USA) with the 500-cycle MiSeq Reagent Kit v2 toconstruct a de novo assembled database via Trinity(Grabherr et al., 2011). For 11 strains of ingrouptaxa (Table 1), 28S rRNA sequences were extracted
from the database.
Phylogenetic analysis
The 14 strains analyzed here (Table 1) were composed of 11 strains belonging to Reticulata groupand three species served as the outgroup. The outgroup strains were selected following Matsuzaki et─ 14 ─

Microb. Resour. Syst. June 2019Vol. 35, No. 1

Table 1 List of taxa/strains included in the phylogenetic analyses of
EMBL/GenBank accession numbers of 18S RNA, 28S RNA,
Taxa

Straingroup (Fig. 1) and DDBJ/
andsequences

Accession Number
18S rRNA 28S rRNA
atpB
psaA

psaB

Ingroup
SAG 12.96

Cr. chlorococcoides

SAG 15.82
SAG 16.82
SAG 72.81

AJ410451,
AB624556
AJ410449,
AB624555
AJ410450,
AB624557
U70785,
AB624558

LC438810† AB624581 AB624584 AB624596
LC438801†,
AB624580 AB624583 AB624595
AB906359
LC438809† AB624582 AB624585 AB624597
LC438806†,
AB084309 AB624586 AB084343
DQ015725

Cr. reticulata

AF517090,
LC438803† AB084316 AB624589 AB084352-3
AB624563
SAG 29.83
U70791,
LC438808†,
AB084312 AB624587 AB084346-7(=UTEX 1970) AB624560 AF395508
AF517091,
LC438805† AB084314 AB624588 AB084349
SAG 32.86
AB624561

Cr. rosae

LC438804†,
SAG 51.72
AB624565
AB084315 AB624590 AB084350-1(=UTEX 1337)
KC196731

Cr. typhlos

LC438802†,
SAG 26.86
AB624566 LC360479, AB084307 AB624591 AB084341(=UTEX 1969)
AF395497

Cr. difformis

NIES-2215

AB701536 LC438811† LC438815† LC438818† LC438814†

Cr. typhlos

NIES-2243

AB701538 LC438807† LC438816† LC438817† LC438813†

Cr. augustae

SAG 5.73

AJ410452

Cr. serbinowii

UTEX 492

U70795,
LC360478† AB084317 AB624594 AB084354
AB624568

Gloeomonas anomalipyrenoides

NIES-447

AB504776,
LC437681† AB084313 AB624593 AB084348
AB624567

SAG 26.90

Outgroup
LC360474.1 AB504757 AB624592 AB504769†

al. (2012). The new nucleotide sequences are available in DDBJ/EMBL/GenBank (Table 1).
The 18S rRNA, 28S rRNA, atpB, psaA and psaB
sequences were aligned using ClustalW (Thompson
et al., 1994). The 18S rRNA sequence data were
found to correspond to positions 127-1626 of the 18S
rRNA sequence of Volvox carteri F. Stein strain
UTEX 1885 (Rausch et al., 1989). The 28S rRNA
sequence data correspond to positions 279-2095 of
the 28S rDNA sequence of Oryza sativa Linnaeus(Takaiwa et al., 1985). The analyzed atpB, psaA andpsaB exons correspond to positions 199-1326, 5801365 a n d 271-1155 o f t h e C h l o r e l l a v u l g a r i s
Beijerinck atpB, psaA and psaB genes, respectively(Wakasugi et al., 1997). Because the combined
sequences from 18S rRNA, 28S rRNA atpB, psaA

Sequenced in the present study

and psaB were the same between SAG 15.82 andSAG 16.82, they were treated as a single operationaltaxonomic unit (OTU).
The combined 6,115-bp data matrix for the 18S
rRNA, 28S rRNA, atpB, psaA and psaB sequencesfrom the 13 OTUs (available from TreeBASE:
http://www.treebase.org/treebase-web/home.html;
study ID: S23720) was subjected to Bayesian inference (BI), maximum likelihood (ML), maximum parsimony (MP) and neighbor-joining (NJ) analyses. The
substitution model for each BI partition was HKY +G for 18S rRNA; GTR + I + G for 28S rRNA; GTR +I + G for the first codon position of the plastid genes
atpB, psaA and psaB; F81 + I + G for the second
codon position of the plastid genes; or GTR + G for
the third codon position of the plastid genes, select─ 15 ─Taxonomy of two Chloromonas strains

Makino et al.

ed using MrModeltest 2.3 (Nylander, 2004). BI was
performed using MrBayes 3.2.6 (Ronquist et al.,2012), as described previously (Nakada et al., 2008),
with 1,000,000 generations of Markov chain Monte
Carlo iterations and discarding the first 25% asburn-in. The ML analysis was conducted using
PAUP* 4.0b10 (Swofford, 2002) with the GTR + I + G
model selected by Modeltest 3.7 (Posada & Crandall,1998), to estimate the bootstrap values (BVs;
Felsenstein, 1985) based on 1000 heuristic search
replications using the tree-bisection-reconnection(TBR) branch-swapping algorithm. Bootstrap valuesof the MP analysis were also obtained using
PAUP* 4.0b10 based on 1000 replicates of the MP
analysis and randomly adding 10 replicates from aheuristic search using the TBR branch-swapping
algorithm, and NJ analysis, with ML distances deter-

mined using the appropriate substitution model.

RESULTS AND DISCUSSION

Molecular phylogenetic analysis
The BI tree constructed using the dataset composed of 18S rRNA, 28S rRNA, atpB, psaA and psaB
gene sequences is shown in Figure 1.
The Reticulata group was composed of two clades.
One clade contained“Cd. subangulosa”strain NIES2243 and Cr. typhlos strain SAG 26.86, and its monophyly was strongly supported by 1.00 posterior
probability in BI and 100% BV in the ML, MP and
NJ analyses. The other clade was composed of fourother species, supported by 51-71% BV, and was
subdivided into two lineages. One lineage is arobust monophyletic group (supported by 1.00 posterior probability and 80-87% BV) composed of theFig. 1 P hylogenetic positions of()
strain NIES-2215 andstrain NIES-2243 withingroup of the genus(Table 1). The
tree was constructed by Bayesian inference based on 6,115 base pairs from combined 18S and 28S
rRNA, and atpB, psaA and psaB genes. Corresponding posterior probabilities (PP; 0.95 or more)are shown at top left. Numbers shown in top right, bottom left and bottom right indicate bootstrap values (50% or more) from maximum likelihood, maximum parsimony and neighbor-joining
analyses respectively. Branch lengths are proportional to the evolutionary distances which areindicated by bar top left to the tree.
─ 16 ─

Microb. Resour. Syst. June 2019Vol. 35, No. 1

four Cr. chlorococcoides strains and the three Cr.
reticulata strains. The other lineage was composedof“Cd. gerloffii”strain NIES-2215 and Cr. rosae,
although its monophyly was only found in BI treewith low support values (<0.95 posterior probability).
Thus, it was not clear which species or group is thesister group to the clade composed of Cr. chlorococ︲
coides and Cr. reticulata. However, it is clear fromthe present tree that“Cd. gerloffii”strain NIES2215 was not closely related to any other included
species in the Reticulata group, and“Cd. subangulo︲sa”strain NIES-2243 was sister to Cr. typhlos strain
SAG 26.86.
In the present phylogenetic tree, Cr. typhlos(including“Cd. subangulosa”strain NIES-2243) has
pyrenoids and represents a weakly supported basal
lineage within the Reticulata group, and other species within the group either contain (Cr. chlorococ︲
coides and Cr. difformis “Cd.
[gerloffii”]) or lack (Cr.
reticulata and Cr. rosae) a pyrenoid. Thus, the mostrecent common ancestor of the Reticulata group

Figs. 2‑5

might have had a pyrenoid in the chloroplast.
Subsequently, the pyrenoid may have independently
been lost twice in ancestors of Cr. rosae and Cr.
reticulata. The Reticulata group is a small clade
within Chloromonadinia (Yumoto et al., 2013), while
pyrenoid loss may have occurred more than once
during the evolution of this group. Thus, theReticulata group represents a model for studying
the pyrenoid evolution. More OTUs possibly
belonging and closely related to the Reticulata
group, such as Cr. arctica Barcytė & Hodač (Barcytė
et al., 2018a) and Cr. svalbardensis Barcytė & Hodač(Barcytė et al., 2018b), are needed to understand themore detailed evolutionary events of pyrenoids within this group.
Morphology
Line drawings of vegetative cells of the twostrains are shown in Figures 2-5.
“Cd. gerloffii”strain NIES-2215 (Figs. 6-12):
Vegetative cells were unicellular, biflagellate, andLine drawings of light microscopic characteristics of vegetative cells in two strains ofgroup. 2, 3. Chloromonas difformis Tomo. Makino, Matsuzaki & Nozaki nom.
nov. strain NIES-2215 (formerly labeled“Chlamydomonas gerloffii”). 4, 5. Chloromonas typh︲l o s ( G e r l o f f ) M a t s u z a k i , Y . H a r a & N o z a k i s t r a i n N I E S -2243 ( f o r m e r l y l a b e l e d“Chlamydomonas subangulosa”). 2, 4. Optical sections of cells. 3, 5. Surface views of cells.
─ 17 ─

Taxonomy of two Chloromonas strains

Figs. 6‑12

Makino et al.

Tomo. Makino, Matsuzaki & Nozaki. strain NIES2215 (formerly labeled). 6-9. Nomarski interference
micrographs of vegetative cells, shown at the same magnification. Scale bar=5 μm. 6.
Optical section of a cell. 7. Surface view of a cell. Arrowheads indicate irregular perforations and incisions on the chloroplast surface. 8. Surface view of a cell showing a shortrod-shaped eyespot. 9. Optical section of a dividing cell showing rotation of parental protoplast and apparently transverse first division. 10-12. Transmission electron micrographsof vegetative cells. 10. Longitudinal section of a cell. Scale bar=2 μm. 11. Pyrenoid showing
thylakoid penetration into pyrenoid matrix (arrows). Scale bar=1 μm. 12. Eyespot composed of three layers of globules. Scale bar=1 μm. ch, chloroplast; cv, contractile vacuole; e,eyespot; n, nucleus; pa, papilla; py, pyrenoid; s, starch grain.

broadly ellipsoidal in shape, measuring 10-18 μm
long and 8-15 μm wide. The cell had a single nucleus, a prominent papilla at the base of the flagella,
and a massive urn-shaped or stellate chloroplastwith a single and somewhat indistinct pyrenoid in
the middle of the cell (Fig. 6). The chloroplast had
irregular perforations and incisions on the surface,
but the lobes of the chloroplast were indistinct (Fig.
7). The eyespot was shaped as a short rod or ellipsoid and was positioned in the anterior 1/3 to 2/5 ofthe cell (Figs. 6, 8). The pyrenoid was spherical or
ellipsoidal and positioned in the center of the chloroplast. The outline of the pyrenoid was obscure
under light microscopy. The cytoplasm contained
many globular vacuoles distributed in the incisions
and perforations of the chloroplast. The papilla washemispherical, often with a flat top face, and thenucleus was positioned in the cytoplasm anterior to
the pyrenoid. The length of the flagella was nearly

1.5 times that of the cell. Two contractile vacuoles
were located near the flagellar base. Asexual reproduction occurred via zoospore formation, producing
four biflagellate zoospores within the parental cellwall. Before the first cell division, the protoplast
rotated 90° (Fig. 9). Under TEM, the pyrenoid
matrix was surrounded by starch granules, whichwere thin, meager and often separated from one
another (Figs. 10, 11). The pyrenoid matrix was
penetrated from various directions by thylakoid
lamellae through the spaces between associatedstarch granules (Fig. 11). The eyespot was composed of three layers of globules (Fig. 12).
According to Ettl (1965, 1983), Cd. gerloffii is a
species of the pyrenoid-containing genus“Chlamydomonas”and is characterized by a broadly
ellipsoidal cell shape and indistinct structures of thechloroplast lobes and pyrenoid with peripheral
starch granules. The present phylogenetic analyses

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Microb. Resour. Syst. June 2019

Figs. 13‑19

Vol. 35, No. 1

strain NIES-2243 (formerly labeled). 13-16. Nomarski interference micrographs of vegetative cells, shown at thesame magnification. Scale bar=5 μm. 13. Optical section of a cell. 14. Surface view of acell. Arrowheads indicate irregular perforations and incisions on the chloroplast surface.
15. Surface view of a cell showing a rod-shaped eyespot. 16. Optical section of a dividing
cell showing rotation of parental protoplast and apparently transverse first division.
17-19. Transmission electron micrographs of vegetative cells. 17. Longitudinal section ofa cell. Scale bar=2 μm. 18. Pyrenoid showing thylakoid lamellae penetrating into pyrenoid
matrix (arrows). Scale bar=1 μm. 19. Eyespot composed of two layers of globules. Scalebar=1 μm. ch, chloroplast; cv, contractile vacuole; e, eyespot; n, nucleus; pa, papilla; py,pyrenoid; s, starch grain.

demonstrated that Cd. gerloffii represents a distinct
phylogenetic position separated from the other four
species within the Reticulata group. Recently,
Barcytė et al. (2018a) reported a new species, Cr.
arctica, which is sister to Cd. gerloffii strain CCAP
11/72 (=NIES-2215) in the 18S rRNA gene tree.
However, Cr. arctica lacks pyrenoids in the chloroplast and exhibits enough differences in its nuclear
rDNA ITS2 sequence to be considered distinct fromCd. gerloffii at or above the species level (Barcytė etal., 2018a). Although Barcytė et al. (2018a) showedthat Cr. paraserbinowii (Skuja) Gerloff & H. Ettl

strain SAG 71.72 and Cd. hydra H. Ettl strain SAG
4.73 belong to the Reticulata group, the former species lack pyrenoids and the latter species is clearly
different from Cd. hydra based on light microscopic
characteristics (Ettl, 1983). Therefore, Cd. gerloffii
should be recognized as a distinct species within thegenus Chloromonas. However, the epithet gerloffii
is unavailable in Chloromonas because Cr. gerloffii
H. Ettl (1963) was described for different species.
Thus, we propose the new name Cr. difformis toencompass Cd. gerloffii under the genus
Chloromonas. Within the Reticulata group, Cr. dif︲─ 19 ─

Taxonomy of two Chloromonas strains

Makino et al.

formis can be distinguished from two other pyrenoid-containing species based on pyrenoid morphology. The pyrenoid matrix in vegetative cells of Cr.
chlorococcoides and Cr. typhlos is entirely surrounded by thick starch plates with no space between
starch granules except for the spaces through which
thylakoid lamellae penetrate (Matsuzaki et al., 2012).
In contrast, the starch plates were thin and exhibited spaces between starch granules surrounding partof the pyrenoid matrix in Cr. difformis (Figs. 10, 11).
This ultrastructural difference in the starch plates
may reflect the difference in pyrenoids observed bylight microscopy. Under light microscopy, the pyrenoids are prominent or distinct in the former two
species (Matsuzaki et al., 2012), whereas the Cr. dif︲formis pyrenoids are somewhat indistinct (Ettl 1983;
Fig. 6).
“Cd. subangulosa”strain NIES-2243 (Figs. 13-19):
Vegetative cells were unicellular, biflagellate, andellipsoidal in shape, measuring 13-18 μm long and6-12 μm wide. The cells had one nucleus, a prominent papilla at the base of the flagella, and a parietal
chloroplast with a single pyrenoid positioned in the
middle of the cell (Fig. 13). The chloroplast was cupor urn-shaped, with irregular perforations and incisions on the surface (Fig. 14). The eyespot wasshaped as a short rod or ellipsoid and positioned in
the anterior 1/3 of the cell (Fig. 15). The pyrenoid
was prominent, spherical or ellipsoidal in shape, andlateral in position. The papilla was hemispherical,
often with a flat top face, and the nucleus was positioned in the cytoplasm anterior to the pyrenoid.
The flagella were the same length as the cell. Twocontractile vacuoles were located near the base of
the flagella. Asexual reproduction occurred via zoospore formation, producing four biflagellate zoospores within the parental cell wall. Before the firstcell division, the protoplast rotated 90° (Fig. 16).
TEM demonstrated that the pyrenoid matrix was
surrounded entirely by starch plates, and thylakoid
lamellae penetrated into the pyrenoid matrix fromvarious directions (Figs. 17, 18). The eyespot wascomposed of two layers of globules (Fig. 19).
Ettl (1983) classified Cd. subangulosa into the“Euchlamydomonas”group because of its basal
pyrenoid in the cup-shaped chloroplast. CCAP
11/28 was referred to be derived from one of theoriginal author of Cd. subangulosa “John”(; George1976). However, significant morphological differences were recognized between the original descriptionof Cd. subangulosa (Fritsch & John, 1942) and thepresent observations of NIES-2243. According to
Fritsch & John (1942), this species has a basal pyrenoid, and the first division is longitudinal. However,the present cultured material has a lateral pyrenoid
in the urn-shaped chloroplast, and the first division
was apparently transverse after 90° rotation of the
protoplast (Figs. 4, 5, 13-16). Moreover, the cell size
according to the original description (Fritsch & John,
1942) conflicts with that of our observations. Fritsch& John (1942) reported that the cells are 16-20 μm
long, whereas in our observation of NIES-2243, thecells were 13-18 μm long. These morphological features were essentially the same between strains
NIES-2243 and CCAP 11/28, which were recently
sent directly from CCAP, and the sequences ofnuclear rDNA ITS2 in NIES-2243 and CCAP 11/28
were exactly the same (DDBJ/EMBL/GenBank
accession numbers LC439250 and LC439251, respectively). Thus, CCAP 11/28 was misplaced with a different strain or misidentified as Cd. subangulosa(George 1976).
“Cd. subangulosa”strain NIES-2243 and Cr. typh︲los strain SAG 26.86 formed a small robust clade
within the Reticulata group. However, some morphological differences were recognized between
these two strains. NIES-2243 has one eyespot percell, whereas Cr. typhlos lacks eyespots (Matsuzaki
et al., 2012). Moreover, on average, the ratio of thecell width to length is lower in NIES-2243 than in
Cr. typhlos strain SAG 26.86. The average ratio of
the cell length to width (based on observations of 25
cells from each strain) is 1.39 in SAG 26.86 and 1.53
in NIES-2243. However, NIES-2243 is highly similar
to Cr. typhlos strain SAG 26.86 in other morphological attributes (Matsuzaki et al., 2012). Both strains
have a hemispherical prominent papilla at the baseof the flagella, a parietal chloroplast with irregularperforations and incisions on the surface and a single lateral pyrenoid positioned in the middle of thecell, and a nucleus positioned anterior to the pyrenoid (Figs. 4, 5, 13, 14) (Matsuzaki et al., 2012).
Because of these morphological characteristics, thestigma-containing species Cd. media G.A. Klebs issimilar to NIES-2243 (Klebs, 1896; Ettl, 1983).
However, Cd. media is different from Cr. typhlos
a n d N I E S -2243 d u e t o i t s l a r g e c e l l l e n g t h .
Vegetative cells of Cd. media measure 18-20 μm
long (Klebs, 1896; Ettl, 1983), whereas Cr. typhlos(including SAG 26.86) and NIES-2243 have vegeta-─ 20 ─

Microb. Resour. Syst. June 2019Vol. 35, No. 1

tive cells measuring 6-18 μm long (Ettl, 1983;
Matsuzaki et al., 2012). In addition, the culture
strain NIES-2743, recently determined to form thebasis of the epitype of Cd. media, has a relatively
smooth chloroplast surface, and its phylogenetic
position is distantly separate from the genus
Chloromonas (Nakada et al., 2012). This species wasrecently transferred to the genus Microglena as M.
media (G.A. Klebs) Nakada (Nakada & Tomita, 2014).
Thus, NIES-2243 is assigned here to the monophyletic species Cr. typhlos. Cr. typhlos now exhibits morphological variability, particularly in terms of thepresence or absence of an eyespot in the chloroplast.
Such eyespot variability has been reported in another monophyletic species of the unicellular
Volvocales, Chlorogonium euchlorum (Ehrenberg)
Ehrenberg (Nozaki et al., 1998) and among strains of
the unicellular volvocalean species Gloeomonas
anomalipyrenoides Nakada, Matsuzaki & Nozaki(Nakada et al., 2015).
Recently, Boldina (2017) reported light and electron microscopy of a Russian strain of Cr. typhlos.
Interestingly, this strain has a stigma as in NIES2243. However, its phylogenetic position was not
resolved. Further molecular phylogenetic studies
using this Russian stain will resolve more accurate
taxonomic status of Cr. typhlos.

TAXONOMIC TREATMENT

Chloromonas difformis Tomo. Makino, Matsuzaki &Nozaki nom. nov.
Replaced synonym: Chlamydomonas gerloffii H. Ettl
1965: 298, fig. 15 (holotype); pl. 30: figs. 1-3.
Priority for Chloromonas gerloffii H. Ettl 1963: 398.
Etymology: the name“difformis”meaning“with different forms from others”refers to the unique anddistinct features of this species within the Reticulata
group, such as a basal or central pyrenoid surrounded by meager starch plates, many globular vacuoles
in the cytoplasm, and three layers of eyespot globules.
DISTRIBUTION: Czech (Ettl, 1965)STRAIN EXAMINED: NIES-2215 (=CCAP 11/72,
authentic strain of Cd. gerloffii strain; George, E.A.,
1976).

ACKNOWLEDGMENT

This work was supported by a Grant-in-Aid for
Scientific Research (No. 16H02518 to HN) from
MEXT/JSPS KAKENHI.

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Chloromonas 属（ボルボックス目，緑藻綱）の Reticulata グループに所属する Chlamydomonas と表示されている NIES 保有 2 株の分類学的再同定
牧野朋代 1），松﨑

令 2），鈴木重勝 2），山口晴代 2），河地正伸 2），野崎久義 1）

東京大学大学院理学系研究科生物科学専攻，2）国立環境研究所生物・生態環境系センター

1）

Yumoto et al.（2013, Microbiol. Cult. Coll.）は 18S rDNA 系統から Chlamydomonas（Cd.）属と表示されている NIES 保
有の 2 株（“Cd. gerloffii”NIES-2215 と“Cd. subangulosa”NIES-2243）が Chloromonas（Cr.）属のタイプ種を含むクレード（Reticulata グループ）に所属することを明らかにしたが，詳細な研究は実施されなかった．本研究ではこれら 2 種の光学・透過型電子顕微鏡による観察と複数遺伝子による分子系統解析を実施した．その結果，NIES-2215 は Reticulata グループの他の 4 種と形態と系統で異なり，Chloromonas 属に所属すると判断したが，先行名として Cr. gerloffii H. Ettl 1963 が存在するため，新名 Cr. difformis Tomo. Makino, Matsuzaki & Nozaki を提唱した．NIES-2243 は眼点をもつという点以外はCr. typhlos と形態的に類似し，Cr. typhlos SAG 26.86 と単系統群を構成したため，Cr. typhlos と再同定した．

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