Chlorella variabilis and Micractinium reisseri sp. nov.

Phycological Research 2010; 58: 188–201
Chlorella variabilis and Micractinium reisseri sp. nov.
(Chlorellaceae, Trebouxiophyceae): Redescription of the
endosymbiotic green algae of Paramecium bursaria
(Peniculia, Oligohymenophorea) in the 120th year
pre_579
188..201
Ryo Hoshina,1 Mitsunori Iwataki3 and Nobutaka Imamura2*
1
Department of Biomedical Science, College of Life Sciences, 2Department of Pharmacy, College of Pharmaceutical
Sciences, Ritsumeikan University, Shiga, and 3Institute for East China Sea Research, Nagasaki University, Nagasaki,
Japan
SUMMARY
Symbiotic algae of the ciliate Paramecium bursaria
(Ehrenberg) Focker are key species in the fields of
virology and molecular evolutionary biology as well as in
the biology of symbiotic relationships. These symbiotic
algae were once identified as Zoochlorella conductrix
Brandt by the Dutch microbiologist, Beijerinck 120
years ago. However, after many twists and turns, the
algae are today treated as nameless organisms. Recent
molecular analyses have revealed several different algal
partners depending on P. bursaria strains, but nearly all
P. bursaria contains a symbiont belonging to either the
so-called ‘American’ or ‘European’ group. The absence
of proper names for these algae is beginning to provoke
ill effects in the above-mentioned study areas. In the
present study, we confirmed the genetic autonomy of
the ‘American’ and ‘European’ groups and described
the symbionts as Chlorella variabilis Shihira et
Krauss and Micractinium reisseri Hoshina, Iwataki
et Imamura sp. nov., respectively (Chlorellaceae,
Trebouxiophyceae).
Key words: Chlorella variabilis, ITS2, Micractinium reisseri, Paramecium bursaria, species concept.
INTRODUCTION
The unicellular ciliate Paramecium O. F. Müller (Peniculia, Oligohymenophorea) is one of the most studied
protists (Fokin et al. 2004). Within this genus, P. bursaria (Ehrenberg) Focker is an invaluable asset. Paramecium bursaria is a single-celled protozoan that
maintains several hundred algal cells within its own
cytoplasm, lending it a green color (Karakashian et al.
1968). Thus, P. bursaria is known as the green Paramecium. The alga lives inside the ciliate, providing it
with photosynthate, while the ciliate provides the alga
with protection from other protozoans or viruses and
chauffeurs it to brightly lit areas for optimal photosynthesis (Hoshina & Imamura 2009a). Therefore, P. bursaria is a ciliate that, like corals and lichens, has
established a mutualistic relationship with an algal
species. Unlike in corals or lichens, even though P.
bursaria is intrinsically a heterotrophic protist, the
daughter cells inherit the same symbionts that were
retained by the mother cell (Siegel 1960).
Although the systematics of the chlorophytes
remains somewhat disorganized, researchers have
determined the progenitor, identified the known
species, and described new species for the Paramecium symbiont. The genus Zoochlorella Brandt was
introduced in 1882 by Brandt, who so designated algae
isolated from the green coelenterate Hydra viridis Linnaeus (Z. conductrix Brandt) and sponges (Z. parasitica
Brandt). The P. bursaria symbiotic alga was later
assigned to Z. conductrix by Beijerinck (Beijerinck
1890), although Brandt (1882) appeared to have
regarded the symbiont as the same as that possessed by
hydra. Today, the first-described Zoochlorella is considered synonymous with the well-known genus Chlorella
Beijerinck (Silva 1999). Although Zoochlorella is an
older name than Chlorella, the genus Chlorella was
determined to be conserved with the type species C.
vulgaris Beijerinck, and consequently, Zoochlorella was
rejected (Appendix IIIA, Greuter et al. 2000).
The symbiotic alga was later renamed as an independent species, ‘C. paramecii’ nom. nud., by Loefer.
However, he did not publish a description for this
nomenclature, for which reason Shihira and Krauss
(1965) later rejected ‘C. paramecii’ and instead
*To whom correspondence should be addressed.
Email: [email protected]
Communicating editor: M. Hoppenrath.
Received 1 September 2009; accepted 25 February 2010.
doi: 10.1111/j.1440-1835.2010.00579.x
© 2010 Japanese Society of Phycology
Taxonomy of the photobionts of Paramecium
described a new species, C. variabilis Shihira et Krauss,
based on authentic strains no. 130 from the Indiana
Algal Culture Collection (which was later moved to the
Culture Collection of Algae at the University of Texas
[UTEX]; the strain is currently not available) and no.
211/6 from the Cambridge Collection (which was later
moved to the Culture Collection of Algae and Protozoa
[CCAP], UK; this strain is also currently not available),
which were claimed to be Loefer’s ‘C. paramecii’.
However, strain 211/6 showed distinct biochemical and
physiological characteristics compared with the other
strains of P. bursaria symbionts (later identified as Auxenochlorella protothecoides [Krüger] Kalina et Punčochářová, Douglas & Huss 1986; Kessler & Huss 1990),
raising questions as to whether it was a symbiont at all.
Consequently, the species name C. variabilis is no
longer in use. A duplicate of 211/6 was transferred to
the Culture Collection of Algae at the University of
Göttingen (SAG), Germany, as strain 211-6. This strain,
however, showed symbiotic characteristics (Kessler &
Huss 1990).
Modern observations of the symbiotic algae are represented by the studies conducted by German researchers (Professors E. Kessler, W. Reisser, V.A.R. Huss, and
their coworkers). These researchers have examined
algal strains isolated from P. bursaria collected in
Germany and the United States, strains Pbi and
NC64A, respectively (NC64A(M) and NC64A(P) appear
in some literature; the former is regarded as the true
NC64A, and the latter is unlikely to be of symbiotic
origin; Douglas and Huss 1986). Their studies demonstrated that the ‘American’ and ‘European’ algal
strains each possess certain distinct characteristics.
The general conclusion of these taxonomic studies
was that no reason exists to distinguish the symbiotic
algae from the genus Chlorella. Rather, one may reasonably assume that these symbionts are derived
from free-living Chlorella spp. but with some evolved
characteristics.
A series of symbiont analyses resulted in the discovery of the Chlorella virus, which specifically infects the
symbiotic alga NC64A and other ‘American’ symbionts
but does not infect ‘European’ symbionts or free-living
Chlorella; likewise, a distinct virus specifically infects
Pbi and other ‘European’ symbionts (Reisser et al.
1988). Thus, these viruses were designated the NC64A
and Pbi viruses, respectively. Reisser et al. (1990) confirmed that each virus is able to identify its host species
based on a key factor present in the algal cell wall.
Takeda (1995) analyzed the cell wall sugar composition
of symbiotic algae. He noted that the characteristics of
the symbiont cell wall indicated a relationship with
Chlorella species (especially C. kessleri Fott et Nováková); however, the distinct proportion of compositional
sugars indicated that the symbiotic algae belong to a
new species. Thus, the question of whether symbiotic
© 2010 Japanese Society of Phycology
189
algae represent an independent species has become an
increasingly important focus in more recent studies.
Isozyme distribution patterns for several enzymes have
demonstrated uniformity within the ‘American’ vs.
‘European’ algae and multiplicity among different
groups (i.e. ‘American’, ‘European’, and free-living
Chlorella spp.; Linz et al. 1999). Similar results were
also obtained through the analysis of universal primer
polymerase chain reaction (UPPCR) fragmentation patterns (Kvitko et al. 2001).
Recent molecular analyses have revealed many facts
about P. bursaria symbiotic algae: (i) P. bursaria almost
always contains single cloned algae as naturally occurring photobionts; (ii) in most cases, the symbionts are
either ‘American’ or ‘European’, but in two exceptional
cases, Chlorella vulgaris and Coccomyxa sp. have been
identified; (iii) the ‘American’ and ‘European’ groups
are characterized by different length small subunit
(SSU) rDNA due to the varying number of group I intron
insertions (three-intron or single-intron); (iv) each group
(‘American’ or ‘European’) of algae has a highly uniform
SSU rDNA (including introns)-ITS1-5.8S rDNA-ITS2
sequence; (v) ‘American’ and ‘European’ sequences
differ by only seven or eight nucleotides in the SSU
rDNA (exon), whereas the ITS sequences exhibit significant differences (approximately 20%); (vi) both the
‘American’ and ‘European’ groups belong to the Chlorella clade (sensu Krienitz et al. 2004); (vii) each group
is equivalent to a species discrete from any known
free-living species; (viii) ancestral P. bursaria may have
obtained the ‘American’ and ‘European’ algae separately; and (ix) the affiliation of SAG 211-6 to the
‘American’ type has also become apparent (Hoshina
et al. 2004, 2005; Gaponova et al. 2007; Summerer
et al. 2008; Hoshina & Imamura 2008a; Luo et al.
2010).
Both the ‘American’ and ‘European’ groups have
attracted attention because of their particularly evolved
group I introns (Hoshina & Imamura 2008b, 2009b).
Johansen and Haugen (2001) proposed a nomenclature
system for the rDNA group I introns based on the host
species name (one-letter abbreviation of the genus
name and two-letter abbreviation of the specific
epithet) and insertion site; this system has been well
accepted in the arena of intron study. However, because
of the lack of a species name for the symbionts, this
rule cannot be applied to their introns. In virology, the
Chlorella virus has developed into a very important
taxon possessing a large dsDNA genome that encodes
many proteins with some unique features. The virus led
to the establishment of the viral genus Chlorovirus
(family Phycodnaviridae) based on three criteria:
viruses that infect P. bursaria ‘American’ symbionts,
viruses that infect P. bursaria ‘European’ symbionts,
and viruses that infect the green hydra symbionts (for a
review on the Chlorella virus, see Van Etten 2003). The
190
namelessness of these algal symbionts has coerced
virologists into using strange terminologies, such as the
NC64A virus and the Pbi virus. The greatest harm
associated with the anonymity is the irresponsible
species identifications that subsequently generate a
chain reaction of misidentification and further disorder.
In fact, three sequence entries of ‘American’ symbionts
are registered as ‘Chlorella vulgaris’ (AB191205-207),
and according to these misidentified entries, further
misidentifications of the symbionts have already
occurred (Kodama et al. 2007; Kodama & Fujishima
2008, 2009a,b). Albeit an exceptional case, true C.
vulgaris has been found from one P. bursaria strain
(Hoshina & Imamura 2008a), which further complicates matters. The underlying problem is clear. Each
‘American’ and ‘European’ symbiont must be assigned
a species name as soon as possible to avoid further
taxonomical confusion.
The P. bursaria symbionts will fascinate protozoologists in the decades ahead and will remain important
study species for molecular evolutionary biologists and
virologists. The current International Code of Botanical
Nomenclature notes that ‘The purpose of giving a name
to a taxonomic group is not to indicate its characters or
history, but to supply a means of referring to it and to
indicate its taxonomic rank’ (Preamble, McNeill et al.
2006). Today, most, if not all, of the endosymbiotic
algae of P. bursaria seem to be autonomous from other
known species. Therefore, today, 120 years after Beijerinck identified a symbiont alga from P. bursaria, we
redescribe the ‘American’ and ‘European’ algae to
provide a means of referencing them correctly.
MATERIALS AND METHODS
Culture
Cells of P. bursaria symbiont strains ATCC 50258
(NC64A) and CCAP 211/83 (Pbi) were cultured in C
medium (Ichimura 1971) with 2.3 mM casamino
acids. Cells were maintained under fluorescent illumination (16 : 8 h light : dark (LD), 50 mmol photons
m-2 s-1) at 25°C.
Microscopy
Cells of symbiotic algae were observed under light
microscopy (BX51; Olympus, Tokyo, Japan), and photos
were taken with an Olympus model DP50 digital
camera. For transmission electron microscopy, the cultured cells were prepared and investigated according to
Iwataki et al. (2002) and were examined using a JEM
1010 (JEOL, Tokyo, Japan) at an accelerating voltage of
80 kV.
R. Hoshina et al.
DNA extraction, amplification,
and sequencing
For DNA extraction, we used a DNeasy plant mini kit
(Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions. We designed two reversedirection primers, Ls3Ce2 (5′-CGA ACC ACG GCT GAA
TCT-3′) and Ls3Ce3 (5′-CGA ACC ACG GCT GAA TCT
C-3′); both anneal to the 3′ side of the H2836 helix
(named large subunit (LSU) rRNA in Escherichia coli).
The Pbi rRNA cistron was amplified using PCR with the
following eight primer pairs: SR-1/SR-5, SR-4/SR-9,
SR-8/SR12, INT4F/HLR3R, HLR0F/LR5, HLR5F/LR8,
HLR7F/HLR9R, and HLR9F/Ls3Ce3. The primers used
for PCR and sequencing are described in Hoshina et al.
(2004) and Hoshina and Imamura (2008b). The LSU
rDNA 3′ end of NC64A was amplified with the primer
pair HLR10Fk/Ls3Ce2. The PCR products were confirmed using agarose gel electrophoresis, purified via
polyethylene glycol precipitation, and then sequenced
directly. A fragment of the HLR9F/Ls3Ce3 (Pbi)
product could not be read by direct sequencing; thus,
this region was re-amplified with the primer pair
HLR10F/LR12k, which was subcloned into the pGEM-T
Easy Vector (Promega, Madison, WI, USA). The subcloning procedure defined polymorphisms with one
nucleotide indels within the L2449 intron, indicating
that the sequence was made unreadable by one of the
nucleotide indels.
SSU rDNA + ITS2 phylogeny
We collected chlorellacean strains with published SSU
rDNA and ITS2 sequences from GenBank in May 2009.
Redundant strains of some species, for example, data
from more than 10 strains of ‘American’ symbionts,
were omitted. We used two sequence datasets of
X74001 (SSU rDNA) with AY323463 (ITS2), and
FM205860 (contains both SSU rDNA and ITS2) for
strain 260 of the Culture Collection of Autotrophic
Organisms (CCALA), Czech Republic.
The SSU rDNA sequences were initially aligned
using Clustal X version 2.0.10 (Larkin et al. 2007) and
then manually aligned taking into consideration secondary structure models for Chlorella vulgaris (Huss &
Sogin 1990). Introns and 5′ and 3′ terminal regions
were removed; thereafter, 1752 aligned sites remained
(these alignment data are available at our website,
http://www.ritsumei.ac.jp/pharmacy/imamura/chlorella.
html/). The ITS2 sequences were folded using Mfold
3.2 (Mathews et al. 1999; Zuker 2003), and the resulting secondary structures were used to assist in the
manual alignment of the ITS2 sequences. We excluded
rapidly evolving helices I and IV as well as the top half
of helix II and the top of helix III because of unreliable
alignment. Consequently, 136 sites remained; this
© 2010 Japanese Society of Phycology
Taxonomy of the photobionts of Paramecium
ITS2 alignment is shown in Supplementary Fig. S1.
Further analyses were conducted using the combined
data from the SSU rDNA and ITS2 alignments (number
of parsimony-informative characters = 100, and 1739
characters were constant).
The phylogenetic analyses were based on maximum
likelihood (ML) methods in PAUP 4.0b10 (Swofford
2003). Based on the Akaike’s Information Criterion, the
best-fit evolutionary models for ML analysis were determined using Modeltest 3.7 (Posada & Crandall 1998),
which selected the TrNef + G + I evolutionary model
with the following parameters: substitution-rate matrix
of AC = 1, AG = 3.1455, AT = 1, CG = 1, CT = 7.5961,
and GT = 1; proportion of sites assumed to be invariable = 0.8376; rates for variable sites assumed to
follow a gamma distribution with shape parameter = 0.9101; and number of rate categories = 4. With
these settings, a heuristic search was performed using
the neighbor-joining tree as the starting tree and a
nearest-neighbor interchange swapping algorithm.
Bootstrap probabilities were computed for 100 replicates with these settings. Further bootstrap analyses
(100 replicates each) were performed using the
neighbor-joining method of Jukes and Cantor, minimum
evolution of maximum composite likelihood with
gamma parameter = 0.8, and maximum parsimony in
MEGA version 4.1 (Tamura et al. 2007). All trees are
shown in Supplementary Fig. S2.
Pairwise analyses among Chlorella-related
species
Small subunit rDNA (exon only) and ITS2 sequences of
selected species of Chlorella, Micractinium Fresinius,
and Meyerella Fawley et K. Fawley were compared. SSU
rDNA sequences of 12 members were initially aligned
using Clustal X and then manually aligned by eye. The
5′ and 3′ terminal regions were removed, and 1742
sites were used. ITS2 sequences were also aligned
manually. Unreliable areas of helices I, II, and IV were
removed, and 193 aligned sites remained (alignment
data are shown in Supplementary Fig. S3).
RESULTS
Chlorella variabilis Shihira et Krauss
1965 Figs 1–3
Solitary cells without mucilaginous covering, planktonic, spherical or ovoid, 2.3–5.8 ¥ 2.5–6.6 mm. Chloroplast single cup- or girdle-shaped, with an ellipsoidal
pyrenoid covered by grains of starch. Thylakoid lamellae penetrating pyrenoid matrix. Asexual reproduction
by autospores. Differs from other species of the genus
by the order of the nucleotides in the SSU rRNA and
ITS2.
© 2010 Japanese Society of Phycology
191
Holotype: figures 3 and 4 in Shihira and Krauss
1965 (page 43)
Synonym: ‘Chlorella paramecii’ Loefer nom. nud.
Type locality: Endocyte of P. bursaria collected from
USA
Distribution: Only known endocyte of P. bursaria
collected from the eastern United States (and unspecified regions), Japan, Shanghai (China), and Melbourne
(Australia)
Micractinium reisseri Hoshina, Iwataki et
Imamura sp. nov. Figs 4–6
Cellulae solitariae, sine tegumento gelatinoso, planctonicae, sphericae, 3.7–8.0 mm in diametro. Chloroplastus unicus parietalis patera vel poculum pyrenoide
ellipsoidea granis amyli tecto. Matrix pyrenoidis penetrantibus lamelli thylacoidum. Propagatio asexualis
per autosporas. Speciebus ceteris generis ordine nucleotidorum in SSU rRNA et ITS2 differt.
Solitary cells without mucilaginous covering, planktonic, spherical, 3.7–8.0 mm in diameter. Chloroplast
single parietal, saucer- or cup-shaped with an ellipsoidal pyrenoid covered by grains of starch. Thylakoid
lamellae penetrating pyrenoid matrix. Asexual reproduction by autospores. Differs from other species of the
genus by the order of the nucleotides in the SSU rRNA
and ITS2.
Holotype: TNS-AL-56965 in TNS (Department of
Botany, National Museum of Nature and Science,
Tokyo), resin embedded CCAP 211/83 (Pbi)
Type locality: Endocyte of P. bursaria collected from
Göttingen, Germany.
Distribution: Only known endocyte of P. bursaria
collected from western European areas: England,
Germany, Austria, and northern Europe, Karelia region
(Russia).
Etymology: Named in honor of the work of Professor
Werner Reisser on symbiotic algae.
As described below, SSU rDNA data of hydra symbionts (primary owner of ‘Z. conductrix’) differ from
those of P. bursaria symbionts. Thus, ‘Z. conductrix’
cannot be used to refer to P. bursaria symbionts. ‘C.
paramecii’ obviously violates current Botanical Code. In
terms of the problematic ‘C. variabilis’, although CCAP
211/6 cannot be confirmed, the duplicate strain SAG
211-6 matches the other ‘American’ symbionts in its
rDNA-ITS level. Apparently, at one of the two collections, the strain was mislabeled at some point after the
transfer to Göttingen. The assumption that the SAG
strain is the Loefer’s isolate is much more reasonable,
as its sequence is identical to that of the ‘American’
symbionts. Thus, we have confirmed SAG 211-6 as the
true authentic strain and the validity of the species
name ‘C. variabilis’. The ‘European’ algae should be
considered a new species of the genus Micractinium
192
R. Hoshina et al.
Fig. 1–6. Morphology and cytology of Chlorella variabilis NC64A and Micractinium reisseri Pbi. Scale bars are 10 mm for light
micrographs (Figs 1,4) and 1 mm for transmission electron micrographs (Figs 2, 3, 5 and 6). 1. Chlorella variabilis, general morphology. 2. Chlorella variabilis, vegetative cell. Arrowhead indicates the thylakoid membranes penetrating the pyrenoid matrix. 3. Chlorella
variabilis, cell during division. 4. Micractinium reisseri, general morphology. 5. Micractinium reisseri, arrowhead indicates the thylakoid
membranes penetrating the pyrenoid matrix. 6. Magnified view of M. reisseri pyrenoid. Arrowhead indicates the double-layered thylakoid.
© 2010 Japanese Society of Phycology
Taxonomy of the photobionts of Paramecium
(see below), and we have named it M. reisseri. Pbi and
NC64A are the most widely used strains, and we chose
strain Pbi as the authentic strain for M. reisseri. Pbi was
isolated in 1974 by Dr Werner Reisser from P. bursaria
collected from a pond in the Old Botanical Garden of
Göttingen University, Germany, and the name was
derived from, simply, ‘Paramecium bursaria isolate’.
This strain is available from CCAP. NC64A first
appeared in a report by Karakashian and Karakashian in
1965. This strain was isolated from P. bursaria syngen
1 collected in North Carolina, USA, and resides at the
ATCC and CCAP. Known strains with affiliations that
were confirmed by DNA sequence analysis are listed in
Table 1.
Morphological and cytological observations
The vegetative cells of C. variabilis in culture were
spherical, ellipsoidal, or ovoid, and 4.0–6.5 mm in
diameter (Fig. 1). Each cell contained a cup- to girdleshaped chloroplast. A pyrenoid was usually seen within
the chloroplast. The mother cells produced four
autospores. The vegetative cells of M. reisseri in culture
were almost spherical and 5–8 mm in diameter (Fig. 4),
somewhat larger than C. variabilis. A single saucer- or
cup-shaped chloroplast per cell was present. A pyrenoid
was usually seen within the chloroplast. The mother
cells produced four autospores. Outer structures, for
example, mucilage, spines, or threads, have never been
observed in either C. variabilis or M. reisseri.
A single-layered cell wall and pyrenoid structure
cleaved by the thylakoid membranes were commonly
observed in C. variabilis and M. reisseri in electron
microscopic observations (Figs 2,3 and 5). The
pyrenoid was surrounded by thinner starch grains in
each species in this study, although several studies
have reported a pyrenoid with thick starch grains when
symbionts inhabit P. bursaria (e.g. Reisser 1987,
1988); this thickness seems to vary according to
culture conditions. Observed C. variabilis maintains
consistency with the original description (Shihira &
Krauss 1965), although these morphological and cytological characters are common in most chlorellacean
taxa.
Sequence of the rRNA cistron
We sequenced the rRNA cistron of the strain Pbi.
Between the primers SR-1 (SSU rDNA 5′ end) and
Ls3Ce3 (LSU rDNA 3′ end), sequences reached 6460
or 6461 bases (due to polymorphism including indels)
including the primer sequences (AB506070/
AB506071). These sequences included two group I
introns at SSU rRNA 651 and LSU rRNA 2449 (the
numbering reflects their homologous positions in the E.
coli rRNA gene; Fig. 7). SSU rDNA (with S651 intron),
© 2010 Japanese Society of Phycology
193
ITS1, 5.8S rDNA, and ITS2 sequences were completely
identical to those we previously introduced as the ‘European’ algae, SW1-ZK, and the algal sequence data
directly amplified from whole P. bursaria (strains CCAP
1660/11, 1660/12) extracts (Table 1). The exonic
region of Pbi LSU rDNA differed at three or four (due to
polymorphisms of SW1-ZK) nucleotide sites compared
with SW1-ZK. The L2449 intronic sequence of Pbi
differed by one to three (due to polymorphisms of Pbi)
nucleotide sites compared with SW1-ZK. Concomitantly with the sequencing of Pbi, we further sequenced
NC64A LSU rDNA with the newly designed 3′ end
primer Ls3Ce2 and added another 270 bp
(AB506072) to our previous NC64A sequences
(AB206549 (SSU rDNA–ITS2) and AB236862 (LSU
rDNA)). Comparisons of the LSU rDNA sequences
revealed a total of 70 nucleotide substitutions (0.0227
substitutions per site) between Pbi and NC64A, 85
substitutions (0.0276) between Pbi and C. vulgaris
NIES-227 (AB237642), and 59 substitutions
(0.0191) between NC64A and C. vulgaris. For the first
800 bp of the LSU rDNA sequences (expected to be a
mutation-intensive region, see Hoshina & Imamura
2008b), we found 41 substitutions (0.0513) between
Pbi and NC64A, 49 substitutions (0.0613) between
Pbi and C. vulgaris, and 39 substitutions (0.0488)
between NC64A and C. vulgaris.
Internal transcribed spacer 2 secondary
structure
Secondary structure diagrams of the C. variabilis SAG
211-6 and M. reisseri Pbi ITS2s are shown in Fig. 8.
Both structures possess ITS2 motifs conserved among
the green algae (Mai & Coleman 1997), i.e. fourfingered hand (four helices), pyrimidine-pyrimidine
bulge on helix II, and the conserved sequence TGGT
(UGGU) on the 5′ side of helix III. Of special note is the
difference in paired nucleotides at the tip of helix III.
Chlorella variabilis exhibits G-C pairing, whereas M.
reisseri exhibits C-G pairing. This compensatory base
change (CBC), i.e. a G-C to C-G change, is the synapomorphic signature of Micractinium (Luo et al. 2006,
2010). We found distinct characteristics in the M.
reisseri ITS2 structure. In all members of the Chlorella
clade, helix II is composed of two double-stranded
regions articulated by an elbow-like bulge. Micractinium reisseri has a significantly large elbow with 10
‘bachelor’ nucleotides, although the other species have
three to six bachelor nucleotides (also refer to alignment data in Supplementary Figs S1 and S3).
Phylogenetic placement
Phylogenetic analyses with the combined dataset of the
SSU rDNA and ITS2 were performed, and an ML tree is
P. bursaria strain
Micractinium reisseri
Algal strain
Göttingen, Germany
Schwarzwald, Germany
Göttingen, Germany
Cambridge, UK
Cambridge, UK
Wildbichl, Austria
Piburger See, Austria
Russia
Karelia, Russia
Karelia, Russia
Karelia, Russia
SSU-ITS1-5.8S-ITS2-LSU
SSU-ITS1-5.8S-ITS2-LSU
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
SSU (partial), ITS1
SSU (partial), ITS1
SSU (partial), ITS1
SSU (partial), ITS1
SSU (partial)
SSU (partial)
Available region
SSU-ITS1-5.8S-ITS2
SSU (partial)
SSU-ITS1-5.8S-ITS2-LSU
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
SSU-ITS1
SSU-ITS1
SSU-ITS1
SSU-ITS1-5.8S-ITS2
SSU-ITS1-5.8S-ITS2
Ohio, USA
USA
Aichi, Japan
Nagano, Japan
(cross breed, Japan-Japan)
Shimane, Japan
Ibaraki, Japan
Hiroshima, Japan
Hiroshima, Japan
Miyazaki, Japan
Oita, Japan
Shanghai, China
Melbourne, Australia
Collection site
SSU, ITS2
SSU-ITS1-5.8S-ITS2-LSU
Available region
USA
North Carolina, USA
P. bursaria collection site
†Algal DNA sequence directly obtained from whole Paramecium extract (Hoshina et al. 2005).
CCAP 1660/11
CCAP 1660/12
SW1
OK1
So13
F36
KM2
Dd1
Bnd1
HB2-2
shiP-7
takaP-3
Cs2
MRBG1
SAG 211-6
ATCC 50258/CCAP
211/84 (NC64A)
ATCC 30562
N-1-A
NIES-2541 (OK1-ZK)
So13-ZK
NIES-2540 (F36-ZK)
KM2-ZK/pbKM2
Dd1-ZK
Bnd1-ZK
HB2-2-1
shiP-7-A4
takaP-3-A2
(uncultured)†
(uncultured)†
CCAP 211/83 (Pbi)
SW1-ZK
SAG 241/80
(uncultured)†
(uncultured)†
PbW
PbPIB
Pbu
OCH
OC-1
OC-6
P. bursaria strain
AB506070-71, FM205852
AB206547, AB437244-56
FM205851
AB206548
AB260894
EF030566, EF030583
EF030565, EF030582
EF030562, EF030579
EF030561, EF030578
AY876298
AY876299
Accession numbers (major ones)
AB206550
AY876293
AB162912, AB437257
AB162913
AB162914
AB162915, EF030567, EF030584
AB162916
AB162917
AB191205
AB191206
AB191207
AB206546
AB219527
AB260893, AB301072, FM205849
AB206549, AB236862, AB506072
Accession numbers (major ones)
Chlorella variabilis and Micractinium reisseri: sources of Paramecium bursaria and algal rDNA accession numbers
Chlorella variabilis
Algal strain
Table 1.
This study; Luo et al. (2010)
Hoshina et al. (2005)
Luo et al. (2010)
Hoshina et al. (2005)
Hoshina et al. (2005)
Summerer et al. (2008)
Summerer et al. (2008)
Summerer et al. (2008)
Summerer et al. (2008)
Gaponova et al. (2007)
Gaponova et al. (2007)
References
Hoshina & Imamura (2008a); Luo et al. (2010)
This study; Hoshina et al. (2005); Hoshina &
Imamura (2008b)
Hoshina et al. (2005)
Gaponova et al. (2007)
Hoshina et al. (2004)
Hoshina et al. (2004)
Hoshina et al. (2004)
Hoshina et al. (2004); Summerer et al. (2008)
Hoshina et al. (2004)
Hoshina et al. (2004)
Unpublished
Unpublished
Unpublished
Hoshina et al. (2005)
Hoshina et al. (2005)
References
194
R. Hoshina et al.
© 2010 Japanese Society of Phycology
Taxonomy of the photobionts of Paramecium
Fig. 7.
195
Survey of nuclear ribosomal RNA cistrons of Chlorella variabilis and Micractinium reisseri with insertion sites of group I introns.
Subgroup IC introns are shown in normal font, and IE introns are in bold. The numbering reflects their homologous positions in the
Escherichia coli rRNA gene: L, large subunit of rRNA; S, small subunit of rRNA. Figure modified from Hoshina and Imamura (2009b).
A
C
G
G
A
U
G
G
A
U
G
G
U
U
C
G
G
U
A
A
C
G
G
G
A
C
G
U
U
U
U
C
C
C
U
A
C
A
C
G
A
U
G
G
G
C
A
A
U
G
G
U
U
C
G
G
U
A
A
C
G
G
G
A
G
C
C
U
G
C
C
G
U
C
G
C
C
C
U G
U
U
G
A
G
C
C
G
C
G
C
GA
AG
C
G
UU
U A
U
C
G
G
C
A
UG
GU
G
U
G
C
GG
C C
C
A
G
C
A
C
C G
A
U
G C
U
A
G C
G C
U
A
G C
G C
C G
U
A
A A
G A A U
AG
C
C
U
G
C
G
U
C
U
U
C
A
C
C
U
G
A
G
A
C
A
C
G
G
A
G
A
C
G
U
U
U
U
C
A
G CC
G
G
AG
AC
UG
C
C
C
U C
U
C
G C
G
C
C G
G
G
CU
G C
C
C
G C
C
U C
C
U
G
G A A GGGC
C
U
G
A
U
C
U
G C
C U U U C CG
U
U
UC
A
U
C
C
A
U
A
U
G C
G C
U
A
C
A
U U
U
U
U
G
C
U
A
C
G
C
A
G
C
C
U
G
C
C
G
U
C
G
C
C
C
U G
G
A
G
C
C
G
C
G
C
G
AG
C
G
CU
U
U A
U
C
G
G
C
A
UGG
C
G
G
C C
C
A
G
A
C
A
U
A
G C
C
G C
G C
G C
G C
C G
G A A C G C
G
A
G
C
U
C
U
C
A
C
G
A
A
C
G
U
A
U
C
G
U
G
G
A
G
A
C
G
U
U
U
U
C
A
G CU
G
G
AG
CC
CG
C
C
C
U C
U
C
G C
G
C
C G
G
G
CU
G C
C
C
U
U
C
CA C
G C
G
C
G
G
A
G
U
U
C
A
U
C
G C
C
U
U
U
A A GU C
C
A
G
C
G C
C
G C
G C
C
U
C
U
A C
G
U U
C
G
Fig. 8. ITS2 secondary structure diagrams for Chlorella variabilis and Micractinium reisseri. Both structures possess ITS2 motifs
conserved among the green algae i.e. four-fingered hand (four helices), pyrimidine-pyrimidine bulge (indicated by ‘py’) on helix II, and the
conserved sequence TGGT (UGGU in boldface) on the 5′ side of helix III. The key compensatory base changes (CBCs) on the tip of helix
III separating Micractinium from Chlorella are boxed. ‘E2’ indicates the elbow-like bulge connecting two double-stranded regions within
helix II. Non-canonical (non-Watson-Crick) pairings (e.g. G-U pairing) are shown by dots. Models are modified from Hoshina and Imamura
(2008a).
shown in Fig. 9 (all other trees in the analyses are
shown in Supplementary Fig. S2). Both C. variabilis
and M. reisseri were included in the Chlorella clade. We
did not include the previously mentioned misidentified
© 2010 Japanese Society of Phycology
‘Chlorella vulgaris’ in the phylogenetic analyses due to
a lack of ITS2 data (AB191205-207, covering SSU
rDNA to ITS1). However, the affiliations are obvious.
‘Chlorella vulgaris’ has three group I introns at S943,
196
R. Hoshina et al.
Fig. 9. A maximum likelihood (ML) tree constructed from a combined analysis of small subunit (SSU) rRNA and ITS2 sequences (length
of 1888 bp). This tree (-ln L = 4148.15) was obtained using the TrNef + I + G evolutionary model. Bootstrap values above the internal
nodes were inferred from ML (left) and minimum evolution of the maximum composite likelihood (right) analyses, whereas the
neighbor-joining method of Jukes and Cantor (left) and maximum parsimony (right) are shown below the nodes; only values above 50%
support are given. Species residing in Paramecium bursaria are in bold.
S1367, and S1512 (true C. vulgaris does not contain
any intronic insertions), and, for example, the ‘C. vulgaris’ HB2-2-1 sequence (AB191205) is completely
identical to that of C. variabilis NC64A. In contrast, the
algal sequence that was directly obtained from P. bursaria CCAP 1660/10 supported its monophyly to true C.
vulgaris (SAG 211-11b is the authentic culture) with
100% bootstrapping in all analyses. Chlorella variabilis
claded with Meyerella planktonica Fawley et K. Fawley,
but M. planktonica was more than 0.01 substitutions/
site away from the branching point. The Chlorella vulgaris – C. lobophora Andreyeva clade and the clade of
C. sorokiniana – Chlorella sp. IFRPD (Institute of Food
Research and Product Development at Kasetsart University, Thailand) strains were moderately to highly supported; however, the monophyly of the genus Chlorella
was not supported regardless of whether C. variabilis
was included. The sequence sets of strain CCALA 260
occupied two different positions: one (FM205860) in
the strain C. sorokiniana – Chlorella sp. IFRPD clade
and the other (X74001 + AY323463) in a clade consisting of Micractinium spp. as a sister of M. reisseri.
Evolutionary divergences among Chlorella,
Micractinium, and Meyerella species
Evolutionary divergences among selected Chlorella,
Micractinium, and Meyerella species are shown in
Table 2. The divergences between SSU rDNA
sequences were very low, limited to 0.0133 substitutions per site (between C. vulgaris and Meyerella planktonica) or less. Although unreliable positions were
removed in the comparisons, ITS2 divergences reached
0.1176 or more (except among C. sorokiniana and
IFRPD strains). These are thought to be crucial data for
the separation of each species. We compared the SSU
r DNA sequences to those of the hydra symbionts. Five
sets of SSU rDNA sequence data exist for the hydra
symbionts, of which the three strains Esh, HvT, and
Ssh (X72706, X72707, and X72854) belong to the
Chlorella clade (Hoshina et al. 2005). These three
sequences do not contain any group I introns, and the
exonic regions differ by 0.0029–0.0040 substitutions
per site (five to eight nucleotide changes) from C.
variabilis and by 0.0012–0.0023 substitutions (two to
© 2010 Japanese Society of Phycology
© 2010 Japanese Society of Phycology
197
Asterisks indicate the strains isolated from green hydra (Huss et al. 1993/94). The number of base substitutions per site from analysis between sequences is shown. All results are based
on the pairwise analyses of 14 sequences for small subunit (SSU) rDNA (lower-left) and 11 sequences for ITS2 (upper-right). Analyses were conducted using the Jukes-Cantor method in MEGA.
All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There were a total of 1742 positions for SSU rDNA
and 193 for ITS2 in the final datasets.
0.0029
0.0058
0.0116
0.0087
0.0122
0.0174
0.2000
0.1641
0.2326
0.2456
0.2057
0.1896
0.2032
0.0017
0.0035
0.0075
0.0110
0.0017
0.0012
0.0029
0.0069
0.0116
0.0017
0.0023
0.0017
0.0035
0.0075
0.0122
0.0058
0.0058
0.0052
0.0046
0.0052
0.0064
0.0058
0.0064
0.0058
0.0064
0.0116
0.0116
0.0000
0.0006
0.0035
0.0006
0.0017
0.0012
0.0017
0.0012
0.0029
0.0069
0.0104
0.0006
0.0035
0.0006
0.0017
0.0012
0.0017
0.0012
0.0029
0.0069
0.0104
0.0029
0.0012
0.0023
0.0017
0.0023
0.0017
0.0023
0.0075
0.0098
0.1422
0.1752
0.1422
0.1360
0.1176
0.1941
0.1752
0.0481
0.0426
0.2284
0.1687
0.0052
0.2354
0.1752
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Chlorella vulgaris SAG 211-11b
Chlorella lobophora SAG 37.88
Chlorella sorokiniana SAG 211-8k
C. sorokiniana CCALA 260
Chlorella sp. IFRPD1018
Chlorella variabilis SAG 211-6
Chlorella sp. Esh*
Chlorella sp. HvT*
Chlorella sp. Ssh*
Micractinium reisseri Pbi
Micractinium sp. CCALA 260
Micractinium belenophorum SAG 42.98
Micractinium pusilum SAG 13.81
Meyerella planktonica 2/24-S-1w
0.0052
0.0040
0.0040
0.0046
0.0063
0.0035
0.0046
0.0040
0.0046
0.0040
0.0058
0.0098
0.0133
0.2158
0.0029
0.0040
0.0035
0.0040
0.0035
0.0040
0.0093
0.0081
0.0012
0.0006
0.0012
0.0006
0.0023
0.0064
0.0110
8
7
6
5
4
3
2
1
Estimates of evolutionary divergence between sequences
Table 2.
0.1141
0.1784
0.1251
0.2227
0.1964
0.1557
0.1493
0.1493
0.1243
0.2963
0.2677
0.2088
0.2020
0.2298
0.2369
0.2828
0.2846
0.2032
0.1964
0.2171
0.2101
0.2341
0.2883
0.1988
0.1919
0.1784
0.1585
0.2241
0.2184
0.1697
0.1631
0.1697
0.1189
13
9
10
11
12
14
Taxonomy of the photobionts of Paramecium
four nucleotides) from M. reisseri (ITS2 sequences of
hydra symbionts were not available). Although these
hydra symbiont sequences may exemplify the diversity
of such entities, they can be discriminated from the P.
bursaria symbionts.
DISCUSSION
Because of its extremely simple morphology with an
exclusively asexual reproductive system, ‘the green
ball’, the genus Chlorella, has caused severe taxonomic
problems. In 1999, Huss et al. restricted the true Chlorella species to C. vulgaris (type species) and three
other species (C. lobophora, C. sorokiniana, and C.
kessleri) due to their comparatively close relationships
based on SSU rDNA phylogeny. Krienitz et al. (2004)
split them into the Chlorella clade (including C.
vulgaris, C. lobophora, and C. sorokiniana) and the
Parachlorella clade (including C. kessleri as a member
of the new genus Parachlorella Krieniz, Hegewald, Hepperle, Huss, Rohr et Wolf) based on SSU rDNA and
ITS2 phylogeny. Each clade includes algae of different
morphologies including colonial or coenobial life-forms;
fusiform, spindle, or needle shapes; and with mucilage,
bristle, spines, or threads (Ustinova et al. 2001; Wolf
et al. 2002; Krienitz et al. 2004). Furthermore, Krienitz et al. (2004) designated the Chlorellaceae for
the Chlorella clade and Parachlorella clade together.
Another new alga, Meyerella planktonica, was
described by Fawley et al. (2005). This species appears
similar to the true Chlorella species in morphology and
is phylogenetically included in the Chlorella clade or is
basal to the clade. However, due to the lack of a
pyrenoid, the authors established a new genus, Meyerella. Consequently, in consideration of the historical
literature, the definition of the genus Chlorella should
be automatically determined by a combination of the
three criteria of morphology, cytology, and molecular
phylogeny, i.e. spherical to ellipsoidal unicellular green
alga lacking any motile stage (without flagella),
autosporulation, possession of a single nucleus, a chloroplast with a pyrenoid whose matrix is divided by the
double-layered thylakoid, a mitochondrion, and a phylogenetic affiliation to the Chlorella clade. Although it
remains unclear whether all members of Chlorella have
a monophyletic origin within this clade, both C. variabilis and M. reisseri meet all of these requirements
(Figs 1–6 and 9). However, most recent studies have
overturned such a generic concept. Luo et al. (2006)
characterized the genus Micractinium, which is closely
related to Chlorella but morphologically different
because of the formation of bristles. However, the
authors emphasized the phenotypic plasticity of bristle
formation, which in some cases could only be induced
by the grazing rotifer Brachionus. Micractinium and
Chlorella could only be genetically differentiated by
198
their variable ITS sequences, specifically by a unique
(for Micractinium) CBC in helix III of ITS2 (Luo et al.
2010; see also Fig. 8 and Supplementary Fig. S3),
which is a characteristic for distinguishing the genus
Micractinium from other genera within the Chlorella
clade. Finally, Luo et al. (2010) extended the generic
concept in Chlorella-related green algae based on phylogeny and synapomorphic nucleotide changes in SSU
rDNA and ITS2. Accordingly, the genus Chlorella now
also contains gelatinous and colonial species such as
Dictyosphaerium Nägeli, whereas the genus Micractinium contains some spherical species as well as
species of the other bristled genus Diacanthos Korshikov (see Fig. 9). Didymogenes Schmidle is another
genus that includes both bristle-bearing and nonbristle-bearing species (Schnepf & Hegewald 1993).
The monophyly of D. anomala (G. M. Smith) Hindák
(bristle-bearing) and D. palatina Schmidle (non-bristlebearing) was partially supported (Fig. 9).
Our phylogenetic analyses indicate that the ‘European’ symbionts are members of Micractinium (Fig. 9).
The ITS2 nucleotide-change characters of ‘European’
symbionts are also congruent (Fig. 8) to those of
Micractinium indicated by Luo et al. (2006, 2010).
These facts strongly indicate the affiliation of ‘European’ symbionts to the genus Micractinium. Meanwhile, we used two sequence datasets for strain CCALA
260. The SSU rDNA (X74001) sequence of CCALA
260 initially appeared in Huss et al. (1999) as ‘Prag
A14’. Subsequently, Krienitz et al. (2004) released the
ITS2 (AY323463) sequence and combined the two
sequences (X74001 + AY323463) to use for phylogenetic analyses. Luo et al. (2010) released a string
sequence of SSU-ITS1-5.8S-ITS2 (FM205860) for the
same strain. Between these SSU rDNA (X74001 vs.
FM205860), two nucleotide changes were present;
however, divergence between these ITS2 sequences
(AY323463 vs. FM205860) reached 0.1631 (data not
shown). One sequence was closer to M. reisseri, but the
other was C. sorokiniana. Mixed-up strains remain a
serious problem plaguing algal taxonomy.
‘American’ symbionts comprise a clade with Meyerella planktonica (Fig. 9). The phylogenetic positions of
Me. planktonica are fluid depending on outer and/or
inner group choice for the analyses (Fawley et al. 2005,
and preliminary data in this study). We believe this
issue is the effect of a long branch attraction artifact
(Felsenstein 1978) caused by the fact that the Me.
planktonica sequence is less similar to any other
sequences; therefore, the monophyly of C. variabilis
and Me. planktonica is unreliable. ‘American’ symbionts can also be separated from Meyerella by the
presence of pyrenoids (Figs 2,3). In the generic concepts based on nucleotide changes in SSU rDNA and
ITS2, the genus Chlorella could not be distinguished by
any synapomorphic character (Luo et al. 2010).
R. Hoshina et al.
Perhaps the genus Chlorella can be regarded as a paraphyletic group maintaining symplesiomorphic statuses
in terms of both cytology and nucleotide lows. Considerable molecular differences still exist for Chlorella-like
organisms, and we believe that the family Chlorellaceae
warrants more genera. However, if one were to establish
a new genus for the ‘American’ symbionts, the genus
would have only one species, and few differences would
exist between the generic and species concepts. Genus
establishment should be postponed until the diversity
and relationships become more obvious. Therefore, at
present, we tentatively use Chlorella for the ‘American’
symbionts.
Chlorella-related species are not distinguishable by
morphology or cytology. For the identification of these
species, physiological characters have often been used
(Huss et al. 1999 and references therein). However,
identical morphology with recondite physiology may
pressure investigators to give up on the identification of
Chlorella species, resulting in the generation of many
unidentified strains. The National Centre for Biotechnology Information (NCBI) Taxonomy Browser, for
example, contains more than 100 unidentified Chlorella strains, some of which may belong to existing
Chlorella species, some of which may not belong to the
genus Chlorella, and some of which may be new species
of Chlorella. Of course, describing new species in this
genus based on traditional means is extremely difficult.
Clearly, the species concept in this genus is also, at
present, facing a major turning point.
Given two organisms, the more they resemble each
other, the more difficult it will be to distinguish whether
they belong to the same species. The internal transcribed spacer 2 is currently attracting attention as a
molecular ‘barcode’ resolving such species problems
(Coleman 2003, 2007; Müller et al. 2007). A remarkable feature of this molecular marker is its high divergence between species. In the ITS2 comparisons
(Table 2), all species are clearly separated from each
other, whereas within species, those divergences are
very small. For example, more than 30 sequences of C.
vulgaris have been published, for which divergence was
only as high as 0.0126 substitutions per site for all 241
nucleotide position comparisons (data not shown). For
M. reisseri, sequence variation has thus far not been
found. Therefore, in species of the Chlorella clade,
ITS2 can be regarded as a highly conserved molecule
within species as well as a highly divergent molecule
between species. In addition to such phylogenetic informativeness, a specific structural feature between two
ITS2 secondary structures, a CBC, can also be used to
distinguish two species from each other (Coleman
2000; Coleman & Vacquier 2002; Behnke et al. 2004;
Young & Coleman 2004; Müller et al. 2007). When
comparing ITS2 helices II and III as highly conserved
regions (Coleman 2003), we always found two or more
© 2010 Japanese Society of Phycology
Taxonomy of the photobionts of Paramecium
CBCs among C. variabilis and other species (see
Supplementary Fig. S3). Helix II of Micractinium
species is structurally variable; for example, M. reisseri
includes a large bulge (Fig. 8), and Micractinium sp.
CCALA 260 has a shortened helix (see Supplementary
Fig. S3). We regard such structural differences as
equivalent to CBCs. Therefore, adequate data exist for
definitive species-level autonomy of C. variabilis and M.
reisseri.
Group I intronic insertions in their rRNA genes comprise another genetic characteristic of C. variabilis and
M. reisseri (Fig. 7). Although group I introns are known
as continually losable mobile genetic elements, in both
C. variabilis and M. reisseri, their sequences, presence
or absence, and insertion positions are stable. Chlorella
variabilis has eight group I introns in its nuclear ribosomal RNA cistron, the highest number in the Viridiplantae (Hoshina & Imamura 2008b). Some introns in
both species insert into unique positions due to their
uniquely evolved infection mechanism (Hoshina &
Imamura 2009b), which will be of considerable help in
identifying these species.
Paramecium bursaria contains green algal symbionts, and this unicellular ciliate is a textbook example
used for microscopic observation in high school science
projects. However, many textbooks now in use are
written as if P. bursaria temporally maintains algae that
were ingested from the outside. Paramecium bursaria,
as a predatory protist with quotidian phagocytotic
behavior, may invite such a misunderstanding. Paramecium bursaria symbionts comprise distinct species
inhabiting the cells of P. bursaria. Both C. variabilis
and M. reisseri demand organic nitrogen compounds
(Kamako et al. 2005) and are sensitive to Chlorella
viruses, the so-called ‘NC64A virus’ and the ‘Pbi virus’,
which are abundant in natural freshwater (Van Etten
et al. 1991; Yamada et al. 1991). As a matter of
course, they have never been collected from nature as
free-living Chlorella. In the present study, we provide
species names (one is a revival of an old name) for
these morphologically indistinguishable algae based on
DNA sequence and structural comparisons. We believe
this work offers important contributions toward constructing a genetic species concept in the fields of
microbial biodiversity and taxonomic analyses, as
well as dispelling misunderstandings about P. bursaria
symbiosis.
Supplementary figures are available at our website,
http://www.ritsumei.ac.jp/pharmacy/imamura/chlorella.
html/.
ACKNOWLEDGEMENTS
Strain Pbi was obtained from Professor James L.
Van Etten (University of Nebraska) with culturing
© 2010 Japanese Society of Phycology
199
instructions provided by Dr James R. Gurnon (University of Nebraska).
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